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10523616 | Two bacterial artificial chromosome (BAC) clones containing genomic copies of the mouse 6Ckine gene were identified in a mouse 129/sv embryonic stem cell genomic library (Genome Systems, Inc.) using PCR primers corresponding to the mouse 6Ckine cDNA: GV100 (5′-CTG CAA GAG AAC TGA ACA GAC-3′) and GV105 (5′-CTT CTG ACT CTC TAG GTC TAC-3′). Several overlapping fragments containing the 6Ckine gene were identified by Southern blot analysis of BAC plasmid DNA using a 275-bp PCR-generated probe (GV100/GV105) labeled with [ 32 P]dCTP (Amersham Corp.; 3,000 Ci/mmol) by random priming (Megaprime DNA Labeling System; Amersham Corp.). They were subcloned into pBluescript (Stratagene Inc.) and mapped by restriction digest. 6Ckine-containing SacI fragments (7.5 kb) from each of the two BAC clones were sequenced using an Applied Biosystems 377 sequencer (Applied Biosystems, Inc.). A pair of PCR primers GV104 (5′-GTA GAC CTA GAG AGT CAG AAG-3′) and GV125 (5′-CGC GGA TCC TTG GAG GAG GAA CCA CAG T-3′), shown in Fig. 2 , were used to amplify 1.35- and 1.2-kb fragments that included part of the 3′ untranslated region (UTR) and ∼1 kb downstream of the gene. PCR conditions were: 94°C for 2 min; 25 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min; and 72°C for 5 min. Both fragments were subcloned into pCR2.1 TA cloning vector (Invitrogen Corp.) and sequenced. Another pair of PCR primers, GV95 (5′-ATG GCT CAG ATG ATG ACT CT-3′) and GV105 (5′-CTT CTG ACT CTC TAG GTC TAC-3′) were used to amplify the 5′ coding region and part of the 3′ UTR of the 6Ckine gene (899 bp) from wild-type and BALB/c- plt (10th backcrossed generation) tail DNA. These fragments were also subcloned into pCR2.1 TA cloning vector (Invitrogen Corp.) and sequenced. Tail DNA was isolated from 129/sv (The Jackson Laboratory) and BALB/c- plt mice. EcoRI and HindIII (New England Biolabs Inc.)–digested mouse tail DNA and BAC DNA were denatured and blotted onto Duralon membrane (Stratagene Inc.) and hybridized with probes A and B . Total RNA was extracted from different tissues of plt and BALB/c mice using Ultraspec RNA Reagent (Biotecx Labs., Inc.), and 20 μg/lane was electrophoresed in a 1% agarose gel. The RNA was blotted onto Duralon nylon membrane (Stratagene Inc.). A 6Ckine probe was labeled with [ 32 P]dCTP (Amersham Corp.; 3,000 Ci/mmol) by random priming (Megaprime DNA Labeling System; Amersham Corp.). Hybridization was carried in QuikHyb hybridization solution (Stratagene Inc.). As part of an effort to generate 6Ckine null mice, a genomic library generated from 129/sv embryonic stem cells was screened by PCR (see Materials and Methods). Two BAC clones were identified, which we designated 6CKBAC1 and 6CKBAC2. Southern blot analysis of EcoRI-digested DNA from each BAC clone revealed two distinct banding patterns . 6CKBAC1 contained bands of 6.0 and 1.35 kb, whereas 6CKBAC2 contained two bands of 3.0 and 1.2 kb. Southern blot analysis of 129/sv mouse genomic DNA showed that all four bands were present. These results immediately suggested the possibility of two distinct 6Ckine genes in the mouse genome. Interestingly, hybridization of plt mouse genomic DNA showed only bands corresponding to those found in 6CKBAC1 , suggesting the possibility that one of the two 6Ckine genes in the plt mouse is altered. To further investigate this possibility and to determine if this was a strain-specific observation, two primers amplifying a segment of the 3′ UTR of the 6Ckine gene were used to amplify genomic DNA from various mouse strains as well as the two BAC clones. DNA from three separate mouse strains, BALB/c, 129/sv, and C57BL/6, all demonstrated two bands of 1.35 and 1.2 kb . 6CKBAC1 showed only a 1.35-kb band and 6CKBAC2 only a 1.2-kb band . These data suggest that wild-type mice have two 6Ckine genes, one of which is represented on 6CKBAC1 and the other on 6CKBAC2. Interestingly, PCR amplification of plt mouse genomic DNA showed only a single 1.35-kb band identical to the size of the band amplified from 6CKBAC1 . This result again suggested that the plt mouse has an alteration in one of the two 6Ckine genes. To conclusively demonstrate that the 6Ckine gene on 6CKBAC1 was indeed different from that residing on 6CKBAC2, SacI fragments (7.5 kb) were isolated from each BAC, subcloned, and completely sequenced. Analysis of the sequence data clearly showed that each BAC contained a distinct 6Ckine gene . Furthermore, although the overall genomic organization of the two 6Ckine genes was similar, consisting of four exons and three introns , there were a number of sequence differences. These differences included numerous single base changes as well as various small (<200 bp) deletions . Within the coding regions of the two genes, only two single nucleotide differences were found, one of which resulted in an amino acid difference at position 65. 6CKBAC1 encodes a leucine at this position, whereas 6CKBAC2 encodes a serine. A series of deletions/insertions in a region of the gene downstream from the polyadenylation signal results in a net difference of 136 bp between the two genes that accounts for the observed difference in the size of the PCR products amplified by GV104/GV125 primers . Sequencing of these two PCR fragments amplified from wild-type genomic DNA shows that the sequence of each exactly matches that of the appropriate BAC, further proving that 6Ckine is encoded by two genes and eliminating the possibility that our previous observations were the result of a BAC cloning artifact. Together, these data clearly demonstrate that there are two 6Ckine genes in mice and that each BAC carries a unique 6Ckine gene. Based on the differences in the amino acid sequence of 6Ckine derived from each gene, we designate the two chemokines 6Ckine-leu (encoded on 6CKBAC1) and 6Ckine-ser (encoded on 6CKBAC2). The initial analysis of the plt mouse DNA suggested that these mice might have an alteration in one of the two 6Ckine genes. To confirm this hypothesis and to characterize these two genes in plt mice, a pair of primers (GV95/GV105) was used to amplify an 800-bp fragment of both genes from wild-type and plt genomic DNA. PCR products were then subcloned and sequenced. DNA amplified from a wild-type mouse would be predicted to consist of sequences encoding both forms of 6Ckine, and indeed, 14/24 subclones derived from the PCR reaction encoded serine at position 65, whereas 10/24 subclones encoded leucine at this position. In contrast, DNA amplified from the plt mouse showed 12/12 subclones encoding leucine at position 65. These data confirm that 6Ckine is encoded by two separate genes in the wild-type mouse genome and suggest that the plt mouse is likely to contain mutations in the serine form of 6Ckine. Furthermore, the fact that two separate primer pairs failed to amplify the 6Ckine-ser gene suggested that the plt mouse has a deletion in this gene. To demonstrate that the plt mouse has indeed deleted one 6Ckine gene, two DNA probes corresponding to the 5′ end of the gene and the 3′ end of the gene were hybridized to DNA from 129/sv and plt mice as well as 6CKBAC1 and 6CKBAC2. Hybridization with probe A revealed two bands of the expected sizes in EcoRI-digested DNA from 129/sv mice . Only one band, corresponding to the leucine form of 6Ckine, was detected in plt mice. A band of the same size was also detected in 6CKBAC1 (encoding 6Ckine-leu) but not in 6CKBAC2 (encoding 6Ckine-ser). Similarly, hybridization with probe B showed a pattern of bands in HindIII-digested 129/sv DNA consistent with the presence of both genes, whereas plt mice showed only bands of the same size as those present in 6CKBAC1. The failure of two independent probes, encompassing at least 7 kb of the 6Ckine gene, to hybridize to plt mouse DNA corresponding to 6Ckine-ser gene shows that the plt mouse has a deletion in the 6Ckine-ser gene and that this deletion likely includes all of the 6Ckine-ser coding region. Our data demonstrates that the plt mouse has deleted the 6Ckine-ser gene but that it retains an intact 6Ckine-leu gene. A previous report has shown that the plt mouse lacks 6Ckine expression in lymphoid tissue, but expression in other tissue types was not reported 15 . As the 6Ckine-leu gene is present in the plt mouse, we thought it possible that these mice might express 6Ckine in nonlymphoid tissue. To examine this possibility, we conducted Northern blot analysis on a variety of both wild-type (BALB/c) and plt mouse organs. Consistent with our previously reported findings 4 , normal mice showed expression of 6Ckine in both lymphoid and nonlymphoid organs . As expected, plt mice were found to lack detectable expression of 6Ckine mRNA in lymphoid organs . Expression of 6Ckine was observed, however, in a number of nonlymphoid organs of the plt mouse . As the 6Ckine-ser gene is deleted in these mice, the observed 6Ckine message likely derives from expression of the 6Ckine-leu gene. 6Ckine/secondary lymphoid organ chemokine has been demonstrated in vitro to be important for lymphocyte adhesion and migration. Recent findings have also suggested a role for this chemokine in dendritic cell migration 8 9 . The lack of 6Ckine expression in the lymphoid organs of the plt mouse, along with the reported phenotype of these mice, suggests that at least some of these observations are also true in vivo. The conclusions drawn from the plt mouse regarding 6Ckine are confounded by the fact that the precise nature of the mutation in these mice remains unknown. We have presented data here that demonstrates that murine 6Ckine is, in fact, encoded by two separate genes and that one of these is deleted in the plt mouse. The two 6Ckine genes are nearly identical in the sequences of their open reading frames and show only one amino acid difference at position 65. Analysis of the public expressed sequence tag (EST) database shows that cDNAs for both forms of mouse 6Ckine are present and, in fact, both forms have been reported and characterized in the literature but not recognized as arising from independent genes. Hedrick and Zlotnik 4 reported the mouse 6Ckine-leu form, and Tanabe et al. 3 reported the mouse 6Ckine-ser form; although a direct comparison of the two mouse 6Ckine proteins had not been made, these two reports showed similar findings regarding their chemotactic activity. Furthermore, computer modeling of the differences between the 6Ckine-ser and 6Ckine-leu proteins based on known chemokine crystal structures does not predict any radical differences in the structures of the two proteins (Murgolo, N., and E. Coates, personal communication). An analysis of the human 6Ckine gene shows that it encodes a leucine at position 65 1 2 6 , and we were unable to find any ESTs corresponding to a human serine form in any of over 300 ESTs examined (Hedrick, J., and L. Wang, unpublished observation). We cannot, however, formally exclude the possibility of a second human gene. Chemokine gene duplication is relatively common, and indeed most members of this cytokine superfamily are thought to have arisen through a series of duplications of “primordial” chemokines. The duplication of mouse 6Ckine is likely to have been a relatively recent evolutionary event, as there have been few changes within the exon/intron regions of the genes. It is also possible, however, that some selective pressure has maintained the two genes in a relatively unchanged state, and thus it will be important to determine the relative contributions of each form of 6Ckine to the process of lymphocyte and dendritic cell trafficking in vivo. | Study | biomedical | en | 0.999996 |
10523617 | The fyn mutation 2 is maintained on a mixed (129 × B6) genetic background, but mice were homozygous for the NKR-P1C allele recognized by the NK1.1 antibody. In some experiments, fyn mutants that had been backcrossed 10 generations to C57Bl/6 were also used. Mice on the C57Bl/6 background that were deficient for either β2-microglobulin (β2M) or Lck were purchased from The Jackson Laboratory. C57Bl/6 and 129Sv mice were bred on site. All mice used in these studies were from 7 to 12 wk of age and were maintained under American Association of Accreditation of Laboratory Animal Care (AALAC) and institutional approved guidelines. Thymocyte and splenocyte suspensions were filtered through mesh to obtain single cell cultures. Spleen suspensions were also depleted of red blood cells by treating with 0.14 M ammonium chloride. Liver lymphocytes were obtained as detailed elsewhere 27 . The following antibodies were used for flow cytometric analysis: anti–TCR-β–FITC (H57-597), anti-NK1.1–PE (PK 136), Fc block (2.4G4), anti-CD4–PE (GK1.5), anti-Ly9.1–FITC (30C7), and anti–heat stable antigen (HSA)–biotin (M1/69) from PharMingen, and Tricolor-strepavidin from Caltag. Three-color immunofluorescence analysis was performed using a FACScan™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software. The Vα14Jα281 rearrangement was detected after RT of 5 μg total RNA using random hexamers and Superscript II reverse transcriptase (GIBCO BRL). Quantitative PCR was performed using the LightCycler (Roche Molecular Biochemicals). In brief, 2 μl of a 1:4 dilution of the RT reaction was amplified in the following reaction mixture: 67 mM Tris, pH 8.8, 16.6 mM ammonium sulfate, 6.7 mM magnesium chloride, 5 mM β-mercaptoethanol, 0.01% gelatin, 10% DMSO, 1 mM dNTPs, 100 ng each primer, 1.5 U Taq, and a 1:20,000 dilution of SYBR green gel stain (Roche Molecular Biochemicals). A standard curve was made using dilutions of an RT reaction from C57Bl/6 splenocytes. The relative value for each sample was then calculated using the LightCycler software. To control for variations in the RT reaction, all PCR reactions were normalized for hypoxanthine phosphoribosyltransferase (HPRT) expression. The following primers were used: HPRT sense, GTAATGATCAGTCAACGGGGGAC; HPRT antisense, CCAGCAAGCTTGCAACCTTAACCA; Vα14 5′, CTAAGCACAGCACGCTGCACA; J281 3′, CAGGTATGACAATCAGCTGAGTCC 9 . To induce cytokines in vivo, mice were injected intravenously through the retroorbital sinus with either 5 μg anti-CD3 (2C11) or PBS. After 1.5 h, the mice were killed, splenocytes were isolated, and RNA was purified. For the in vitro studies, T cells were enriched for by passing splenocytes over a nylon wool column. The suspensions averaged 80–90% T cells. Aliquots of 20 × 10 6 cells were stimulated in a 2-ml volume with 5 μg/ml anti-CD3 (2C11) and 1 ng/ml PMA or 1 ng/ml PMA and 500 nM ionomycin at 37°C. After 2 h, RNA was isolated and 7.5 μg of RNA was analyzed using the RiboQuant RNase protection assay system (PharMingen), probe set m-CK1. The samples were separated on a 5% acrylamide/urea gel and dried at 80°C for 60 min. The gels were then exposed on a PhosphorImager ® (Molecular Dynamics) or directly onto film. Thymocytes from five C57Bl/6 mice were cultured in 6-well dishes at 30 × 10 6 cells/ml in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 25 IU/ml penicillin, 25 μg/ml streptomycin, 50 mM β-mercaptoethanol, 11 μg/ml sodium pyruvate, 3.6 μg/ml asparagine, 0.6 μg/ml folic acid, 11.2 μg/ml arginine, and 150 U/ml human recombinant (hr)IL-2 (Roche Molecular Biochemicals) plus 1% conditioned media from the IL-7–producing cell line, J558. After 5 d, dead cells were removed by centrifugation through Lympholyte-M (Accurate Chemical, Inc.). The cells were then cultured at 2 × 10 5 cells/ml in media with 150 U/ml hrIL-2. After 3 d, the cells were harvested and used for fluorescent sorting after staining with anti-NK1.1–PE and anti–TCR-β–FITC. Protein lysates were resolved by 8% SDS-PAGE, then transferred to polyvinylidene difluoride membrane (NEN). The membrane was blocked using TNB buffer (30 mM Tris, pH 7.6, 75 mM NaCl, and 3% bovine serum albumin) overnight at 4°C. Polyclonal anti-Fyn (Santa Cruz Biotechnology) or anti-Lck (Upstate Biotechnology) was added at concentrations suggested by the manufacturers and incubated for 1 h. The membrane was then incubated with 0.5 μCi/ml iodinated protein A for 1 h at room temperature, and washed with TBST (30 mM Tris, pH 7.6, 75 mM NaCl, and 0.2% Tween 20) for 1 h followed by autoradiography. 6-wk-old 129Sv mice received 700 rads whole body irradiation from a cesium source. 5 × 10 6 C57Bl/6 strain bone marrow cells (Fyn, β2M, or wild-type), plus 1 × 10 6 129Sv bone marrow cells were then injected intravenously through the retroorbital sinus. Thymus and spleen were harvested after 10–12 wk and analyzed by flow cytometry for the presence of NK T cells. The relative contribution of C57Bl/6 and 129Sv cells to the hematopoietic lineages was assessed by monitoring Ly9.1 expression, which is present only on 129Sv-derived cells. Since fyn mutant mice synthesize reduced amounts of IL-2 after cross-linking of the TCR 2 , we measured the induction of T cell–produced cytokines, IL-4 and IL-13, after TCR engagement to determine whether their production was also impaired. Splenic T cells from naive wild-type and fyn mutants were isolated and stimulated in vitro with anti-CD3∈ plus PMA for 2 h. No IL-4 RNA was detected in the fyn mutant cultures , but was present in the wild-type cells. Additionally, induction of IL-13, which shares some common regulatory elements and functions with IL-4 (for a review, see reference 28 ), also does not occur in the mutant cells. Longer incubation with antibody still failed to elicit IL-4 or IL-13 in the mutant cultures (data not shown). To confirm the in vitro data, naive mice were injected with anti-CD3∈ antibody to induce cytokine production. Analysis of spleens from treated mice again revealed that no IL-4 or IL-13 RNA was induced in the fyn mutants, whereas they were readily detectable in wild-type mice . To determine if the failure in IL-4 production was due to defective activation through the TCR, T cells were treated in vitro with ionomycin and PMA to bypass the requirement for surface receptors. Splenic T cells from wild-type mice produced abundant amounts of IL-4 and IL-13 RNA, but again none was induced in fyn -deficient T cells . One interpretation of these data is that Fyn may be required for proper development of the T cell subset(s) capable of rapid IL-4/IL-13 production. The rapid accumulation of IL-4 after TCR engagement has been shown to originate mostly from the NK T cell subset 14 . The lack of IL-4 induction in fyn mutants suggests that this cell type may be missing or nonfunctional. To address this, lymphocytes were isolated from thymus, spleen, or liver, and examined for the presence of NK T cells by flow cytometric analysis . Relative to wild-type animals, the number of NK T cells in fyn −/− mice was reduced 10-fold in the thymus and >5-fold in the spleen and liver. This approached the limits of detection under the conditions used. The NK T cell number was compared with β2M-deficient mice, since this mutation prevents NK T cell development 19 29 . In all tissues examined, the fyn mutant had roughly twice as many NK T cells as β2M mice. These data indicate that loss of Fyn significantly disrupts NK T cell development. Since NK T cells are selected by CD1d, it was determined that fyn mutant mice still express normal levels of CD1d in the thymus (data not shown). This indicates that the fyn mutation does not disrupt NK T cell development by affecting CD1d surface expression. However, it remains a formal possibility that CD1d or the stromal environment may be altered in the mutant and prevent maturation of NK T cells. For example, CD1d uses a tyrosine-based motif for entering the endocytic pathway required for loading antigen and subsequent presentation to NK T cells 30 ; perhaps phosphorylation by Fyn is required for targeting to endosomes. To determine whether Fyn is required in accessory cells of the microenvironment or directly in NK T cells, radiation chimeras were constructed. Since NK T cells are selected by bone marrow–derived cells rather than thymic epithelia, it was necessary to mix bone marrows from wild-type 129Sv mice with either wild-type C57Bl/6 or mice on the C57Bl/6 background that were mutant for fyn or β2M , then inject into irradiated 129Sv mice. Cells derived from the 129Sv bone marrow should provide the appropriate environmental cues necessary for C57Bl/6 wild-type or mutant cells to develop into NK T cells. The 129Sv strain was selected for two reasons. First, the 129Sv strain does not express the NK1.1 marker, so any NK1.1 + TCR-α/β + (NK T) cells detected must be C57Bl/6 derived. Second, the 129Sv and C57Bl/6 strains express different alleles of the Ly9 cell surface marker, and the relative contributions of the two types of bone marrows to the chimera can then be determined by flow cytometry. Thymocytes from each group were analyzed for the presence of NK T cells by flow cytometry. Irradiated mice which received either wild-type or β2M mutant bone marrow developed NK T cells . Since β2M is required for stable surface expression of CD1, this result indicated that the CD1d present on wild-type 129Sv cells was able to complement the defect and allow the β2M-deficient lymphocytes to undergo selection and form NK T cells. In contrast, mice receiving the mixture of 129Sv and fyn mutant bone marrow had no detectable NK T cells. Because the wild-type 129Sv–derived cells were unable to rescue NK T cell development in the fyn mutant chimera, it is likely that this is a cell-autonomous defect in the NK T cell or progenitor. Fig. 3 B shows that all of the chimeras contain C57Bl/6-derived thymocytes at similar levels. Chimerism extended to the periphery, since spleens from all reconstituted mice contained C57Bl/6-derived NK cells and T lymphocytes as well (data not shown). This indicates that the lack of NK T cells in the chimeras made from fyn mutants is not due to poor colonization by fyn mutant bone marrow. Lck appears to be the predominant Src family member required for conventional T cell development. However, from studies using transgenic mice overexpressing Fyn, and mice with mutations in both fyn and lck , it is clear that Fyn can partially compensate for Lck and allow development of double positive and mature single positive T cells, though at a much reduced efficiency 5 6 . One possibility as to why Fyn is required for NK T cell development is that Lck may not be expressed during NK T cell development or in mature NK T cells, leaving Fyn as the only Src family member required for a later expansion or survival of NK T cells. Alternatively, Fyn may have a specific role that Lck cannot compensate for. To distinguish between these two possibilities, the expression of Lck and Fyn was determined in NK T cells. Both conventional and NK T cells were purified from thymocyte cultures, then examined for Lck and Fyn expression by Western blot analysis. As shown in Fig. 4 , equivalent amounts of Lck and Fyn are present in both NK T cells and conventional T cells. This suggests that Fyn and Lck may have nonredundant roles in NK T cell ontogeny, although it is still formally possible that Lck may not be expressed in some progenitor. This seems unlikely, since Lck is expressed throughout normal T cell development as well as in mature T cells and NK cells 31 . These data demonstrate that Lck is expressed in NK T cells as well. To assess whether Lck is required for NK T cell development, lck mutant mice were examined for the presence of this population by flow cytometry . The levels of NK T cells were reduced 10–20-fold relative to wild-type mice, and were equivalent to β2M mutant mice. Thus, both Fyn and Lck are required during NK T cell development. NK T cells are thought to progress through the pre-TCR stage of T cell development due to their requirement for pre–TCR-α 24 . Since Lck is required for proper pre-TCR signaling, it is expected that lack of Lck should have an adverse effect on NK T cell development. NK T cell ontogeny has been largely defined by various mouse mutants. Mice lacking CD1 or β2M have an early block in development as determined by a lack of cells using the invariant Vα14Jα281 TCR, as well as cells that express NK cell markers in conjunction with the TCR. It has been shown that the common cytokine receptor γ chain is required for NK1.1 expression and IL-4 secretion, but not for selection of Vα14Jα281-positive cells 32 . To gauge where the block in development lies in fyn − / − and lck − / − mice, this rearrangement was assayed for by quantitative RT-PCR using the LightCycler quantitative PCR machine . Low levels of this rearrangement were observed in all of the mutant animals due to nonproductive or random rearrangements as demonstrated by detectable Vα14Jα281 rearrangements in NK T cell–deficient mice (e.g., β2M mutants). The usage of this specific rearrangement is reduced 10- and 20-fold in fyn − / − thymus and spleen, respectively. In lck mutant mice, this rearrangement is decreased >20-fold in both the thymus and spleen, similar to the levels observed in β2M mice. These data, combined with the flow cytometric analyses, suggest that the lck mutation leads to an early block similar to that seen in β2M mice. However, the amount of the invariant rearrangement present in fyn −/− mice was about twofold higher than that found in either the lck − / − or β2M − / − mice, confirming the flow cytometric data. This suggests that lack of Fyn leads to drastic though incomplete block in NK T cell development. Fyn may have a role in an early selection event or later expansion occurring after TCR rearrangement. Regardless, it is clear that Fyn is required for more than just NK1.1 upregulation and IL-4 secretion. These studies demonstrate the requirement of the two Src family members, Fyn and Lck, in NK T cell ontogeny. Additionally, these results indicate that the rapid pulse of IL-13 synthesis occurring after TCR ligation is most likely due to NK T cells. While conventional T cell ontogeny is highly dependent on Lck 4 33 34 , the NK T cell subset is the first lymphocyte population that also demonstrates a strict requirement for Fyn during development. These experiments underscore a novel role for Fyn in regulating NK T cells, especially since this kinase is not required in pre-TCR signaling, positive selection, and negative selection of conventional T cells 2 3 35 . This suggests that NK T cells use unique signals in their selection or expansion process. The fyn mutation defines the first intracellular signaling molecule that is selectively required for NK T cell, but not for conventional T lymphocyte or NK cell development. These and future studies will give insights into where this unique lineage diverges from conventional T cell development. | Study | biomedical | en | 0.999997 |
10525529 | PCR reactions were performed with the Pfu polymerase (Stratagene) according to the manufacturer's instructions. The nucleotide sequences of the DNA primers used are available upon request. The full-length hamster COR1(SCP3) cDNA was PCR-amplified and inserted in the EcoRI-BamHI sites of pGAD424 and pGBT9 as described previously . The full-length mouse SYN1(SCP1) cDNA was excised from a pET28a construct with NcoI, ends were filled in with the Klenow fragment of E . coli DNA polymerase I and further digested with XhoI. This fragment was subcloned into the SmaI-SalI sites of pGAD424 and pGBT9 vectors. The full-length mouse DMC 1 cDNA was PCR-amplified from a pET3 construct and subcloned into the EcoRI-BamHI sites of pGBT9 and pGAD424. The full-length mouse RAD 51 cDNA was PCR-amplified from a pET3 construct and subcloned into the SmaI-BamHI sites of pGBT9 and pGAD424. The fragments of mouse DMC1 and RAD51 proteins used in two-hybrid and in vitro protein–protein interaction analyses are shown in Fig. 1 . The cDNA fragment encoding DMC1N was PCR-amplified and inserted into the EcoRI-SalI sites of pGBT9 and pGAD424. The cDNA fragment encoding DMC1C was obtained by removing the 5′ StuI-EcoRI fragment from the construct containing full-length DMC1 cDNA in pGAD424. After digestion with these two enzymes, the ends were blunted with the Klenow fragment of E . coli DNA polymerase I and religated, rendering the pGAD424 vector plus a cDNA fragment that encodes DMC1C. The cDNA fragment encoding RAD51N was PCR-amplified and subcloned into the SmaI-SalI sites of pGBT9 and pGAD424. The cDNA fragment encoding Rad51C was obtained by removing the 5′ EcoRI fragment from the construct of full-length RAD 51 cDNA in pGAD424. After EcoRI digestion, the ends were religated, rendering the pGAD424 vector with a cDNA fragment that encodes RAD51C. All constructs were sequenced with MATCHMAKER5′ primer (Clontech) to verify the correct open reading frames. The method for cotransformation of the GAL4 fusion constructs into the yeast strains HF7c and SFY526 (kindly provided by O. Kovalenko, Yale University, New Haven, CT) was described by Chen et al. 1998 . The selective media required as well as the methods for determining β-galactosidase activity using the filter assay have been described previously . Each one of the interactions reported was tested in at least three independent experiments. For the in vitro protein–protein interaction experiments and polyclonal antibody generation we expressed the proteins involved as (His) 6 or HA fusions. The (His) 6 -tagged proteins were generated in pET29b as COOH-terminal or in pET28a/pET3a as NH 2 -terminal fusions. The COOH-terminal fusion of COR1(SCP3) with the (His) 6 tag was generated by PCR amplification of the coding region of hamster cDNA and cloning into the EcoRI-XhoI sites of pET29a. An NH 2 -terminal (His) 6 fusion of the SYN1(SCP1)/SCP1 was generated in pET28a as previously described from a construct containing the full-length cDNA in pBluescript (provided by J. Sage, University of Nice). The pET3-derived plasmids used for expression of the DMC1 and RAD51 proteins (His) 6 -tagged at the NH 2 terminus were described previously . The HA fusions were generated in E . coli from a modified pET29a vector generated in our laboratory that we named pET29a HA . To introduce the HA tag into the pET29a vector we synthesized two complementary 45-bp oligonucleotides with the following DNA sequences: 5′-GGC CAT ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GGT ACC CGC-3′ and 5′-GCG GGT ACC AGC GTA ATC TGG AAC ATC GTA TGG GTA CAT ATG GCC-3′. After annealing and digestion with KpnI, the resulting product was subcloned into the pET29a (Novagen) expression vector that had been digested with NdeI, ends filled in with the Klenow fragment of E . coli DNA polymerase I and further digested with KpnI. With this procedure, we replaced the NH 2 -terminal S-Tag™ (Novagen) normally present in the pET29 vector, with the HA epitope. The new tag can be removed by thrombin digestion : 20 μg of soluble extract from bacteria induced to express HA-COR1(SCP3) was incubated for 15 min at room temperature with 0.25 U thrombin and thrombin buffer (20 mM Tris-HCl, pH 8.3, 150 mM NaCl, and 2.5 mM CaCl 2 ), or with thrombin buffer alone. The HA-tagged COR1(SCP3) was detected with an anti-HA antibody, while no major band is visible in the Coomassie staining of the same gel. Thrombin treatment abolishes the anti-HA staining , indicating complete removal of the HA tag from the protein. All the constructs made in pET29a HA rendered NH 2 -terminal in-frame fusions of the desired proteins with the HA tag and lacked the (His) 6 tag, as determined by DNA sequencing. The full-length hamster COR1(SCP3) cDNA was excised as an EcoRI-SalI fragment from the pGBT9 construct and subcloned into the EcoRI-XhoI sites of the pET29a HA . The full coding region of mouse SYN1(SCP1) cDNA was PCR-amplified with Pfu polymerase (Stratagene) from a construct in pBluescript provided by J. Sage (University of Nice) and subcloned into the BamHI-XhoI sites of pET29a HA . The full-length mouse DMC 1 and RAD5 1 cDNAs were excised as EcoRI-SalI fragments from the corresponding pGAD424 constructs (described below) and subcloned into the EcoRI-XhoI sites of pET29a HA . To express the RAD51 and DMC1 NH 2 -terminal fragments as HA fusions, the corresponding sequences were PCR-amplified from constructs of the full-length cDNAs in pGAD424 and subcloned into the EcoRI-XhoI sites of pET29a HA . The cDNA fragments encoding RAD51C and DMC1C were excised with EcoRI and SalI from the corresponding constructs in pGAD424 and subcloned into the EcoRI-XhoI sites of pET29a HA . To test protein–protein interactions in vitro, proteins were expressed as (His) 6 or HA fusions. The (His) 6 -tagged protein was expressed in E . coli strain BL21(DE3) upon induction with 1 mM IPTG. After lysis and sonication in sonication buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole), the soluble fraction was incubated with Ni-NTA agarose (Qiagen) and the bound protein was washed three times in sonication buffer with 50–100 mM imidazole. The remaining unbound sites on the beads were blocked by incubation in storage buffer for 2 h at room temperature. The protein bound to the Ni-NTA agarose was stored in 100% glycerol at −20°C. The HA-tagged proteins were expressed in E . coli strain BL21(DE3) after induction with 0.5 mM IPTG. After lysis under nondenaturing conditions and sonication the soluble fraction was isolated by centrifugation at 50,000 g for 1 h and stored at −20°C. The protein-binding reaction contained 1× reaction buffer , 0.05% Tween 20, 50 μg BSA, 20 μl slurry with (His) 6 -tagged protein preequilibrated in reaction buffer (∼10 μg of protein) and 50 μg soluble fraction of the bacterial extract containing the HA-tagged protein. The proteins were incubated for 1 h at room temperature on a Nutator. The beads were sedimented, washed three times with 500 μl reaction buffer with 50 mM imidazole, and resuspended in 2× SDS sample buffer supplemented with 4 M urea. Samples were boiled for 5 min and separated on 10% or 12% SDS–polyacrylamide gels. The (His) 6 -tagged protein was detected in the control reaction using an antibody specific for this protein. The presence of the HA-tagged protein in the eluted samples was detected with a rat monoclonal anti-HA antibody (Boehringer-Mannheim). The mouse DMC1 and RAD51 full-length proteins were overexpressed in E . coli using the pET3 expression system . The (His) 6 -tagged proteins were purified on a Ni-NTA agarose column (Qiagen) and injected into mice and rabbits, respectively. The preimmune sera gave no staining when tested on meiotic prophase chromosomes. The polyclonal anti-DMC1 serum was depleted of anti-RAD51 cross-reactive components by adsorption on Sepharose-bound (Pharmacia) mouse RAD51 protein. The depleted antiserum was used at a 1:200 dilution in blocking buffer (5% skim milk powder, 0.05% vol/vol Triton X-100 in PBS) for Western blotting and 1:2 dilution in antibody dilution buffer (10% vol/vol goat serum, 3% BSA wt/vol, 0.05% vol/vol Triton X-100 in PBS) for immunocytochemistry. Similarly, the rabbit anti-RAD51 polyclonal serum was immunodepleted of components recognizing DMC1 epitopes by adsorption on a DMC1-bound Ni-NTA agarose column (Qiagen). This procedure abolished the cross-reactivity with the DMC1 protein initially observed on Western blots. As an additional specificity test, the two polyclonal sera were each reacted separately with either the mouse DMC1 or RAD51 proteins. The immune complexes were pelleted by centrifugation, and the supernatant used in immunofluorescent staining of spermatocyte nuclei. COR1(SCP3) was overexpressed in an E . coli expression system and purified using a (His) 6 -Tag. The full-length protein was injected into mice to generate a polyclonal serum. COR1(SCP3) localization on meiotic chromosomes was described previously . The localization of RAD51 and DMC1 antigens was determined on spread rat spermatocyte nuclei prepared as described in Tarsounas et al. 1999 . Human CREST sera were used to identify the centromeres. The fluorochromes were visualized with a Polyvar epifluorescent microscope (200 W mercury-vapor light source) and recorded on 400ASA black/white or color slides. For EM, nuclei were attached to a 50-nm-thick plastic carrier film and treated for 5 min at room temperature with DNaseI at a final concentration of 1 μg/ml in MEM (GIBCO BRL). The surface-spread nuclei were incubated with primary antibody for 2 h at 37°C and with secondary antibody (goat anti–mouse conjugated with 5 nm gold, goat anti–rabbit conjugated with 10 nm gold and goat anti–human conjugated with 15 nm gold, 1/50 dilution) for 1 h at 37°C. The immunogold-labeled preparations were postfixed in 4% OsO 4 before observing under the EM. The electron microscope images were recorded at 8,500× magnification on 35-mm film. Testis preparation was performed as described by Tarsounas et al. 1999 . Separation of the spermatocyte fractions by elutriation was performed following the procedure described by Grabske et al. 1975 and Meistrich 1977 , with modifications by Heyting et al. 1985 . 100 ml were collected from each fraction at increasing flow rates from 15 to 30 ml/min (5-ml/min increase at every step). The composition of these fractions in cells at various meiotic stages was assayed by immunofluorescent staining for the COR1(SCP3), SYN1(SCP1), and centromeric marker proteins. The composition varies from early to late prophase I stages (i.e., fraction I contains 45% leptotene cells, 45% zygotene cells, and 10% pachytene cells; fraction IV contains 85% pachytene cells and 15% leptotene/zygotene cells; fraction VI contains 80% diplotene cells and 20% pachytene cells). The number of spematogonia observed was too small to be taken into consideration. The cells in each fraction were centrifuged and resuspended in sample buffer (4 M urea, 10% SDS, 0.25 M Tris-HCl, pH 6.8, 20% glycerol, 0.015% bromophenol blue, 1 mM EDTA, and 10% vol/vol β-mercaptoethanol) to a concentration of 10 6 cells/ml. For Western blot analysis, protein corresponding to equal numbers of cells were loaded in each lane. To determine the chromosomal distribution of RAD51 and DMC1, we generated antibodies against the full-length mouse proteins. As the overall degree of identity at the amino acid level between the two proteins is 54%, each antibody displayed immunoreactivity with the homologous protein on a Western blot . To eliminate the cross-reactivity of the anti-DMC1 antibody with the RAD51 protein, we produced a Sepharose matrix with covalently bound RAD51. The polyclonal anti-DMC1 serum was adsorbed on this matrix twice; the immunodepleted antibody reacted with DMC1 but showed no reactivity with the RAD51 protein . Similarly, the anti-RAD51 antibody was immunoadsorbed twice on a matrix containing the mouse DMC1 protein covalently attached to it. This treatment rendered a specific anti-RAD51 antibody . Equal amounts of proteins purified on Ni-agarose column were loaded in each lane, as shown in the Coomassie staining of the gel . The purified antibodies were further used in immunostaining preparations for light and EM, and Western blotting of the spermatocyte fractions. As an additional method for determining the specificity of the antibodies, we reacted each of them with the RAD51 and DMC1 proteins separately, discarded the immune complexes by centrifugation, and used the depleted sera in immunofluorescence staining of rat meiotic chromosomes . Normal numbers and position of the RAD51 foci on the chromosome cores were observed when the anti-RAD51 antibody was reacted with the mouse DMC1 protein , and when the anti-DMC1 antibody was reacted with the mouse RAD51 protein . Fig. 4A and Fig. C , encompass several nuclei of early prophase I spermatocytes, where the RAD51 and DMC1 proteins are abundant. In Fig. 4A ′ and C′, a section of the A and C figures is enlarged. When the anti-RAD51 antibody was reacted with the RAD51 protein, the ability of this antibody to stain discrete foci on the chromosomes was completely blocked . Similarly, reacting the anti-DMC1 antibody with DMC1 protein abolished the staining by this antibody . These experiments established that each purified antibody recognized only the protein against which it was generated. Surface-spread mouse meiotic chromosomes were reacted with the purified rabbit anti-RAD51 and mouse anti-DMC1 antibodies, each visualized with EM of immunogold particles of different sizes (10 and 5 nm, respectively) attached to the secondary antibodies. The 15-nm gold particles identify the centromere stained with a human CREST serum. The majority of foci observed at various prophase I stages contained a mixture of 5- and 10-nm gold particles . We counted 150 of these EM foci present in leptotene, zygotene, and pachytene cells, and 11% of them contained only one size of grains. This is consistent with the null class estimate predicted from the Poisson distribution of the results (data not shown). It seems reasonable to conclude that the foci contain both sizes of grains, and the presence of foci with a single grain size is due to the random binding of the antibody to the available sites. This distribution pattern of the gold grains indicates that the two proteins colocalize on the chromosomal cores before they synapse and in the confines of the SC, suggesting that they might act in a concerted manner during meiosis. Interestingly, at leptotene, when the cores are in the process of alignment, the gold grains are physically associated with the cores . Later on, at zygotene, the grain distribution coincides extensively with interstitial connections between the homologues , cytologically similar to the axial associations reported in yeast to be the synapsis initiation sites . In lily and mice, these sites most likely identify the early recombination nodules . At pachytene , the grains label electron-dense structures localized in the central region of the SC. Because the foci observed with EM contained both RAD51 and DMC1 recombination proteins, we refer to them as recombination complexes. We attempted to show that at the level of fluorescence microscopy the numbers and relative positions of DMC1 and RAD51 foci are also identical. The distribution of RAD51 foci during successive stages of the meiotic prophase I in mammalian oocytes and spermatocytes has been reported previously . Using double staining of the DMC1 and RAD51 proteins and superimposing the two colors as described in Gasior et al. 1998 , it can be seen that RAD51 and DMC1 foci coincide. A correspondence of 95% was recorded for 10 prophase nuclei. This is in agreement with the colocalization results of EM where 89% of the foci are mixed. As the distribution of COR1(SCP3) during meiosis is well characterized , we used a mouse anti-COR1(SCP3) antibody (red) to identify the prophase I stages and sub-stages. This antibody was added in trace amounts to merely mark the cores, without interfering with the staining of the anti-DMC1 antibody also generated in a mouse. In Fig. 6 A the leptotene nucleus was identified based on the fact that the cores are just beginning to form, while in the adjacent zygotene nucleus, the cores are extensive . The coincident staining with anti-RAD51 and anti-DMC1 antibody shows that the foci are associated with the cores and that the number of foci per nucleus is high at leptotene/zygotene (250–300 foci per nucleus) and low at pachytene (∼100 foci or less per nucleus). To monitor the presence of the two recombinases at progressive stages of meiotic prophase, we have isolated fractions of testicular cells by centrifugal elutriation . In general, this method allows cell fractionation by size. As the size of spermatocytes increases with their progression through meiosis, it is possible to separate prophase I cells in fractions enriched for cells at specific meiotic stages . A sample of each fraction was fixed in 2% paraformaldehyde and used to determine the composition in cells at various meiotic stages, using immunofluorescence staining with anti-SYN1(SCP1), anti-COR1(SCP3), and anti-centromeric antibodies. The prophase I spermatocytes increased from 90% leptotene/zygotene cells in fraction I to 80% diplotene cells in fraction VI (data not shown). To demonstrate that the protein extracts prepared from these fractions give an accurate representation of the stages identified with immunofluorescence staining, we demonstrate the distribution of marker proteins COR1(SCP3) and SYN1(SCP1) in these fractions . In accordance with its distribution on the meiotic chromosomes from early prophase to metaphase I, COR1(SCP3) was detected in all fractions as a constantly present 30-kD polypeptide. A higher molecular mass form of the protein was also detected, which changed from 35 kD in early prophase I to 33 kD in the late stages. Previously, COR1(SCP3) was being referred to as the 30/33-kD polypeptide, because of its constant detection on Western blots of testicular cells as a doublet of these sizes. The results presented here indicate that the COR1(SCP3) protein (estimated M r 28,000) most likely corresponds to the 30-kD polypeptide, while the slower migrating band (35 or 33 kD) is possibly the result of posttranslational modifications such as phosphorylation , which vary with the meiotic stage. SYN1(SCP1) was used as a second marker for the protein composition of the testicular fractions collected. This protein is known to be present on the chromosomes during zygotene when synapsis initiates, and at pachytene in synapsed chromosomes. The protein disappears at the end of pachytene/onset of diplotene when the homologues separate . Consistent with its presence in the meiotic chromosomes, the SYN1(SCP1) protein was only detected in fractions II–IV, and not in the fractions where the initial and final prophase I stages were predominant. The presence of RAD51 and DMC1 was only detected in the first three fractions containing cells in early prophase I stages, in accordance with the immunocytological observation of abundant RAD51/DMC1 foci on the chromosomal cores of early stages of prophase I. We used the yeast two-hybrid system to test possible protein–protein interactions involving mouse RAD51 and DMC1 ( Table A). Homotypic interactions were detected for both mouse proteins, as shown previously for the yeast and human homologues . We tested heterotypic interactions with each protein expressed either as a fusion to the GAL4 activation domain or to the GAL4 DNA-binding domain. Compared with their ability to self-interact, the heterotypic interactions between RAD51 and DMC1 were weaker in the sense that the reporter gene expression ( HIS 3 and lacZ ) was delayed. In the negative controls no expression was detected under identical conditions. The interaction between RAD51 and DMC1 was also tested using an in vitro assay in which the RAD51 protein was expressed in E . coli as a (His) 6 -fusion and bound to Ni-NTA agarose column. The HA-tagged DMC1 from a soluble E . coli extract was incubated with the RAD51 matrix, precipitated, and detected with an anti-HA antibody . These data indicate that the formation of the RAD51/DMC1 complexes can be mediated by direct interactions between these two proteins. As a positive control for this experiment, precipitation of HA-DMC1 with a (His) 6 -DMC1 fusion confirms the homotypic interaction detected in the in vivo two-hybrid assay. Next, we wished to determine which region of the mouse RAD51 and DMC1 proteins was essential for homotypic and heterotypic interactions. We investigated whether the NH 2 -terminal domains , which show the lowest identity between the two proteins, could have distinct abilities in establishing protein–protein interactions. In a two-hybrid assay, we show that the DMC1 COOH-terminal fragment (DMC1C; M r 31,000) containing amino acids 63–340 interacts with the full-length protein (340 amino acids; M r 37,800), while the NH 2 -terminal fragment (DMC1N; M r 7,200) containing amino acids 1–62 does not. This indicates that the COOH-terminal region, or a portion of it, is required for the DMC1 homotypic interaction to occur. Similarly, the COOH-terminal RAD51 fragment (RAD51C; M r 23,000), or a portion of it, mediates RAD51 self-interacting capacity. In addition, the COOH-terminal region of RAD51 is able to interact weakly with the full-length DMC1 in a two-hybrid assay and conversely, the COOH terminus of DMC1 establishes weak interactions with the full-length RAD51 ( Table A). These data suggest that the COOH-terminal regions of the two proteins may be involved in mediating heterotypic RAD51/DMC1 interactions as well. The homotypic interactions observed in the two-hybrid assays were confirmed by the in vitro binding experiments. Fig. 8 B shows that only DMC1C, and not DMC1N, interacts with the full-length DMC1 protein. The truncated derivatives of RAD51 act similarly in the presence of the full-length RAD51 protein. These data suggest that the NH 2 - and COOH-terminal regions of the two proteins behave similarly in mediating self-interactions. To gain further insight into the relationship between the SC and the RAD51/DMC1 recombinases, we tested possible interactions between the SC proteins and the components of recombination complexes. We performed in vivo two-hybrid assays with DMC1, RAD51, and each of the SC components, SYN1(SCP1) and COR1(SCP3), the results of which are presented in Table B. COR1(SCP3) is a component of the chromosomal cores from early prophase I when the cores start to assemble, to pachytene when the cores synapse and form the lateral domains of the SC, and until late diplotene when the homologues separate from each other . Both RAD51 and DMC1 interact with COR1(SCP3) in the in vivo two-hybrid system and in the in vitro coprecipitation assays. This is consistent with the presence of the recombination complexes in association with the cores from leptotene to pachytene, as detected with electron and light microscopy . SYN1 (SCP1) is pachytene-specific, possibly identifying the protein that establishes and maintains synapsis between homologues . RAD51 interacts with this synaptic protein, while DMC1 does not . The third component of the SC tested for interaction with the RAD51/DMC1 recombinases was SCP2. This protein localizes in the cores/lateral domains of the SC and has a temporal and spatial distribution very similar to that of COR1(SCP3) . In a two-hybrid screen of a hamster testis cDNA library for proteins interacting with COR1(SCP3) , we have isolated a 50-kD polypeptide from the COOH-terminal region of the SCP2 protein ( M r 190,000). We tested this fragment in a two-hybrid assay for its ability to participate in direct interactions with the DMC1/RAD51 proteins. No interaction was detected, suggesting that the COOH-terminal SCP2 region which mediates the interaction between the two core components (COR1(SCP3) and SCP2) is not required for the binding of the recombination complexes to the cores. This finding does not exclude the possibility that another region of the large SCP2 protein may be functional in this respect. A major difficulty in studying the distribution of RAD51 and DMC1 in mammalian meiotic chromosomes is that sera raised against the full-length proteins recognize both proteins in immunoblots and presumably in cytological preparations. A polyclonal serum raised against a 15–amino acid NH 2 -terminal peptide of RAD51, while showing a high specificity for RAD51 on Western blots, does not render any signal on meiotic chromosome spreads (data not shown), possibly because the NH 2 terminus of this protein is not available to the antibody. To differentiate between these two proteins, we have used sera that were depleted of the cross-reactive antibodies. The high specificity of the resulting antibodies was apparent in Western blot analyses of bacterially expressed proteins and in spermatocyte immunostaining in which each antibody was prereacted with the antigen or with the homologous protein . Data presented here, therefore, accurately reflect the distribution of the two proteins on the chromosomes. In yeast, Rad51 and Dmc1 proteins form discrete foci along the meiotic chromosome cores with variable degrees of colocalization . We report here the results of a similar study performed on mammalian meiotic chromosomes. The larger size of these chromosomes, as well as the spreading techniques available for their fixation allow a more detailed analysis of the proteins associated with the chromosomal cores. The RAD51 and DMC1 foci detected by immunofluorescence staining of rat spermatocyte nuclei with antibodies against each of the two proteins overlap in 95% of the cases. This observation does not necessarily imply that the two proteins colocalize on the chromosomes . Thus, we attempted to define the relative position of the RAD51 and DMC1 foci using EM with immunogold visualization. RAD51 was visualized with 10-nm gold grains and DMC1 with 5-nm gold grains. The two types of particles occurred in mixed groups in 89% of the cases, while only 11% of the groups were composed of single-size particles. According to the Poisson analysis of our numeric data, the latter focus type is expected to occur by chance alone if the number of labeled proteins is small relative to the total number of protein molecules per focus. Similarly, in immunofluorescence staining of meiotic nuclei a small percentage of foci (∼5%) were composed of only one or the other type of protein. We conclude that RAD51 and DMC1 colocalize in mixed foci on the mouse meiotic chromosomes. The number of RAD51/DMC1 complexes is most abundant at early meiotic stages and decreases thereafter, becoming undetectable at late pachytene/early diplotene. Consistent with this is the detection of the two proteins in spermatocyte fractions containing predominantly early meiotic stages . The downregulation of DMC1/RAD51 during meiotic prophase I is correlated with the progression of recombination. The DMC1/RAD51 complexes are most abundant at the leptotene/zygotene stages, during which homologous recombination is initiated, and less numerous at pachytene, when maturation of the recombination intermediates is postulated to occur. This is consistent with a role for these proteins in the early steps of meiotic recombination, presumably in homology recognition and initiation of strand exchange reactions as previously proposed . We addressed the question whether the RAD51/DMC1 mixed foci observed with immunocytological methods are formed by direct interactions between the mouse RAD51 and DMC1 proteins. The interaction detected between RAD51 and DMC1 in an in vivo two-hybrid assay was weak, as estimated from the delayed expression of the HIS 3 and lacZ reporter genes. One possibility is that the interaction between RAD51 and DMC1 is indirect and requires an adaptor protein. A strong candidate for such an adaptor protein is the yeast Tid1 , a Rad54 homologue which interacts with both Rad51 and Dmc1 in a two-hybrid screen. It is possible that the yeast Tid1 protein can mediate the interaction between the two mouse homologues when they are expressed in the same yeast cell in the two-hybrid assay. Its recruitment, however, may delay the establishment of an interaction between the two proteins and, consequently, the expression of the reporter genes. The second possibility is that each of the two proteins (RAD51 and DMC1) form homotypic interactions very easily, especially in the presence of the DNA substrates as may be the case in vivo, and, therefore, little protein is left available for the heterotypic interaction to occur . Using biochemical assays, we showed that RAD51 and DMC1 can be coprecipitated in vitro . This suggests that the mixed RAD51/DMC1 foci detected immunocytologically may assemble through direct interactions between the two proteins. Determining the regions of RAD51 and DMC1 involved in homotypic interactions will help understand the assembly of the recombination complexes on the chromosomes. We used truncated derivatives of the two proteins in two-hybrid and in vitro binding experiments, and showed that a version of RAD51 or DMC1 bearing a deletion at the NH 2 terminus still allows establishment of heterotypic and homotypic interactions. Biochemical studies have shown that RAD51 assembles nucleoprotein filaments with single- and double-stranded DNA and DMC1 may have similar properties . Our EM data indicating colocalization of these proteins on the meiotic chromosomes and their ability to establish direct heterotypic protein–protein interactions suggest the possibility that RAD51/DMC1 form mixed filaments associated with DNA at meiosis. Moreover, we scored 150 EM foci containing >1,500 gold grains and found that 46% of the grains correspond to RAD51 and 54% to DMC1 (data not shown). Therefore, the possibility that these filaments have a constant stoichiometry of RAD51 to DMC1 molecules is not excluded. We show here that interactions between RAD51/DMC1 complexes and SCs are established by direct protein–protein interactions between their components. These are detected in an in vivo two-hybrid system and in vitro binding analyses using bacterially expressed proteins. Coimmunoprecipitation of these proteins from testis extracts is technically unattainable due to the high insolubility of the SC components in these extracts (Tarsounas, M., P. Moens, and R.E. Pearlman, unpublished data). The interactions detected between SC components and the two recombinases support the cytological observations from immunofluorescence and EM, where the RAD51/DMC1 complexes are detected in association with the chromosomal cores at the very early prophase I stages or with the SCs at pachytene . Based on these data, it is possible that the SC components may provide the structural frame that stabilizes complexes formed of recombination proteins and DNA . Several lines of evidence support this hypothesis. In mammals, SC formation is obstructed in the absence of Atm . This structural defect may cause the mislocalization of RAD51/DMC1 complexes detected in the Atm −/− spermatocytes . In yeast, the core-associated Red1 protein with a meiotic distribution similar to that of COR1/SCP3 is necessary for recombination and specifically for normal levels of interhomologue joint molecule formation . Using genetic analyses and EM of spread yeast chromosomes, Rockmill et al. 1995 have shown that Rad51 and Dmc1 proteins are required to establish the axial associations between homologues, which represent the synapsis initiation sites. Here we show that in rat chromosome spreads the RAD51/DMC1 complexes coincide with cytologically similar axial associations. The gold grains may detect a nucleoprotein filament formed at the site of a DSB after resection of the ends as predicted . This nucleoprotein filament bridges the two homologues and possibly plays a role in homology recognition as well. By these criteria, a RAD51/DMC1-coated ssDNA visualized in association with single cores at leptotene/early zygotene may recognize homologous DNA sequences on another chromosome and establish the axial associations visualized in Fig. 5 B, as previously suggested . This hypothesis is also supported by the recent observation that RAD51 foci are associated with ssDNA in meiotic cells . At pachytene, the gold grains appear in the central region of the SC . The recombination complexes may be anchored there by a direct interaction of RAD51 with the synaptic protein SYN1(SCP1) which also localizes in the central region of the SC . We detected such a direct interaction between RAD51 and SYN1(SCP1) in the two-hybrid and in vitro binding analyses. The significance of this is unclear at present. It is possible, however, that the few recombination complexes present at pachytene are anchored in the central region of the SC through a direct interaction with SYN1(SCP1). In addition to the RAD51/DMC1 recombinase, other proteins involved in DNA repair (e.g., MLH1, BRCA1, BRCA2), as well as sensors of DNA damage (e.g., HRAD1, ATR) have been detected in association with the meiotic chromosome cores and/or the SCs . This suggests a concerted action of all these proteins in the repair of meiotic DSBs and maintenance of genomic integrity in the germ line. Determining the molecular details of this process requires further experimentation. | Study | biomedical | en | 0.999997 |
10525530 | HEp-2 carcinoma human primary fibroblasts (HF) have been previously described . Fibroblasts from Bloom syndrome patient were obtained from the Coriell Institute for Medical Research; mouse primary embryonic fibroblasts (MPEF) and PML−/− mouse primary embryonic fibroblasts , N-myc-amplified neuroblastoma NGP cells , were maintained in DME supplemented with 10% FCS and antibiotics. Human embryonic NT2 cells and the retinoic acid–differentiated NT2 cells were provided by V. Lee (University of Pennsylvania) . All cells were grown at 37°C in a humidified 5% CO 2 atmosphere. For immunohistochemical staining, cells were grown on round coverslips in 24-well plates (Corning Glass, Inc.) until ∼80% confluent before fixation. For cell fusion experiments, both cell types were plated on glass coverslips at a 1:1 ratio. The following day, they were treated with prewarmed 50% PEG-6000 (Serva Co.) for 2 min and washed three times with complete culture medium. Immunostaining was performed 16 h after fusion. Unfused cells in the PEG-6000–treated cells and unmixed fused cells were used as negative controls. During microscopic analysis, DNA staining was used to identify the nuclei of mouse and human cells. ND10 were visualized using the following antibodies: mAb 138 labels NDP55 , whereas mAb 5E10 reacts with PML . Isotype-matched mAbs of unrelated specificity were used as controls. Polyclonal rabbit antiserum against PML and rabbit antibodies against Sp100 were obtained from Dr. J. Frey . A human antiserum specific for Sp100 from patients with primary billiary cirrhosis was found positive for mouse ND10. Human serum 602 recognized the centromeres in immunohistochemistry and the centromeric recombinant protein CENP-C in Western blots . Rabbit antibodies against human Daxx were produced using recombinant RGS-His-tagged human Daxx purified on NTX resin (Qiagen). Antibodies against RGS-HIS were purchased from Qiagen. A different antibody against hDaxx was produced in mouse against the hDaxx COOH-terminal 480–740 amino acid fragment. In addition, a rabbit anti–Daxx antibody was purchased from Santa Cruz Biotechnology, Inc. Antibodies against SUMO-1 were obtained from Dr. P. Freemont (I.C.R.F. London) . Anti-CBP, antibodies were purchased from Santa Cruz Biotechnology, Inc., and those against RGS-His from Qiagen (Valencia, CA). Rabbit anti-BLM has been described . 2 d after plating on round glass coverslips, cells were fixed at room temperature for 15 min with freshly prepared 1% paraformaldehyde in PBS and treated as previously described . Cells were analyzed using a Leica confocal laser scanning microscope. Leica image enhancement software was used to balance signal strength and eightfold scanning was used to separate signal from noise. Because of the variability among cells in any given culture, the most prevalent cells were photographed and are presented as small groups of nuclei or single nuclear images at high magnification. The interaction between Daxx and PML or Sp100 was measured in a yeast two-hybrid assay using β-galactosidase activity as a reporter of protein–protein interaction. Sp100 and PML were cloned in the pAS1 vector that contains the TRP1 gene , so that these proteins are fused to GAL4-DBD (a gift from R. Evans, Salk Institut). The pAS1-PMLΔ coil deletion mutant was generated by digestion of pAS1-PML with endonuclease Bss HII and self-ligation, which resulted in a deletion of 214–329 amino acids of the PML protein. hDaxx cDNA and the respective mutants were produced by PCR using the high fidelity Vent polymerase (New England Biolabs) and pQE-30 hDaxx as a template and cloned into the BamHI site downstream of NLS-VP16 driven by the ADH promoter into pVP16 , which carries the LEU2 gene. pSD5 (a gift from S. Berger, The Wistar Institute) containing the HIS2 gene and GAL4 binding site upstream of the bacterial lacZ gene was used as a reporter plasmid. Plasmids were transfected into the trp1 derivative of yeast strain PSY316 (MATα his 3-200 leu 2-3, 112 lys 2 ura 3-53) . β-galactosidase activity was assessed and normalized to protein concentration as described . The data represent results of three independent experiments. To analyze the intranuclear distribution of various Daxx mutants, we first constructed a pET plasmid, encoding GFP with a nuclear localization signal from SV40 large T-antigen (amino acid PKKKRKV). Two synthetic oligonucleotides (5′-AATTCTCCTAAGAAGAAGCGTAAGG-3′ and 5′-TCGACCTTACGCTTCTTCTTAGGAG-3′) were annealed and inserted into the COOH-terminal end of the GFP open reading frame between the EcoRI and SalI sites on pEGFP-C1 (CLONTECH Laboratories). Different deletion mutants of hDaxx were constructed by subcloning into BamHI cut pET vector. The respective fragments of BamHI cut PCR products were amplified with the sense primer 5′-CACACGGATCCGCCACCGCTAACAGC-3′ and the antisense primers: 5′-GTGGTGGATCCCTCCTCTGATTGCTTCCTGG-3′ for Daxx 1–595 amino acids; 5′-GTGGTGGATCCATCAGAGTCTGAGAGCACGATG-3′ for Daxx 1–740 amino acids; or BglII-BamHI cut PCR fragment amplified with 5′-CACACAGATCTGATTCTGGTCCCCCCTGC-3′ as the sense and antisense primers 5′-GTGGTGGATCCATCAGAGTCTGAGAGCACGATG-3′ for Daxx 624–740 amino acids using high fidelity Vent polymerase (New England Biolabs) and pQE-30hDaxx as a template for the Daxx gene. All constructs were verified by sequencing. Plasmids PML-K65,160,490R (referred to here as PMLΔSUMO), PML-K65R, and PML-K65,160R , based on the pcDNA3 plasmid (Invitrogen), expresses RGS-His-fused PML mutants with corresponding lysines substituted by arginines. Transient transfections were carried out using the DOSPER reagent (Boehringer) according to the manufacturer's recommendations. To address the question, which proteins are essential for the formation or maintenance of ND10, we identified cell lines that do not express certain ND10-associated proteins. The first cell line identified came from the observation that the BLM protein, a member of the DExH box containing DNA helicases, was located in discrete nuclear domains in most cells . Immunostaining of BML−/− fibroblasts derived from BLM syndrome patients did not reveal any discrete domain staining, confirming specificity of BML localization at such sites in normal cells. These patients are homozygous for the mutation in the BLM gene, which leads to the early truncation of the BLM helicase. Double labeling of primary human fibroblasts with antibodies against PML, a constitutive ND10-associated protein, and antibodies against BLM showed that the two antibodies labeled ND10 . To determine whether the absence of BLM helicase affects ND10, BML−/− fibroblasts were double labeled with antibodies against several ND10-associated proteins (PML, Sp100, SUMO-1, and CBP). All were detected in ND10 . Thus, BLM helicase is not essential for the formation of ND10. In previous studies, we found that nuclei of most brain cells do not contain Sp100 accumulations . Therefore, we checked for Sp100 expression in human NT2 embryocarcinoma cells, which can be induced to differentiate into nerve-like cells by retinoic acid . Using HEp-2 cells as a control, we tested by Western blot analysis for the presence of Sp100 and PML in NT2 cells. Since Sp100, as well as PML, have interferon response elements in their promoter regions , we stimulated the cells for 24 h with interferon α to upregulate a potentially undetectable level of expression. Both cell lines contained PML , although the amount of PML was higher in the HEp-2 cells. Interferon treatment dramatically increased the amount of PML (compare lanes 5 and 6 with 7 and 8). The unspecific band marked by an asterisk indicates equal loading. No Sp100 was found in NT2 cells even upon interferon upregulation (lanes 1 and 2), whereas in HEp2 cells interferon treatment resulted in a massive enhancement of the Sp100 signal (lanes 3 and 4). The results of Western blot analysis were confirmed by immunostaining with two different anti–Sp100 antibodies of human and rabbit origin. No Sp100 accumulations were seen in NT2 cells with or without interferon activation . However, most NT2 cells contained ND10 as judged by the presence of PML at specific sites. Analysis using the entire panel of ND10-specific antibodies revealed the presence of all corresponding antigens at the PML-positive sites. The negative Sp100 phenotype was also retained after differentiation induced by retinoic acid treatment and in NGP neuroblastoma cells (data not shown). Thus, we concluded that Sp100 was absent in NT2 cells and, that the presence of Sp100 was not essential for the maintenance of ND10. The question whether PML is important for ND10 assembly and maintenance was tested in PML−/− MPEF cells. To obtain a reasonable assurance that ND10 are present or absent in PML−/− MPEF cells, we needed a panel of antibodies that reacted with several mouse ND10-associated proteins. We had identified a rabbit antibody that reacted with human and mouse PML and labeled ND10 in mouse fibroblasts . This antibody was used to identify antibodies that were specific for mouse proteins located in ND10. Screening several Sp100-positive human autoantibodies, we identified one that interacts with mouse Sp100 . Antibody specificity was confirmed by Western blotting using recombinant Sp100. In addition, the anti-NDP55 mAb labeled ND10 . Also, SUMO-1, which can modify both PML and Sp100, was detected in mouse ND10 . To show that Sp100, NDP55, and SUMO-1 are concentrated in ND10, they are presented as separate green images below the merged one in the upper row. This panel of four antibodies recognized mouse ND10 and was used to probe for the presence of ND10 in PML−/− MPEF. When PML−/− MPEF were tested for the distribution of the different ND10-associated proteins, neither Sp100, NDP55, nor SUMO-1 was seen in typical ND10. Instead, these proteins appeared throughout the nucleus without any ND10 accumulations . The distribution pattern of three ND10-associated proteins, in the absence of PML, suggested PML as a likely candidate for establishing ND10 integrity. To test this possibility, we expressed PML in the PML−/− MPEF cells by transient transfection and probed for the location of Sp100, NDP55, and SUMO-1. At low expression levels, PML appeared in domains with the frequency and distribution of ND10 in wild-type cells . Upon accumulation of PML, larger aggregates with a lower frequency appeared and sometimes cytoplasmic accumulations were seen. All tested mouse ND10 proteins were segregated into PML-positive structures. These observations established that PML is essential for the assembly of ND10 and for the segregation and accumulation of ND10-associated proteins. It has been shown before that PML cannot interact with Sp100 directly . However, it can recruit Sp100 into ND10 upon transient expression in PML−/− MPEF cells. We concluded that some adapter proteins must mediate this interaction and searched for new proteins that were part of ND10 and interacted with PML. In previous studies, we had cloned human Daxx through its ability to bind to the steroidogenic factor-1–like binding site in the human steroidogenic acute regulatory protein gene promoter DNA sequence . Rabbit antibodies produced against this protein showed an ND10-like nuclear distribution. When compared with PML, Daxx colocalized perfectly in ND10 . Since Daxx interacts with the death domain of Fas and, therefore, was anticipated to be a cytoplasmic protein, we confirmed the ND10 location of Daxx with two other independently generated antibodies to ensure that the antigen detected in ND10 was truly identical to Daxx. Both a commercial rabbit antibody and a mouse antibody produced against a recombinant hDaxx fragment reacted with the same structure as did PML antibodies. Specificity of Daxx antibodies were confirmed by Western blot analysis of in vitro translated hDaxx (not shown). These experiments strongly argued against the possibility that antibodies identified a spurious localization. In addition to Fas, Daxx was shown to interact with CENP-C and to be located at centromeres in a cell cycle–dependent fashion . When we used human autoantibodies against centromeric proteins together with Daxx antibodies in wild-type mouse fibroblasts, we observed that a few sites did appear to colocalize. However, in most cases Daxx was situated beside centromeres or was not apparent at centromeres . We concluded that Daxx is at its highest concentration in ND10 and is, therefore, a novel ND10-associated protein. To investigate the possibility that the localization of Daxx reflects an interaction between Daxx and another ND10-associated protein, we employed the yeast two-hybrid assay. We determined whether Daxx could interact with Sp100 and/or PML fused to GAL4DB. Daxx was fused to VP16 and the VP16-producing plasmid was used as a negative control. To quantitate the strength of interaction, we used the liquid β-galactosidase assay. Using this assay, we did not observe any evidence of interaction between Daxx and Sp100. In contrast, Daxx strongly interacted with PML ( Table ). To assess the specificity of this interaction, we mapped the region of Daxx that is required for interaction with PML (see Table for details). The PML coil-coiled region deletion mutant can still interact with Daxx, although it can also interact with Vp16 alone . Unexpectedly, two of the Daxx NH 2 -terminal deletion mutants (amino acids 488–740 and 625–740) interacted with PML approximately threefold more strongly than the full-length molecule. The NH 2 -terminal region of Daxx can lower the strength of its interaction with PML, probably as a result of protein folding. In contrast, the COOH-terminal deletion mutant (amino acids 1–625) and the smallest COOH-terminal construct (amino acids 661–740) failed to interact at all. Another Daxx deletion mutant (amino acids 433–740) showed an ∼20-fold weaker interaction than the Daxx 488–740 amino acid mutant. The weaker signal might reflect the exposure of the acidic amino acid–rich region between amino acids 434 and 485, diminishing the interaction. Thus, the PML interaction domain, as defined by the yeast two-hybrid assay, lies between amino acids 625 and 740, and amino acids 625–661 are essential for this interaction. Together, these data demonstrate that Daxx interacts with PML and suggests that the spatial colocalization of PML and Daxx in ND10 reflects an interaction between these two proteins. The results of the yeast two-hybrid assay indicated that Daxx and PML interacted, and the colocalization of the two proteins in ND10 was consistent with physiological association. To test whether various deletions in Daxx would affect Daxx localization in the context of mammalian cells, we fused the mutants with GFP-NLS. In agreement with indirect immunofluorescence results, the GFP-Daxx fusion protein was found to accumulate efficiently at PML-positive sites . In contrast, the COOH-terminal deletion mutant (amino acids 1–595), which lacked the PML-interacting region, was diffusely present throughout the nucleus and was not accumulated at PML-positive sites . The results from the yeast two-hybrid interaction were also confirmed by the finding that the Daxx COOH-terminal region (amino acids 624–740) alone was sufficient to localize GFP to ND10 . These data suggest that Daxx interaction with PML is necessary for ND10 localization. If Daxx is accumulated in ND10 through interaction with PML, one would predict a different Daxx distribution in cells without PML. In normal mouse fibroblasts, Daxx colocalized specifically with PML in ND10 . However, in the absence of PML (mouse PML−/− fibroblasts), we found the localization of Daxx to be quite different. Daxx was localized in patches which, when counterstained for DNA, proved to be condensed chromatin . Therefore, in the absence of ND10, condensed chromatin is an alternative nuclear compartment of Daxx accumulation. To investigate the relationship between PML and Daxx further, we tested whether PML could recruit endogenous Daxx into ND10 when transiently expressed in PML−/− MPEF cells. As shown in Fig. 3H–J , the untransfected PML−/− MPEF cells exhibited the patchy Daxx distribution characteristic of its condensed chromatin location. In PML-transfected cells , Daxx was not seen at condensed chromatin but, instead, now colocalized almost exclusively with PML. These PML-induced structures were found to also contain the other ND10-associated proteins and were, therefore, considered to be ND10. To test if PML could restore Daxx accumulation at ND10 without the strong overexpression induced by transient transfection, we performed a cell fusion experiment where PML−/− MPEF cells were fused with human fibroblasts as a source of PML. Species specificity of the cells was determined by DNA distribution, which showed strongly condensed chromatin for mouse cells . In unfused mouse cells , Daxx appeared at its highest concentration only in condensed chromatin. But in another mouse cell (upper left cell), which became positive for human PML as a result of fusion with HF (upper right cell), Daxx started to appear in additional domains that colocalized with PML. In this experiment we could not distinguish between human and mouse Daxx. However, the finding that mDaxx, after hPML overexpression in PML−/− MPEF cells was recruited to ND10 , made it likely that in the fusion experiment mDaxx could be accumulated into PML-positive structures. Therefore, we concluded that physiological quantities of PML can induce ND10 formation in nuclei that normally do not have them. PML has been shown to be modified by SUMO-1 at least at three sites. The SUMO-1 modification seemed necessary for the deposition of PML at ND10 . Therefore, we were interested in determining whether this modification affected interaction with Daxx. Double labeling of HEp-2 cells for SUMO-1 and overexpressed PML showed that PML in the nucleus colocalized with SUMO-1 in large aggregates, and that the cytoplasmic PML aggregates did not stain for SUMO-1. This suggested that only nuclear PML was modified by SUMO-1 . We tested whether elimination of SUMO-1 modification sites in PML influenced SUMO-1 aggregation in PML-positive sites. SUMO-1 modification sites have been identified previously at lysines 65, 160, and 490 . We transfected HEp-2 cells with RGS–His-tagged PML mutants containing successively decreasing numbers of SUMO-1–modified lysines and analyzed the localization of these overexpressed PML mutants and endogenous SUMO-1. We observed that a decrease of SUMO-1 accumulation in PML domains paralleled the number of mutated lysines. Shown in Fig. 5B and Fig. F , are cells transfected with the RGS–His-tagged mutant PMLΔSUMO, in which all three lysine residues are substituted with arginine. Upon high PMLΔSUMO accumulation, similar enlarged and later distorted ND10 appear as seen for the wild-type PML overexpression, except that these accumulations did not label with SUMO-1 antibodies like the wild-type PML accumulations . Contrary to published reports, all PML mutants were accumulated at ND10 (recognized by the location of endogenous SUMO-1) at low level of expression . This PMLΔSUMO accumulation indicates that either SUMO-1 modification is not necessary for ND10 targeting of PML and/or is due to potential dimer formation between PMLΔSUMO and wild-type PML, where the wild-type PML would serve as an ND10 targeting vehicle. We concluded that SUMO-1–modified PML appears only in the nucleus, and confirmed in situ that PMLΔSUMO is not SUMO-1–modified. We tested whether endogenous Daxx recruitment into ND10 was influenced by the level of PML SUMO-1 modification. HEp-2 cells were transfected with PML and PMLΔSUMO expression plasmids and tested for the location of endogenous Daxx. We found that Daxx is accumulated in domains formed by wild-type PML but not in those formed by PMLΔSUMO . These data demonstrate that Daxx accumulation at ND10, and potentially PML-Daxx interaction, depends on SUMO-1 modification of PML . If PMLΔSUMO does not recruit endogenous Daxx into domains, both proteins should not colocalize upon overexpression. When we cotransfected Daxx and PML into HEp-2 cells, we found that the two proteins colocalized in the nucleus . However, PMLΔSUMO and Daxx formed separated aggregates upon overexpression . Taken together, these results show that the SUMO-1 modification of PML determines the ability of PML to segregate Daxx into ND10. The nucleus has been increasingly segmented into different domains that relate to the traditional nuclear functions of replication and transcription. These domains are defined by specific chromosomal territories , replication sites , transcription sites , or domains that contain excess splicing components . Like the nucleolus, coiled bodies have been suggested to reflect high rates of transcription because of the localization of certain genes at these sites . Nuclear domains such as ND10 and Gemini have gained attention through their connection to specific diseases or viral infections . For ND10, a function in transcription also has been postulated . The role ND10 plays as a structure remains unclear, although we have suggested that these domains function as nuclear depots for a number of proteins . Such a model dissociates the function of the respective ND10-associated proteins from their location at ND10. It also suggests that physiologically relevant interactions of these proteins with other proteins might occur at different locations. Recruitment of proteins from nucleoplasm to ND10, leading to the changes in the intranuclear protein balance, may affect cellular functions. Modification of ND10 in a number of pathological processes strongly suggests that investigating the ND10 assembly mechanism is an essential step towards understanding the function of this nuclear structure. Using cells that lack either Sp100 or the newly described ND10-associated protein BML, we demonstrated that neither the lack of Sp100 nor the absence of BLM affected the structure of ND10. In contrast, cells lacking PML exhibited dispersion of all ND10-associated proteins. ND10 could be reconstructed by the introduction of PML into PML−/− cells either by transfection or, at more physiological concentrations of PML, through the fusion with PML-containing cells. This reconstruction includes the recruitment of all ND10 proteins, including Sp100, which does not interact with PML, suggesting the presence of mediator proteins. Our observation establishes that PML is the essential protein for ND10 assembly under physiological conditions. The destruction of ND10 induced by the herpes virus immediate early gene products supports the notion that PML plays a central part in the maintenance of ND10. Particularly the direct interaction of IE1 with PML may deprive ND10 of PML and so lead to their destruction. Consistent with this effect, constitutive expression of IE1 in astrocytoma cells and human fibroblasts also results in the loss of detectable ND10 (Ishov, A.M., unpublished observation). The formation of ND10 has been found to accompany APL remission in promyelocytes . After retinoic acid treatment of APL-derived NB4 cells, the dominant negative PML–retinoic acid receptor α (PML-RARα) fusion protein is selectively hydrolyzed through the proteosome pathway, releasing wild-type PML . This, in turn, might be the reason for ND10 formation in RA-treated NB4 cells, which normally have only dispersed ND10-associated proteins. Therefore, the recovery of ND10 may be a consequence of PML availability, which leads to the segregation of other ND10 proteins. The central role of PML in ND10 formation suggests the presence of proteins that are accumulated at ND10 through interaction with PML. We found that the recently cloned DNA-binding protein Daxx was highly concentrated in ND10. Moreover, we found that Daxx interacted with PML in the yeast two-hybrid assay, mapped the interaction domain of Daxx, and demonstrated that Daxx localization at ND10 depended on the presence of a PML interaction domain as well as SUMO-1 modification of PML. The discrepancy between previously reported Daxx interactions with Fas , CENP-C , and ND10 localization of this protein suggests that Daxx does not accumulate together with all interaction partners, but is preferentially accumulated at ND10. In the absence of PML in PML−/− MPEF cells, ND10 are destroyed. Therefore, ND10-associated proteins are expected to be found at their alternative binding locations. Most of these proteins were dispersed throughout the nucleus and, thus, were not amenable to microscopic analysis. Only Daxx was detected at higher concentrations in the areas of condensed chromatin. Daxx was removed from these chromatin regions through the introduction of PML by transient transfection concomitant with the formation of ND10. Therefore, these chromatin regions have a substantial amount of unsaturated Daxx binding sites. We propose that Daxx acts at sites other than ND10 by interactions with DNA or other proteins at a variety of cellular locations. Whether the balance of Daxx between ND10 and condensed chromatin can be modified under physiological conditions is not yet conclusively established; however, such a balance may constitute a potential control mechanism. A key finding of our study was that the Daxx accumulation in ND10 is dependent on SUMO-1 modification of PML. In the yeast two-hybrid system, SMT3, the yeast homologue of SUMO-1 , may have facilitated the PML–Daxx interaction. Moreover, conjugation of SMT3 to other yeast proteins is facilitated by Ubc9 , the yeast homologue of human Ubc9, which is involved in the SUMO-1 modification of PML . Failure to coimmunoprecipitate in vitro translated Daxx with recombinant PML (not shown) is consistent with the idea that SUMO-1 modification of PML mediates this interaction. The SUMO-1 modification level of PML might be a determinant of the amount of Daxx recruited and the avidity with which Daxx is retained at ND10. Therefore, the regulated posttranslational modification of PML may balance the amount of Daxx available in the nucleus. Contrary to previous reports , we found that SUMO-1 modification may not be essential for the deposition of PML at ND10, but rather results in the Daxx accumulation at this domain. The central role of PML in ND10 formation suggests the presence of a protein network where some adapter proteins can mediate recruitment of non-PML–interacting proteins to ND10. Daxx may act as such an adapter and recruit other ND10-associated proteins that do not bind directly to PML (Negorev, D., unpublished results). An emergent hierarchical model for ND10 formation is presented schematically in Fig. 6 . The appearance of ND10 after mitosis must result from a nucleation event possibly through homo- or heteromultimerization of PML. This event may take place at specific nuclear deposition sites, as postulated earlier . Transcriptional activation, for instance by interferon, can upregulate PML expression , nucleating additional aggregation sites. SUMO-1 modification-demodification of PML (third level) may lead to a reversible accumulation of Daxx to ND10 (fourth level), increasing or decreasing the availability of this protein for alternative binding partners (DNA, CENP-C, Fas, Pax3, DNA methyltransferase), and thus regulate corresponding functions. The complexity and plasticity of such a supramolecular regulatory mechanism are evident and envisioned structurally as a network of interacting proteins with PML at its core. | Study | biomedical | en | 0.999996 |
10525531 | All strains used were derived from Saccharomyces cerevisiae wild-type DF5α and its derivative kap122 Δ (MAT α, lys2-801 , leu2-3 , 112 , ura3-52 , his3-200 , trp1-1 (am) , pdr6::URA3) . Yeast strains were grown at 30°C in yeast extract/peptone/glucose (YPD); all yeast manipulation was performed according to described protocols . For deletion of PDR6 in wild-type strain DF5 α, the HIS3 gene was used as a selective marker to replace the PDR6 open reading frame by integrative transformation in a haploid DF5α strain. HIS3 replacement cassette was generated by PCR amplification of markers from pRS 306 with primers that contained 60 nucleotides flanking the PDR6/KAP122 open reading frame from 5′ and 3′ ends . HIS3 marker was switched to URA3 marker by recombination (plasmids were gifts of Dr. F.R. Cross, Rockefeller University). Deletion of genes was confirmed by PCR on total yeast DNA with internal primers. Carboxy-terminal genomic KAP122 -PrA fusion constructs were created by integrative transformation of PCR-amplified constructs with four and a half IgG-binding domains of protein A immediately upstream of PDR6/KAP122 stop codon, followed by a not-in-frame HIS5 ( Schizosaccharomyces pombe ) selection marker (donated by Dr. R. Beckmann, Rockefeller University). TOA1 and TOA2 protein A tagging was similar to that of KAP122 . Primers used for PCR amplification of protein A/ HIS5 cassette contained 60 nucleotides directly upstream of the stop codon of the relevant gene for the 5′ primer and 60 nucleotides 150 bp downstream of the stop codon for the 3′ primer. Haploid yeast cells were transformed by electroporation. This resulted in expression of the chimeric protein A fusion construct under the control of the endogenous promoter. Fractionation and immunoisolation of protein A fusion proteins were performed as described . For a typical isolation, 500 ml of postribosomal supernatant (cytosol) was prepared from a 6-liter YPD culture with a density of 1.7 at A 600 . Cytosol was incubated with 200 μl of rabbit IgG-Sepharose beads at 4°C overnight. After washing with transport buffer (TB: 20 mM Hepes-KOH, pH 7.5, 110 mM KOAc, 2 mM MgCl 2 , 1 mM DTT, 0.1% Tween 20), bound proteins were eluted with a step gradient of MgCl 2 from 50 to 4,500 mM. Proteins were precipitated, resolved by SDS-PAGE on a 4–20% acrylamide gel (Novex), and stained with Coomassie blue. Proteins of interest were excised and prepared for MALDI-TOF spectrometry and/or sequencing. Yeast cells were fixed in 3.7% formaldehyde for 15 min and cell walls were digested. Indirect immunofluorescence was carried out according to published protocols . Protein A moieties of fusion proteins were detected with rabbit IgG that had been preadsorbed to wild-type yeast spheroplasts; Nab2p was detected by rabbit polyclonal antiserum to Nab2p , Npl3p was detected by mouse monoclonal antiserum to Npl3p , the appropriate Cy3-conjugated anti–rabbit or anti–mouse IgG was used for visualization. Nuclei were visualized with the DNA binding stain 4′,6-diamidino-2-phenylindole (DAPI). A Nup1p fragment containing the FXFG repeat region (amino acids 432–816) and a Nup2p fragment containing the FXFG repeat region (amino acid 186–561) were expressed as glutathione S-transferase (GST) fusion proteins as described . Proteins of bacterial lysates were separated by SDS-PAGE and transferred to nitrocellulose. Overlay assays were performed as described . Yeast cytosol from the Kap122p-PrA–expressing strain was diluted 1:1 with TB-5% milk and incubated on the blot overnight at 4°C. Bound proteins were detected with rabbit antibodies to mouse IgG and HRP-conjugated donkey anti–rabbit antibodies and enhanced chemiluminescence. TOA1 , TOA2 , and KAP122 open reading frames were amplified from yeast genomic DNA by PCR using synthetic oligonucleotide primers with incorporated restriction sites for subcloning into relevant Escherichia coli expression vectors. TOA1 was subcloned into NdeI and BamHI sites of kanamycin resistance–conferring pET 28a (Novagen, Inc.) to allow expression of Toa1p with an amino-terminal His tag. TOA2 was subcloned into NcoI-BamHI sites of ampicillin resistance–conferring pET 19b (Novagen, Inc.) to allow coexpression of Toa2p with Toa1p. E . coli strain BL21 gold (Stratagene) was cotransformed with both the His 6 -Toa1p– and the Toa2p-expressing plasmids, and E . coli cells coexpressing both subunits of TFIIA were grown at 37°C in LB medium containing 60 μg/ml kanamycin and 100 μg/ml ampicillin to a density of 0.7 at A 600 . Protein expression was induced by 1 mM isopropyl-β- d- thiogalactopyranoside (IPTG) for 10 h at 17°C. E . coli were lysed in TB with added protease inhibitor cocktails (Boehringer Mannheim) using the French pressure cell. The bacterial lysate was incubated with Talon™ resin (CLONTECH Laboratories) for 30 min at room temperature (RT). Talon™ beads were washed with lysis buffer (TB-0.1% Tween 20) and TB buffer containing 2 mM ATP, 10 mM Mg(OAc) 2 , and 10 mM imidazole. For protein binding assays, the His 6 -Toa1p/Toa2p complex was used either still bound to Talon™ beads or was eluted with TB containing 80 mM imidazole. KAP122 was subcloned into BamHI-EcoRI sites of the derivative of pGeX4T3 (Pharmacia Biotech) with additional Tev protease cleavage site , to allow expression of Kap122p with a potentially cleavable amino-terminal GST tag. Kap122p-expressing E . coli cells were grown in LB medium containing 100 μg/ml ampicillin to a density of 0.7 at A 600 and GST-Kap122p expression was induced by 1 mM IPTG for 3 h at 30°C. GST-Kap122p fusion protein was purified from bacterial lysates according to the manufacturer's protocol (Pharmacia Biotech). Binding assays were performed in TB with the addition of 10% glycerol and 0.1% casaminoacids (Invitrogen Corp.) to prevent nonspecific interactions. 10 μl of glutathione-Sepharose beads containing either 2 μg immobilized GST-Kap122p or 2 μg immobilized GST were incubated with 5 μg of purified His 6 -Toa1p/Toa2p complex in a total volume of 100 μl for 1 h at RT. Beads were collected by centrifugation at 1,000 g for 30 s, washed five times by mixing with 1 ml of TB followed by sedimentation, and were resuspended in 15 μl of sample buffer. Proteins in one half of each sample were resolved by SDS-PAGE on a 4–20% acrylamide gel (Novex, Inc.) and stained with Coomassie blue. 200 μl of GST-Kap122p E . coli lysate was incubated with 10 μl Talon™ resin at RT for 1 h to deplete the lysate of endogenous bacterial proteins that interact nonspecifically with Talon™ beads. Beads were sedimented at 1,000 g for 30 s, and the lysate was passed through a Micro Bio-Spin ® chromatography column (Bio-Rad Laboratories) to exclude any remaining Talon™ beads. 50 μl of TB containing 50% glycerol and 0.5% casaminoacids was added to 200 μl of precleared lysate and the mixture was incubated for 1 h at RT with 2 μg of His 6 -Toa1p/Toa2p complex immobilized on 10 μl of Talon™ beads or with 10 μl of empty Talon™ beads as a control. At the end of incubation, beads were sedimented and washed five times with 1 ml of TB. Talon™ beads with bound proteins were resuspended in sample buffer and half of each sample was resolved by SDS-PAGE on a 4–20% acrylamide gel (Novex, Inc.) and stained with Coomassie blue. Recombinant Saccharomyces cerevisiae Ran was prepared and loaded with GDP or GTP as described . Protein A–tagged Toa2p with bound Kap122p was immunoisolated from a postribosomal supernatant as described above. After washing, IgG-Sepharose beads were resuspended in TB and divided into several equal fractions. These fractions were incubated either with TB alone, or with 4 μM RanGDP or RanGTP for 60 min at room temperature. 1 mM GTP was included, except when using RanGDP. Beads were transferred to a column, and the drained liquid, together with a subsequent 100-μl TB wash, was collected; this constituted the released material. After washing columns with TB, remaining bound proteins were eluted with 1,000 and 4,500 mM MgCl 2 step gradient. Fractions eluted at 1,000 and 4,500 mM were combined and comprised the bound material. Proteins were precipitated, resolved by SDS-PAGE, and stained with Coomassie blue. 15 μl of Talon™ beads with 3 μg of immobilized His 6 -Toa1p/Toa2p complex were washed five times with 1 ml of TB, incubated with precleared GST-Kap122p lysate, as described above, to obtain a GST-Kap122p/His 6 -Toa1p/Toa2p complex. Three equal aliquots of beads were incubated with either 4 μM RanGTP, 4 μM RanGDP, or TB alone for 2 h at RT in a final volume of 40 μl. 1 mM GTP was included, except when using RanGDP. Beads were sedimented by centrifugation and 30 μl of supernatant were collected and passed through Micro Bio-Spin ® chromatography column (Bio-Rad Laboratories) to exclude any contaminating Talon™ beads. The drained liquid represented the released material. Beads were washed five times with 1 ml of TB and resuspended in 15 μl of sample buffer. Proteins in one half of each bound and released sample were resolved by SDS-PAGE on 4–20% acrylamide gel (Novex, Inc.) and stained with Coomassie blue. In all cases described here, we found that the genomic replacement of a gene by its protein A–tagged version did not result in any apparent changes in growth rates under the conditions used in our experiments and when compared with those of an otherwise isogenic strain. This indicated that PrA tagging did not apparently interfere with the function of the essential proteins that were tested here; however, this cannot be confirmed for the Kap122p-PrA strain because of the absence of a discernible phenotype of the KAP122 deletion strain. Immunofluorescence microscopy of a KAP122-PrA haploid strain showed that Kap122p-PrA is located both in the cytoplasm and the nucleus , which is consistent with the localization of other protein A–tagged Kaps and their function in shuttling between the nucleus and the cytoplasm. Karyopherins have been previously shown to interact with peptide repeat–containing nucleoporins . To test whether Kap122p interacts with nucleoporins, we expressed FXFG repeat–containing fragments of the nucleoporins Nup1p and Nup2p in E . coli , separated proteins of bacterial lysates by SDS-PAGE, transferred them to nitrocellulose, and performed overlay assays with Kap122p-PrA cytosol. Kap122p-PrA interacted strongly and specifically both with Nup1p and Nup2p fragments . This strongly suggests that Nup1p and Nup2p are among the nucleoporins that bind to Kap122p. Kaps that function in protein import form stable complexes with their import substrates in the cytoplasm . To isolate such complexes, we prepared a postribosomal cytosol fraction from the KAP122-PrA haploid strain and incubated it with IgG-Sepharose. Bound proteins were eluted with a step gradient of MgCl 2 , separated by SDS-PAGE, and stained with Coomassie blue . The Kap-bound substrates usually elute at MgCl 2 concentration between 100–1,000 mM MgCl 2 , whereas the PrA-tagged Kap elutes at 4.5 M MgCl 2 . Indeed, a major band with an apparent M r of 150 kD, which likely represented Kap122-PrA, eluted at 4.5 M MgCl 2 . The numerous minor bands in this fraction, as well as in fractions eluting at lower MgCl 2 concentrations, are likely degradation products of Kap122-PrA retaining their PrA moiety, as confirmed by immunoblotting with rabbit IgG (data not shown). A number of proteins were present primarily in the 100- and 250-mM MgCl 2 eluates and these are putative substrates for Kap122p. One of these bands migrating at ∼14 kD was excised, analyzed by mass spectrometry and peptide microsequencing after digestion with trypsin , and thereby identified as the small subunit (Toa2p) of the general transcription factor IIA (TFIIA). These data suggested that Toa2p might be an import substrate for Kap122p. To determine whether Toa2p is indeed a transport substrate for Kap122p, we examined the localization of Toa2p in wild-type (WT) and KAP122 deletion ( kap122 Δ) strains. There were no apparent differences in growth rates between kap122 Δ and an isogenic wt strain in YPD medium at 30°C. For these experiments, TOA2 was genomically tagged with PrA in isogenic wt and kap122 Δ strains. Immunofluorescence microscopy showed that in wild-type cells Toa2p-PrA was located primarily in the nucleus, whereas in the kap122 Δ cells Toa2p-PrA was mislocalized largely to the cytoplasm . The diminished nuclear localization of Toa2p-PrA in the kap122 Δ strain is consistent with Kap122p representing the principal Kap for import of Toa2p. Localization of several other substrates, whose transport is mediated by distinct Kaps, was not affected by KAP122 deletion. The nuclear import of Npl3p, mediated by Kap111p and Nab2p, mediated by Kap104p , appeared similar in both wild-type and kap122 Δ strains , showing that deletion of KAP122 does not generally affect nucleocytoplasmic transport. Yeast TFIIA has been shown to consist of two subunits: a small subunit (Toa2p, calculated M r 13.5 kD) and a large subunit (Toa1p, calculated M r 32 kD) that migrates as a 43-kD protein in SDS-PAGE . Separately expressed Toa1p and Toa2p are insoluble and unable to complement transcription systems unless both subunits are renatured together ; this suggests that both subunits of TFIIA are unlikely to exist as separate entities in the cell . Crystallographic data of TFIIA bound to a DNA–TATA-binding protein (TBP) complex revealed that the two subunits are intertwined with each other to form a heterodimer, and that the heterodimer interacts with TBP and also with the phosphate backbone of DNA . Hence, it is clear that in the nucleus Toa1p and Toa2p interact with the DNA–TBP complex as a heterodimer. Our data above suggested that, in the cytoplasm, Toa2p existed in a complex with Kap122p-PrA and it was possible that Toa1p was part of this complex. As Toa2p and Toa1p bind as a dimer to the DNA–TBP in the nucleus, we investigated whether a Toa1p–Toa2p complex existed also in the cytoplasm. Therefore, we analyzed a postribosomal cytosol from a haploid TOA2-PrA strain by IgG-Sepharose affinity chromatography. A band migrating at ∼110 kD was eluted between 100 and 1,000 mM MgCl 2 , and this band was confirmed to be Kap122p by mass spectrometric analysis. As expected, Toa2p-PrA eluted at 4.5 M MgCl 2 . However, there was another major band, migrating at ∼45 kD, which coeluted with Toa2p-PrA . By mass spectrometric analysis this band was identified as Toa1p. The elution of Toa1p at 4.5 M MgCl 2 suggested that Toa1p and Toa2p form a tight cytoplasmic complex that interacts with Kap122p. To determine the cytoplasmic binding partners of Toa1p-PrA, we analyzed the postribosomal cytosol of a TOA1-PrA strain by IgG-Sepharose affinity chromatography. A band migrating at ∼110 kD and eluting between 100 and 1,000 mM MgCl 2 was identified by mass spectrometric analysis as Kap122p. Toa2p, identified by mass spectrometric analysis, coeluted with the Toa1p-PrA at 4.5 M MgCl 2 , confirming the data in Fig. 6 that these two proteins form a tight cytoplasmic complex that interacts with Kap122p. Numerous degradation products of Toa1p-PrA, retaining their PrA moiety and identified by immunoblotting with rabbit IgG, were also visible in this fraction (data not shown). The apparent sensitivity of Toa1p to proteolysis could explain less than equimolar quantities of Toa1p that were isolated with either Kap122p-PrA or with Toa2p-PrA . Toa1p was similarly sensitive to proteolysis when coexpressed with Toa2p in E . coli . These smaller fragments of Toa1p that we observed when Toa1p was isolated from yeast or expressed in E . coli , may correspond to p55-related fragments observed when a human homologue of Toa1p was expressed in bacteria . As expected, immunofluorescence microscopy of Toa1p-PrA in a wt and a kap122 Δ strain gave similar results to those obtained for Toa2p-PrA : the primarily nuclear localization of Toa1p-PrA in the wt strain is diminished in the kap122 Δ strain in favor of a diffuse cytoplasmic localization . Toa1p and Toa2p are essential for viability, whereas Kap122p is not. One solution to this apparent paradox is that one or several Kap(s) other than Kap122p can import these proteins into the nucleus in a kap122 Δ strain. However, the immunofluorescence data of Fig. 4 and Fig. 8 suggested that if Toa1p–Toa2p were imported by alternative Kaps, this import would be less efficient than that mediated by Kap122p. Nevertheless, to search for alternative Kaps, a cytosol from a kap122 Δ /TOA2-PrA strain was analyzed by IgG-Sepharose affinity chromatography . As expected, Toa2p-PrA was eluted together with Toa1p (and its numerous degradation products) in the 4.5-M MgCl 2 fraction . However, fractions eluted between 100 and 1,000 mM MgCl 2 did not show any visible bands in the 100-kD region and above, where Kaps migrate. Hence, if Kaps other than Kap122p can import the Toa1p–Toa2p, they might do so with lower efficiency than Kap122p and, therefore, are likely to be below the limits of detection of this assay. Import complexes of substrate–Kap are dissociated by RanGTP . To investigate whether this is also the case for the Toa1p/Toa2p/Kap122p complex, we used postribosomal cytosol from a TOA2-PrA strain to prepare an IgG-Sepharose–bound complex of Toa2p-PrA/Toa1p/Kap122p. This complex was incubated with either transport buffer alone, RanGDP, or RanGTP, and the material that was released from the IgG-Sepharose after completion of incubation was collected. Thereafter, the remaining IgG-Sepharose–bound proteins were eluted at 1.0 and 4.5 M MgCl 2 . Proteins were analyzed by SDS-PAGE and Coomassie blue staining . Incubation with RanGTP clearly led to the release of most of the Kap122p (compare lanes 1–3). In contrast, incubation with RanGDP did not result in dissociation of Kap122p (lanes 4–6). These data indicated that the cytoplasmic Toa2p-PrA/Toa1p/Kap122p complex, like other import substrate–Kap complexes is sensitive to dissociation by RanGTP but not by RanGDP. To determine whether Kap122p is able to interact directly with a Toa1p–Toa2p complex, we coexpressed both His 6 -Toa1p and Toa2p in E . coli . The TFIIA subunits formed a soluble complex and were purified from bacterial lysate via the His 6 affinity tag at the amino terminus of Toa1p. Kap122p with an amino-terminal GST tag was also expressed in E . coli . We tested for binding between GST-Kap122p and the His 6 -Toa1p/Toa2p complex when either was immobilized on GSH-Sepharose or Talon™ beads, respectively. We found that purified and immobilized His 6 -Toa1p/Toa2p complex bound GST-Kap122p present in a preincubated bacterial lysate depleted of its Talon™-binding proteins . Incubation of empty Talon™ resin with E . coli lysate containing GST-Kap122p did not result in any Kap122p binding . Likewise, GST-Kap122p, immobilized on GSH-Sepharose, bound the purified soluble His 6 -Toa1p/Toa2p complex . A control using immobilized GST alone showed no binding of His 6 -Toa1p/Toa2p . These experiments using E . coli -expressed Toa1p–Toa2p and Kap122p show that Kap122p interacts directly with Toa1p–Toa2p complex. We investigated whether the complex of recombinant GST-Kap122p/His 6 -Toa1p/Toa2p is sensitive to dissociation by RanGTP but not RanGDP. To this end, we prepared a Talon™-bound complex of GST-Kap122p/His 6 -Toa1p/Toa2p recombinant proteins . Beads were divided into three equal fractions and incubated with RanGTP, RanGDP, or TB alone. At the end of incubation, released material was collected, beads were extensively washed with TB, and bound and released proteins were analyzed by SDS-PAGE and Coomassie blue staining . Incubation with RanGTP resulted in almost complete release of GST-Kap122p from Talon™-bound His 6 -Toa1p/Toa2p complex . Incubation with RanGDP did not release GST-Kap122p from the Toa1p–Toa2p complex , neither did control incubation with TB in the absence of Ran . These results confirm the specificity of the interaction between recombinant Kap122p and the recombinant Toa1p–Toa2p complex as well as the sensitivity of this interaction to dissociation by RanGTP. Based on sequence similarity with karyopherin βs, the product of the PDR6 gene of S . cerevisiae was previously suggested to be a member of the Kap β family . In this paper, we report that the hitherto uncharacterized product of the PDR6 gene does indeed function as a Kap and, therefore, named it Kap122p. We show that Kap122p functions in the nuclear import of the complex of large and small subunits, Toa1p, and Toa2p, of TFIIA. The relationship between the observed drug resistant phenotype of PDR6 and the function of Kap122p/Pdr6p in nuclear import of TFIIA (or of other proteins) remains to be elucidated. We found that Kap122p is localized both in the cytoplasm and the nucleus, which is consistent with its function of shuttling between these two compartments. Cytosolic Kap122p exists as a complex with the small and large subunit of TFIIA. The precise stoichiometry of this complex remains to be determined. Based on biochemical and crystallographic data, it is unlikely that the two subunits exist as separate entities . Therefore, one possibility is that Kap122p also functions as a chaperone, and that immediately after synthesis in the cytoplasm, each subunit associates with Kap122p. In this scenario, each subunit would contain a Kap122p cognate NLS. Each of the subunit/Kap122p heterodimers would associate, via interaction between the two subunits, to form a tetramer, which is imported into the nucleus. Alternatively, only one of the subunits may contain a Kap122p-cognate NLS. After synthesis, this subunit could associate with Kap122p and with the other subunit to form a heterotrimer that would be imported into the nucleus. After import, in each of these two scenarios, RanGTP would dissociate the TFIIA heterodimer from Kap122p. Our data here argue against a third possibility, namely that a subunit/Kap122p heterodimer is imported separately because we found a stable interaction between the two subunits in the cytoplasm. In fact, while the Toa1p–Toa2p complex was dissociated from Kap122p by MgCl 2 concentrations between 100 and 1,000 mM, the interaction between the two subunits resisted dissociation at these MgCl 2 concentrations . PrA-tagged Kap122p was found to be associated in the cytoplasm with other proteins . We do not yet know the identity of these proteins and whether they represent contaminants or alternative import substrates for Kap122p. However, it is clear that these other proteins are not part of the Toa1p/Toa2p/Kap122p complex as they were not copurified in a reverse pullout with PrA-tagged Toa1p or Toa2p . As in the case of other PrA-tagged Kaps, several degradation products of Kap122p-PrA retaining their protein A moiety and eluting predominantly in the 4,500 mM MgCl 2 fraction were observed and confirmed by immunoblotting (data not shown). We were able to reconstitute the Kap122p/Toa1p/Toa2p complex from recombinant proteins . Moreover, this complex was sensitive to dissociation by RanGTP but not RanGDP . These findings support the conclusion that Kap122p interacts directly with the Toa1p–Toa2p complex and that this interaction, like Kap122p/Toa1p/Toa2p interaction in the yeast cytosol, is sensitive to dissociation by RanGTP but resists dissociation by RanGDP. It is known that RanGTP dissociates import substrates from Kaps by binding to the Kap . The X-ray crystal structure of RanGTP complexed to Kap β2 (transportin) and Kap β1 (importin) has been determined . Ran binding to Kap122p/Pdr6p has been previously investigated and no Ran binding was detected . Consistent with this report, we have so far not been able to demonstrate binding of RanGTP to Kap122p in overlay or solution binding assays (data not shown). This might indicate that RanGTP binds Kap122p with very low affinity. Stable binding of RanGTP to Kap122p may require additional proteins. However, the Kap122p/Toa1p/Toa2p complex isolated from yeast or reconstituted from recombinant proteins could be dissociated by RanGTP but not RanGDP. These data confirm that RanGTP is directly involved in dissociating Kap122p from the Toa1p–Toa2p complex and provide support for the existence of functionally relevant interaction between RanGTP and Kap122p. It appears that Kap122p is the principal Kap dedicated to the import of TFIIA because in a strain where KAP122 had been deleted there was a significant mislocalization of the two TFIIA subunits from the nucleus to the cytoplasm . Surprisingly, KAP122 deletion is not lethal, whereas deletion of either of the two TFIIA subunits is. Therefore, Kaps other than Kap122p are likely to be involved in nuclear import of the TFIIA subunits. However, so far we have failed to identify alternative Kaps by cytosolic pullout experiments with Toa2p-PrA in a kap122 Δ strain . It is likely that import of the TFIIA subunits by these putative alternative Kaps proceeds with much lower efficiency than import by Kap122p, based on the significant reduction in the nuclear localization of the TFIIA subunits observed in a kap122 Δ strain. Nevertheless, import of these two essential proteins in the absence of Kap122p appears to be sufficient, as there is no apparent difference in the growth rate between the kap122 Δ and an isogenic wt strain. Alternative Kap(s) may be difficult to detect in an immunoisolation assay as they may bind to the two TFIIA subunits with lower affinity. There are precedents for essential proteins being imported by several Kaps of which the principal one is not essential. For example, ribosomal proteins have been shown to be imported by the abundant Kap123p . However, Kap123p is not essential, but the essential Kap121p can back up ribosomal protein import in a kap123 Δ strain . Alternatively, the Toa1p–Toa2p heterodimer is small enough (<60 kD) that it might diffuse through the NPC. Kap122p is the third Kap that so far has been shown to be dedicated to the import of a general transcription factor, the others being Kap119p, which is involved in the nuclear import of the nonessential transcription factor TFIIS , and Kap114p, which is involved in the nuclear import of the TATA binding protein . The advantages of maintaining dedicated Kaps for the import of specific transcription factors are likely to be in the regulatory realm and remain to be elucidated. | Study | biomedical | en | 0.999995 |
10525532 | Neuroblastoma B104 cells and CHO cells were grown in 6% newborn calf serum in DME. Oligodendrocytes from shiverer mice were isolated after 12–14 d of growth in culture as described previously . Restriction enzymes and RNA polymerase were obtained from New England BioLabs, Promega, Stratagene, and Epicentre Technologies. RNA and protein molecular markers were obtained from GIBCO BRL. Texas red–conjugated dextran (TRD) was obtained from Molecular Probes, Inc. Rabbit polyclonal antibody to ribophorin I (9R1) was obtained from R. Gould (New York State Institute for Basic Research and Developmental Disabilities, Staten Island, NY). Characterization of antibody 9R1 is described in Kreibich et al. 1983 . Mouse monoclonal antibody to hnRNP A2 (EF-67) was obtained from W. Rigby (Dartmouth Medical School, Lebanon, NH). Recombinant hnRNP A2 was obtained from R. Smith (University of Queensland, Queensland, AU). Full-length cDNA for S65T mutant GFP cloned in pRSETB, was obtained from Dr. R.Y. Tsien (University of California, San Diego, CA). pGEM1A, containing the SP6 promoter and poly A, was obtained from Dr. G. Carmichael (University of Connecticut Health Center, Farmington, CT). The entire S65T mutant GFP cDNA was subcloned into the BamH1 site of pGEM1A to create pGFP′A. The RTS sequence (GCCAAGGAGCCAGAGAGCATG) was inserted into AvaI/SacI cut pGFP′A and HindIII-XbaI cut pGFP′A to create pGFP′3RTS and pGFP′5RTS, respectively. To create pGFP′53RTS, the RTS was inserted into both AvaI-SacI and HindIII-XbaI cut pGFP′A. To create dicistronic constructs, full-length cDNA for BFP, cloned in pQBI50 purchased from Quantum Biotechnologies Inc., was amplified by PCR using oligonucleotides that placed XbaI and XmaI cloning sites at the 5′ and 3′ ends, respectively, and then was inserted into XbaI-XmaI cut pGFP′A and pGFP3′RTS to replace the GFP ORF with the BFP ORF, creating pBFP and pBFP3′RTS. The IRES from EMCV and S65T GFP ORF cloned in pTR5-DC/GFP (a kind gift from D.D. Moser, Biotechnology Research Institute, Montreal, Canada) were amplified by PCR using oligonucleotides that placed XmaI cloning sites at both 5′ and 3′ ends, and then was inserted into XmaI cut pBFP and pBFP3′RTS to create pBDCG and pBDCG3R, respectively. GFP mRNA and GFP/RTS mRNA, containing the RTS in the 3′UTR, 5′UTR, or both, were prepared by in vitro transcription using AmpliScribe SP6 Transcription Kits (Epicentre Technologies) using pGFP′A, pGFP′3RTS, pGFP′5RTS, and pGFP′53RTS, linearized with SapI as template DNA, and m7G(5′)ppp(5′)G added to the transcription mixture to produce capped mRNAs. To assay translation in vivo, capped GFP′A mRNA and GFP′3RTS mRNA at equivalent concentrations were microinjected into neuroblastoma B104 cells, CHO cells, or shiverer oligodendrocytes as described previously . TRD was coinjected to provide a measure of the injected volume. Injected cells were incubated at 37°C for 20–24 h. The expressed GFP/TRD fluorescence was visualized by dual channel confocal microscopy, using a Zeiss LSM 410 confocal laser scanning system (Zeiss) with an infinity-corrected 60× 1.4 NA objective. Unless otherwise indicated, images were collected within the dynamic range of the frame store such that pixel values outside the cell were >0 and pixel values inside the cell were <255. GFP and TRD pixel values were integrated over the nucleus to avoid autofluorescent signal in the cytoplasm. The concentration of injected RNA in each cell was calculated based on the initial concentration of RNA and TRD in the pipet and the integrated TRD pixel values in the nucleus. Translation efficiencies were determined by calculating the GFP/RNA ratio for each cell. To assay translation in vitro, nuclease-treated rabbit reticulocyte lysate and wheat germ extracts were purchased from Promega and reactions were performed according to the manufacturer's instructions. Translation was monitored by [S 35 ]methionine (Amersham) incorporation. Translation products were separated by 12% SDS-PAGE. After electrophoresis, the gels were fixed, soaked in EN 3 HANCE (New England Nuclear), dried, and subjected to autoradiography to visualize labeled GFP polypeptide. The intensities of specific bands were integrated using ImageQuaNT. Fluorescein-labeled GFP RNA and GFP/3′RTS RNA, prepared as previously described , were microinjected into the cytoplasm of neuroblastoma B104 cells. Images of injected cells were collected with a cooled CCD camera at 10-min intervals. Total intracellular fluorescence intensities were integrated using the ImageQuaNT program. B104 cells or oligodendrocytes were incubated in defined culture medium containing 8 μM final concentration of phosphorothioate oligonucleotide obtained from the Molecular Core at UCHC) as described previously . The media containing the oligonucleotide was changed every 8 h for a period of 24 h. Antisense oligonucleotide (CTTTTCTCTCTCCATCGCGGA) was complementary to a region around the translation start site of human hnRNP A2 mRNA. The corresponding sense oligonucleotide was used as a control. Expression of hnRNP A2 in oligodendrocytes was evaluated by immunofluorescence (IF) using monoclonal antibody to hnRNP (1:100). Expression of hnRNP A2 in B104 cells was evaluated by Western blotting with antibody to hnRNP A2 (1:1,000) and rabbit polyclonal antibody to ribophorin I (1:1,000) as a control for loading. The NaeI-HindIII fragment from pBluescript SK II(+) was inserted into NaeI-HindIII cut pGFP′A and pGFP′A/3′RTS to create pGT7 and pG3RT7, respectively. Digoxigenin-labeled GFP mRNA and GFP/3′RTS mRNA were generated by in vitro transcription using BsaWI cut pGT7 and pG3RT7 as a template. The RNA transport assay was performed as previously described . A translation enhancer is a cis-acting RNA sequence that increases the translation efficiency of the mRNA containing it. Translation efficiency is a measure of the amount of protein translated from a certain amount of RNA. To determine translation efficiency for a particular RNA in vivo, it is necessary to measure both the concentration of that RNA and the amount of protein expressed from it in individual cells. Conventional transfection techniques are of limited use for this purpose because the amount of DNA or RNA introduced or expressed is variable and difficult to measure in individual cells. In some cells, the level of RNA may be saturating, in which case the rate of translation may be determined not by the concentration of RNA but by the activity of rate limiting components of the translation machinery (such as eIF4E) in the cell. Therefore, translation efficiency can only be accurately measured in cells where the exogenous RNA is present in the cell at subsaturating concentrations. Furthermore, cells with saturating levels of RNA generally have high levels of protein expression and, therefore, a disproportionate contribution to the level of protein expression measured for the population as a whole. This tends to obscure the contribution of cells with subsaturating levels of RNA and lower levels of protein expression which are the very cells that are informative for determining translation efficiency for a particular RNA. For these reasons it was necessary to develop a method to introduce subsaturating amounts of RNA into cells and to measure both RNA concentrations and protein translation levels in individual cells. A ratiometric translation efficiency assay was developed using mRNA encoding green fluorescent protein (GFP) from Aequorea victoria as a reporter for translation and TRD (10 kD) as a reporter for the volume of microinjected RNA. Capped, polyadenylated mRNA encoding GFP was coinjected with TRD into the cytoplasm of B104 cells which were used instead of oligodendrocytes because they lack long processes so that translation of the injected RNA can be analyzed without the confounding variable of RNA transport. After incubation to allow time for GFP expression, injected cells were analyzed by dual channel confocal microscopy. TRD dispersed rapidly throughout the cytoplasm and nucleus, and total fluorescent intensity in each cell remained constant over 24 h (data not shown). GFP, which also dispersed throughout the cytoplasm and nucleus, was first detected by 2 h after injection, increased to a maximum by 9 h, and remained constant up to 24 h (data not shown). The relative amount of RNA injected into each cell was calculated based on the integrated TRD fluorescence intensity in the injected cell. The amount of GFP expression was quantified by integrating GFP fluorescence intensity. Since both TRD and GFP enter the nucleus by diffusion, fluorescence intensities were integrated in optical sections through the nucleus to avoid autofluorescent signal from the cytoplasm. The intensity of TRD fluorescence in each cell provides a measure of the volume of RNA injected. The intensity of GFP fluorescence provides a measure of the amount of protein translated from the injected RNA. The ratio of GFP to RNA provides a measure of the translation efficiency in each cell. The RTS is a cis-acting sequence that mediates intracellular trafficking of MBP and other mRNAs in oligodendrocytes. To determine its effect on translation efficiency the RTS was inserted into the 3′UTR of GFP. Representative cells injected with GFP/RTS RNA or GFP RNA are shown in Fig. 1 . The intensity in the red channel (which measures TRD concentration which is proportional to RNA concentration) was comparable in the cells injected with GFP/RTS RNA and GFP RNA indicating that comparable volumes of RNA were injected into these cells. The intensity in the green channel (which measures GFP concentration) was greater in the cells injected with GFP/RTS RNA compared with cells injected with GFP RNA , indicating that the RTS increases GFP expression. The values for intracellular RNA concentration (in nM) and GFP (expressed in arbitrary fluorescence intensity units) obtained from multiple individual cells are shown in Fig. 1 B. Overall, the level of GFP expression was greater in cells injected with GFP/RTS RNA compared with cells injected with GFP RNA. There was considerable variability in both the amount of RNA injected and the level of GFP expression per cell. Some of the variability in GFP expression may reflect injection of RNA into subcellular compartments with different translational activities. For example, RNA injected into the nucleus is not efficiently exported to the cytoplasm and, therefore, not translated efficiently. To quantify translation efficiency the ratio of GFP/RNA was calculated and plotted versus the amount of injected RNA for each cell. Fig. 1 C shows translation efficiencies for cells injected with different amounts of GFP/RTS RNA and GFP RNA. In cells injected with GFP RNA, translation efficiency was relatively constant (mean translation efficiency = 1.0 ± 0.3) over a wide range of injected RNA, indicating that the endogenous translation machinery in the cell is not saturated by the injected RNA. Cells injected with very low levels of GFP RNA (<5 nM) expressed very low levels of GFP. In some cases the level of GFP expression, while detectable, was too low to quantify accurately. These cells were not included in the graph. In cells injected with low amounts of GFP/RTS RNA (<5 nM), translation efficiency was enhanced (>10-fold in several cells and up to 16-fold in one cell) relative to GFP RNA. At higher amounts of injected RNA (>10 nM), translation efficiency for GFP/RTS RNA was comparable to GFP RNA. These results indicate that the RTS enhances expression of GFP mRNA at low RNA concentrations but the enhancer function is saturated at high RNA concentrations suggesting that RTS-mediated enhanced expression requires component(s) that are present in limiting amounts in the injected cells. Increased GFP expression mediated by the RTS could be due to either enhanced translation efficiency or enhanced RNA stability in the injected cells. To measure the relative stabilities of GFP/RTS RNA and GFP mRNA in microinjected cells, a novel in vivo fluorescence dequenching assay was used. Fluorescein-UTP was incorporated by in vitro transcription into the body of GFP/RTS and GFP RNA. In the intact RNA, fluorescence is reduced by intramolecular quenching between proximate fluorophores. The fluorescently labeled RNA was microinjected into cells and intracellular fluorescence was measured as a function of time after injection. In this assay, degradation of the fluorescent RNA causes an increase in total fluorescence due to relief of intramolecular quenching (dequenching) in the injected RNA. Complete degradation of the RNA results in an increase of ∼50% in the fluorescent quantum yield due to dequenching. The amount of dequenching provides a measure of the intracellular stability of the injected RNA. As shown in Fig. 2 , there was no significant dequenching over a period of 40 min after injection with either GFP/RTS RNA or GFP RNA, indicating that the two RNAs are both relatively stable within this time frame. The assay was not extended beyond 40 min because translation of the injected RNA could produce GFP fluorescence that would interfere with measurement of the injected fluorescein-labeled RNA. The dequenching assay provides a measure of the rate of RNA degradation in the cell in the time period immediately after injection. Since there is a lag of up to 4 h between synthesis of GFP and appearance of GFP fluorescence in the cell, and since most accumulation of GFP fluorescence occurs within a few hours after injection, it follows that overall accumulation of GFP fluorescence reflects GFP translation within the first few hours after injection. Since there was no detectable difference in stability between GFP RNA and GFP/RTS RNA in the period immediately after injection, it is unlikely that the differential GFP expression observed with GFP/RTS RNA compared with GFP RNA is attributable to differences in initial RNA stability. Therefore, increased GFP expression must be due to enhanced translation efficiency mediated by the RTS. To determine if the translation enhancer function of the RTS is dependent on its position in the mRNA, chimeric GFP mRNAs were synthesized containing the RTS inserted into either the 3′UTR, the 5′UTR, or both. Because there is an AUG within the RTS, insertion into the 5′UTR of GFP RNA may change the translation initiation site which could result in a 10–amino acid extension at the NH 2 terminus of GFP. NH 2 -terminal extensions do not generally affect GFP fluorescence . Chimeric RNAs were microinjected into B104 cells and translation efficiencies were determined at subsaturating RNA concentrations as described in Fig. 1 ( Table ). Translation was enhanced 4.19-fold with the RTS in the 3′UTR, 4.82-fold in the 5′UTR, and 3.27-fold with copies in both the 3′ and 5′UTRs. This indicates that the translation enhancer function of the RTS is position and copy number independent. The marginal decrease in translation enhancer activity with two copies of the RTS compared with a single copy may be due to titration of some limiting factor required for enhanced translation. To determine if the translation enhancer function of the RTS is cell type specific, translation efficiencies for GFP/RTS RNA and GFP RNA were compared in rat B104 cells, CHO cells, and mouse oligodendrocytes in primary culture ( Table ). In CHO cells, the level of GFP fluorescence was more variable than in B104 cells. In some cells, the level of GFP fluorescence was so high that the image was saturated. These cells were not included in the calculation of translation efficiency because the intensity values were not within the dynamic range of the assay, resulting in an underestimate of the actual translation efficiency of GFP/RTS RNA in CHO cells. The variability in GFP expression in CHO cells suggests that factors required for the translation enhancer function of the RTS are expressed at variable levels in these cells. Wild-type mouse oligodendrocytes express high levels of endogenous MBP mRNA, which contains the RTS and which could compete with the microinjected exogenous GFP/RTS RNA. To minimize this potential competition, oligodendrocytes from shiverer mutant mice, which do not express endogenous MBP mRNA , were used for these experiments. The RTS enhanced translation 4.19-fold in B104 cells, 2.56-fold in CHO cells, and 3.38-fold in oligodendrocytes ( Table ), indicating that the translation enhancer function is cell type independent, although the magnitude of the effect may vary among different cell types depending on the capacity of the translation machinery or the level of endogenous RTS-containing RNAs. The experiments in Fig. 1 and Table were performed with monocistronic RNA containing a 5′ cap. The results indicate that the RTS enhances cap-dependent translation. To determine if IRES-dependent translation is also enhanced, a dicistronic mRNA encoding blue fluorescent protein (BFP) in a cap-dependent cistron and GFP in an IRES-dependent cistron was constructed. The RTS was inserted into the 3′UTR of the dicistronic mRNA as shown in Fig. 3 A. Translation efficiencies for BFP and GFP were determined by microinjection into B104 cells. Triple channel images of representative injected cells are shown in Fig. 3 B. In the red channel, TRD intensities are comparable for cells injected with RTS and nonRTS RNA, indicating that the volume of injected RNA was comparable in the two cells shown. In the green channel, GFP intensities are comparable, indicating that IRES-dependent translation is not enhanced by the RTS. In the blue channel, BFP intensity is greater in the cell injected with RTS RNA compared with the cell injected with nonRTS RNA, indicating that cap-dependent translation is enhanced by the RTS. Translation efficiencies for cap-dependent and IRES-dependent translation in cells injected with various amounts of RNA were calculated from the ratio of blue to red and green to red intensities, respectively. In the case of cap-dependent translation, at subsaturating concentrations of RTS RNA, translation efficiency was enhanced 3.61-fold relative to nonRTS RNA . This is consistent with the previous results for cap-dependent translation of monocistronic GFP RNA . In the case of IRES-dependent translation, translation efficiency was comparable between RTS and nonRTS RNA indicating that the translation enhancer function of the RTS is specific for cap-dependent translation and does not affect IRES-dependent translation. In addition, the observation that IRES-dependent translation efficiency was relatively constant over a wide range of RNA indicates that the machinery in the cell required for IRES-dependent translation is not saturated by high concentrations of exogenous RNA. HnRNP A2 binds specifically to the RTS in vitro . To determine if hnRNP A2 is required for either the RNA transport or translation enhancer functions of the RTS cells were treated with antisense oligonucleotide designed to hybridize with the translation start site of hnRNP A2 and suppress its expression. Control cells were treated with the corresponding sense oligonucleotide. The level of hnRNP A2 in oligodendrocytes was estimated by IF with monoclonal anti-hnRNP A2 antibody. In untreated oligodendrocytes , hnRNP A2 is detected at high levels in the nucleus and also in granules in the perikaryon and processes indicating that hnRNP A2 is present in both the nucleus and the cytoplasm. To visualize the hnRNP A2 distribution in the cytoplasm, which is lower intensity than in the nucleus, images were collected under conditions where the nuclear signal was saturated. Both the nuclear and cytoplasmic staining are specific for hnRNP A2 and were not observed with control antibody (data not shown). In oligodendrocytes treated with hnRNP A2 sense oligonucleotide , hnRNP A2 staining in both the cytoplasm and nucleus was comparable to the untreated cell indicating that hnRNP A2 expression is not affected. In oligodendrocytes treated with hnRNP A2 antisense oligonucleotide , hnRNP A2 staining was reduced compared with either the untreated cell or the sense-treated cell, indicating that hnRNP A2 expression was suppressed. It appears that nuclear hnRNP A2 is decreased to a greater extent than cytoplasmic hnRNP A2, suggesting that in oligodendrocytes the nuclear pool of hnRNPA2 turns over more rapidly than the cytoplasmic pool. Since the image for untreated and sense-treated cells is saturated in the nuclear compartment, the extent of hnRNP A2 suppression in antisense-treated cells cannot be quantified from these images. However, in images collected under conditions where the nuclear compartment was not saturated (where cytoplasmic hnRNP A2 is not visualized; data not shown), suppression of nuclear hnRNP A2 in antisense-treated cells was >90%. The effect of suppressing hnRNP A2 expression on RNA transport in oligodendrocytes was determined by microinjecting digoxigenin-labeled RNA into antisense-treated cells and measuring the percentage of cells in which the injected RNA was transported to the peripheral processes . GFP/RTS RNA was transported in >70% of untreated or sense-treated oligodendrocytes but in <40% of antisense-treated oligodendrocytes, while GFP RNA was transported in <20% of treated or untreated cells. These results indicate that the RNA transport function of the RTS in oligodendrocytes is at least partially dependent on hnRNP A2 expression. The level of hnRNP A2 in B104 cells was estimated by Western blotting with anti-hnRNP A2 antibody with anti-ribophorin antibody as a loading control . In untreated cells, hnRNP A2 is detected as a major band with an apparent molecular mass of ∼36 kD, whereas ribophorin I is detected as a band with an apparent molecular mass of ∼65 kD. In some samples, the hnRNP A2 antibody detects two additional bands with molecular masses slightly larger than the major band at 36 kD, which may represent splicing variants. In cells treated with sense oligonucleotide, the relative intensities of the hnRNP A2 and ribophorin bands are comparable to untreated cells, whereas in cells treated with antisense oligonucleotide the intensity of the hnRNP A2 band is decreased relative to ribophorin. This indicates that treatment with antisense oligonucleotide suppresses hnRNP A2 expression in B104 cells. The effect of suppressing hnRNP A2 expression on the translation enhancer function of the RTS was determined by measuring translation efficiencies for GFP/RTS and GFP RNA injected into antisense-treated B104 cells at subsaturating concentrations of RNA . Compared with GFP RNA, the translation efficiency of GFP/RTS RNA was enhanced by 4.19-fold in untreated B104 cells, 2.0-fold in sense-treated cells, and was slightly inhibited (0.94-fold) in antisense-treated cells. These results indicate that the translation enhancer function of the RTS in B104 cells is dependent on hnRNPA2 expression. The partial reduction of translation efficiency in sense-treated cells may be due to nonspecific toxic effects of the oligonucleotides on the cells. To determine if the RTS enhances translation in vitro, GFP/RTS and GFP mRNAs were translated in a rabbit reticulocyte lysate translation system and in wheat germ extract (data not shown). In both systems, the translation efficiencies were comparable for the two RNAs over a range of RNA concentrations , indicating that the translation enhancer function of the RTS requires factors that are lacking in rabbit reticulocyte lysate and wheat germ extract. Addition of recombinant hnRNP A2 to rabbit reticulocyte lysate had little effect on translation of GFP RNA but enhanced translation of GFP/RTS RNA in a dose-dependent manner up to a maximum stimulation at 1 μg . This experiment was repeated several times with different batches of reticulocyte lysate and different preparations of hnRNP A2. As shown in Fig. 5 D, the extent of stimulation by hnRNP A2 was variable, but in each experiment, translation of GFP/RTS RNA was enhanced relative to GFP RNA. These results indicate that the translation enhancer function of the RTS is hnRNP A2 dependent in rabbit reticulocyte lysate. The variability in the extent of stimulation may reflect variation in the activities of different preparations of recombinant hnRNP A2 or variation in the activities of different batches of reticulocyte lysate. Addition of recombinant human hnRNP A2 to wheat germ extract had no effect on translation of RTS RNA (data not shown), suggesting that the interaction between hnRNP A2 and component(s) of the translation machinery that enhances translation in rabbit reticulocyte lysate is specific to mammalian systems. The RTS, originally identified as a cis-acting RNA trafficking sequence in MBP mRNA, is shown to function as an enhancer of cap-dependent translation in vivo and in vitro. This represents the first specific translation enhancer identified in a mammalian system. The translation enhancer function of the RTS is saturable with increasing amounts of RNA, position, copy number, and cell type independent, and cap and hnRNP dependent. A speculative model illustrating the proposed role of RTS/A2 cis / trans determinants in RNA transport and translation activation is shown in Fig. 6 . According to this model, the RNA granule is transported along microtubule tracks using kinesin as a molecular motor . Association of the RNA granule cargo with the kinesin motor during transport requires RTS/A2 determinants. Once the RNA granule reaches its destination, translation is activated through interactions between RTS/A2 determinants and components of the translation machinery, possibly eIF4E at the 5′ cap of the mRNA. This provides an explanation for why the translation enhancer function of RTS/A2 is specific for cap-dependent translation and saturable at high concentrations of RNA, since eIF4E is required for cap-dependent initiation and is present in limiting amounts in most cells . The proposed interactions between RTS/A2 and kinesin during transport and between RTS/A2 and eIF4E during translation may be direct or indirect, mediated through unknown adapter molecules. The model does not specify a detailed molecular mechanism for either the transport or translation enhancer functions of RTS/A2 determinants. However, several possible mechanisms are suggested by structural features of the RTS and properties of hnRNP A2. One possible mechanism is based on sequence homology between the RTS and the consensus Kozak sequence. In the scanning model of eukaryotic translation initiation, the 40S ribosomal subunit binds initially at the 5′ end of mRNA and scans in the 3′ direction, initiating translation at the first AUG codon in a favorable context . In vertebrates, efficiently used AUG start codons are embedded in a consensus sequence motif, termed the Kozak sequence. As shown in Table the consensus Kozak sequence is partially homologous to the RTS suggesting that the Kozak sequence and the RTS use similar mechanisms for enhancing translation. There are several caveats to this hypothesis. The Kozak sequence is position dependent, since it always encompasses the initiation codon, whereas the RTS is position independent, although it does contain an AUG. In addition, the Kozak sequence is known to interact with two proteins (∼50 and 100 kD) from HeLa cells , while the RTS interacts with hnRNP A2 (36 kD), suggesting that the Kozak sequence and the RTS use different trans-acting factors to enhance translation. A second possible mechanism involves the RNA helix destabilizing and annealing properties of hnRNP A2 . The presence of secondary structure in the 5′UTR of mRNA tends to inhibit translation. After association of the eIF4F complex (consisting of initiation factors eIF4A, eIF4E, and eIF4G) with the cap of the mRNA, the interaction of eIF4B and the RNA helicase eIF4A causes ATP-dependent melting of secondary structure in the 5′UTR. This activity is believed to facilitate binding of the 40S preinitiation complex to the mRNA. Recently, eIF4B was shown to stimulate eIF4A-mediated melting of RNA secondary structure in vitro . In a similar fashion, binding of hnRNP A2 to the RTS might promote unwinding of secondary structure in the mRNA to reduce a kinetic barrier to formation of the translation initiation complex. Melting of RNA secondary structure by hnRNP A2 could facilitate binding of the 40S preinitiation complex to the mRNA and scanning the 5′UTR in translation initiation. Besides melting secondary structures, the RNA annealing activity of hnRNP A2 could also play a role in recognition of the start codon. Base pairing between the AUG initiation codon and the anticodon of the initiator tRNA, which is a major determinant for AUG codon recognition, could be facilitated by the RNA annealing activity of hnRNP A2, thus, enhancing translation efficiency. A third possible mechanism involves potential eIF4E-binding motifs in hnRNP A2. A recently determined x-ray structure of murine eIF4E, bound to a cap analogue (7-methyl-GDP), provides a basis for analyzing contacts between eIF4E and proteins that interact with eIF4E during translation initiation . The eIF4E-binding sites in eIF4E-binding proteins, 4E-BP1, 4E-BP2, and eIF4G, share a common motif, Y-X-X-X-X-L-L, suggesting that these proteins compete for the same site in eIF4E. The RBD1 domain of hnRNP A2 contains a peptide sequence, Y-E-Q-W-G-K-L, which is similar to the putative eIF4E binding motif and might be involved in eIF4E binding. This could affect formation of the initiation complex. It will be important to determine whether there is direct biochemical interaction between hnRNP A2 and eIF4E. Whatever the molecular mechanism(s) for translation activation by the RTS and hnRNP A2, the fact that the same cis / trans determinants that mediate multiple steps in the RNA trafficking pathway also enhance translation underscores the integral role of translation regulation in RNA trafficking. The RTS and hnRNP A2 comprise the first translation enhancer identified in a mammalian system. This may prove useful in applications where maximal expression is critical. | Study | biomedical | en | 0.999997 |
10525533 | All constructs were made by site-directed mutagenesis using PCR with Vent polymerase (New England Biolabs). The final constructs for in vivo expression were subcloned into the vector pECE and verified by sequencing. To construct chimeric proteins with conflicting signals, the coding sequence for the 98 NH 2 -terminal residues of influenza hemagglutinin (strain A/Jap/305/57 [H2N2]) was fused via a linker sequence (Gly-Ser) in front of codon 4 of the cDNA of the asialoglycoprotein (ASGP) receptor subunit H1 . The codon for methionine in position 95 was mutated to one for isoleucine to eliminate potential internal translation initiation. The resulting sequence encoded a chimeric protein with two ER targeting signals separated by a spacer of 128 residues. By PCR with this sequence as a template, 5′ truncations were prepared consisting of a 5′ Bgl II site, the COOH-terminal 100, 80, 60, 40, or 20 residues of the spacer sequence, and the signal and the COOH-terminal remainder of H1. In front of these sequences, the cDNAs of the signal sequences of hemagglutinin (H), human prepro-vasopressin-neurophysin II (V), bovine preprolactin (P), and the ASGP receptor H1 (with the wild-type hydrophilic sequence, A, or with a 30 residue deletion, ΔA) , as shown in Fig. 1 C, were ligated by BamH I/Bgl II (Gly-Ser) to produce five series of plasmids. The constructs were named to indicate the origin of the first signal, the length of the hydrophilic spacer sequence, and the origin of the second signal (e.g., V40A). The series without the second signal (H20− etc.) was generated by deleting the entire coding region of the H1 signal from the premade constructs harboring two conflicting signals (H20A etc.) by PCR. To generate potential N -glycosylation sites in the spacer sequence, the codons for IleThrLeu (ITL), MTM, or GS were inserted into the spacer sequence after the codon for Asn-13 of the H1 sequence of the spacer sequence between the signals. To destroy the glycosylation site NITL, the codon for Thr was mutated to one for Asn. To test a different spacer sequence with a natural glycosylation site (NFT), the residues 26–59 of V40A were replaced by residues 61–100 of H1, resulting in V45(NTF)A. The glycosylation site was mutated to QTF in V45(QTF)A. To generate the truncated constructs H55, H75, H95, and H115, the 5′ sequences of constructs H40(NIT)A, H60(NIT)A, H80(NIT)A, and H100(NIT)A encoding the hemagglutinin signal and the spacer sequences were fused via two methionine codons and a Kpn I site encoding Gly-Thr to the 14 COOH-terminal residues of H1, which are recognized by the antibody anti-H1C. Constructs V55, V75, V95, and V115 were constructed accordingly. Cell culture reagents were from Life Technologies, Inc. COS-1 cells were grown in modified MEM supplemented with 10% FCS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 7.5% CO 2 . Transient transfection was performed with lipofectin (Life Technologies, Inc.) according to the manufacturer's instructions in 6-well clusters, and the cells were processed the second day after transfection. For in vivo labeling, transfected cells were incubated for 40 min in methionine-free medium, labeled for 30 min at 37°C with 100 μCi/ml [ 35 S]methionine in starvation medium, transferred to 4°C, washed twice with PBS, lysed, and immunoprecipitated using a rabbit antiserum directed against a synthetic peptide corresponding to residues 277–287 near the COOH terminus of the ASGP receptor H1 (anti-H1C). The immune complexes were isolated with protein A–Sepharose (Amersham Pharmacia Biotech) and analyzed by SDS-PAGE and fluorography. For deglycosylation, the immune complexes were released from protein A–Sepharose by boiling in 50 mM Na citrate, pH 6, 1% SDS, and incubated with 1 mU endo-β- d -N-acetyl glucosaminidase H (endo H) for 5 h at 37°C, before gel electrophoresis. Quantitation was performed using a PhosphorImager (Molecular Dynamics, Inc.). For constructs with the vasopressin signal, the results were corrected for the loss of a methionine within the signal peptide upon signal cleavage. Alkaline extraction was performed as described previously . In brief, cells were homogenized and incubated in Hepes buffer, pH 11.5, for 15 min on ice. One half of each sample was centrifuged through a sucrose cushion, and the membrane pellet and the supernatant were immunoprecipitated separately and compared with the immunoprecipitate of the untreated second half of the sample (total). In vitro translation was performed with standard procedures using rabbit reticulocyte lysate and dog pancreas microsomes . Protease protection assay was performed as described by Wahlberg and Spiess 1997 , except that the scraped cells were homogenized in a cell cracker (from EMBL) as described by Leitinger et al. 1995 . To characterize the in vivo topogenesis of membrane proteins and to test the linear insertion model, we constructed a series of chimeric polypeptides containing an NH 2 -terminal cleaved signal sequence and an internal type II signal-anchor sequence. An NH 2 -terminal portion of influenza virus hemagglutinin with its cleavable signal sequence was fused to the NH 2 terminus of subunit H1 of the ASGP receptor, a type II membrane protein. By successive truncation of the hydrophilic sequence separating the two hydrophobic signals, a series of constructs with spacers of ∼20, 40, 60, 80, and 100 residues was created (H20A, H40A, etc.) . In the wild-type context, each of the two signals efficiently directs translocation of its COOH-terminal end across the membrane. According to the linear insertion model, the NH 2 -terminal hemagglutinin signal will target the polypeptide to the ER membrane and induce translocation of the spacer sequence. The internal H1 signal will then enter the translocon as a stop-transfer sequence and halt further translocation, leaving the COOH terminus of the polypeptide, including the only two potential sites for N -glycosylation, in the cytosol . If, in contrast, the topogenic information of the internal signal was dominant, the COOH-terminal portion of H1 would be translocated and glycosylated, whereas the NH 2 -terminal signal would be forced to insert with the opposite N exo /C cyt orientation or would fail to insert at all . The constructs were transiently expressed in COS-1 cells, labeled for 30 min with [ 35 S]methionine, immunoprecipitated, and analyzed by SDS-gel electrophoresis and fluorography . Consistent with the linear insertion model, the constructs H100A and H80A yielded single products which were not glycosylated, as revealed by their resistance to deglycosylation by endo H. However, the constructs with shorter spacers between the signals, H60A, H40A, and H20A, produced an increasing fraction of an additional species of lower electrophoretic mobility. The shift in mobility was due to glycosylation, as shown by endo H digestion. Both the glycosylated and the unglycosylated products were stably integrated into the ER membrane, since they were recovered in the membrane pellet after alkaline extraction . Under the same conditions, a control protein lacking a membrane anchor (H20−), was completely extracted into the soluble fraction. The results demonstrate that the internal signal, when separated by <80 residues from the NH 2 -terminal signal, did not always function as a stop-transfer sequence, but was able to direct translocation of its COOH terminus across the membrane and to anchor the protein in a N cyt /C exo orientation. Whereas the sequence context of the internal H1 signal is the same in all constructs, that of the hemagglutinin signal is always different. As a result, the flanking charges, which are known to influence the topological preference of signal sequences, are not constant. The charge difference between the 15 COOH-terminal and the 15 NH 2 -terminal flanking residues, Δ(C–N), as defined by Hartmann et al. 1989 , is −0.5 for the hemagglutinin signal in its wild-type context. Δ(C–N) is more negative in H40A (−1.5), H60A (−1), and H80A (−1). Thus, the observed insertion pattern cannot be explained simply by an effect of the flanking charges on the orientation of the NH 2 -terminal signal. Only in H20A, the construct with the shortest spacer, in which the flanking segments of the two signals largely overlap, is Δ(C–N) positive for the hemagglutinin signal (+2) and might favor translocation of the NH 2 terminus. To test experimentally the insertion behavior of the hemagglutinin signal in the different chimeras without the potential interference by a second signal, a series of constructs was made lacking the hydrophobic part of the internal signal (H20−, H40−, etc.). In all cases, the COOH-terminal sequence was completely translocated and glycosylated , demonstrating that different flanking sequences did not affect the functionality of the hemagglutinin signal as a secretory signal. It required the competition by the second signal to cause insertion of H20A, H40A, and H60A with a translocated COOH terminus. To determine the contribution of the NH 2 -terminal signal on topogenesis, the hemagglutinin signal in the H#A constructs was replaced by the cleavable signals from preprolactin (P#A) or the vasopressin precursor protein (V#A), or by the signal-anchor sequence of the ASGP receptor H1, either with (A#A) or without the complete 40-residue NH 2 -terminal, hydrophilic domain (ΔA#A) . The constructs were expressed in COS cells and analyzed by gel electrophoresis and fluorography . The fraction of glycosylated products corresponding to the topology with a translocated COOH terminus was quantified . The constructs with the preprolactin signal yielded fewer COOH-terminally translocated proteins than those with the hemagglutinin signal: a maximum of 35% for P20A and none for P60A. Since the presequence of preprolactin is relatively long, signal cleavage could be observed for P20A. The unglycosylated, cleaved products displayed a slightly higher electrophoretic mobility than the glycosylated, uncleaved products after endo H digestion, consistent with their expected topologies . The constructs with the signal of vasopressin generated more glycosylated products than the other series: ∼90% for V20A and ∼50% with a spacer of 60 residues (V60A). However, with spacers of 80 or 100 residues, no glycosylated products were generated for any of the constructs. Based on these results, the strength of the different secretory signals, i.e., the ability to dominate the insertion process, can be ranked P > H > V. In the construct series A#A and ΔA#A, the first copy of the internal signal-anchor of the ASGP receptor H1 within the polypeptide appeared to dictate the topology, since glycosylated products were only generated with the shortest spacer length. In A20A and ΔA20A, the positive flanking charges at the NH 2 terminus of the second signal are also part of the COOH-terminal flanking region of the first, and are thus likely to weaken the first signal. Deletion of most of the 40-amino acid hydrophilic NH 2 terminus in ΔA20A increased the fraction of glycosylated products from ∼55 to ∼90%. Thus, the larger NH 2 terminus of A20A inhibits the topology with a translocated COOH terminus. Two mechanisms could explain the observed behavior of conflicting signals in the insertion process: competition between the signals for the recruitment of SRP in the cytosol , and/or competition for the preferred topology within the translocon . According to the first model, the kinetics of SRP binding to the first signal are slow enough that the second signal may emerge from the ribosome before SRP was recruited to the first. The two signals then compete directly for SRP binding. If the first signal binds SRP, it will initiate translocation of the spacer sequence. If the second signal does, it will induce translocation of the COOH terminus, whereas the first signal will remain in the cytosol or will subsequently insert into the membrane with an N exo /C cyt orientation. This model predicts, in agreement with our results, that the ability of the second signal to compete for SRP decreases with the length of the spacer, and depends on the relative affinities of the two signals for SRP. This model requires that at the time when the second signal emerges from the ribosome, there are still polypeptides that have not been targeted to the ER. To test this condition, we generated the constructs consisting of the signal of vasopressin or hemagglutinin, followed by 55, 75, 95, or 115 residues, including a diagnostic glycosylation site (V55, H55, etc.). Since at least 35 residues of a nascent polypeptide are hidden within the ribosome and inaccessible to SRP, translation will reach the stop codon and trigger disassembly of the ribosome when the first 20, 40, 60, or 80 residues following the signal sequence have emerged from the ribosome. This corresponds to a moment in the translation of the constructs V20A, V40A, V60A, and V80A (or of the corresponding constructs H#A) when the second hydrophobic sequence is just beginning to emerge from the ribosome . When the stop codon is reached, only polypeptides that have already been targeted to the ER membrane will produce glycosylated products; all others will be released into the cytosol and remain unglycosylated. In vitro translation in reticulocyte lysate with dog pancreas microsomes added either during or after translation confirmed that these constructs could not be posttranslationally translocated (data not shown). As shown in Fig. 6 B, even the shortest construct with a vasopressin signal, V55, was >90% glycosylated (lanes 1 and 2), indicating that the binding of SRP to the vasopressin signal and its targeting to the ER membrane was fast. Only few polypeptides of the constructs V#A thus had the opportunity to be targeted by the second signal. In contrast, the polypeptides with a hemagglutinin signal produced significant fractions of unglycosylated products: ∼40% of H55, ∼25% of H75, and still ∼10% of H95. Therefore, SRP recruitment is considerably slower than with a vasopressin signal. This suggests that the second signal in the H#A constructs might be able to compete for SRP binding. However, the numbers of untargeted polypeptides cannot account for the observed populations of H20A, H40A, and H60A with a translocated COOH terminus . Most importantly, a larger fraction of the V#A than of the H#A constructs inserted with a translocated COOH terminus, even though targeting by the vasopressin signal is faster. Therefore, the different topologies are more likely the result of competition between the signals within the translocon than of the kinetics of SRP binding and targeting by the first signal. According to the second model , the spacer sequence of all polypeptides is exposed to the ER lumen, at least transiently. To probe the lumenal exposure of the spacer, a diagnostic N -glycosylation site was introduced. In the construct V40(NIT)A, a potential glycosylation site NIT was generated by insertion of three additional codons at asparagine 13 of the spacer segment. As shown in Fig. 7 A, lanes 3 and 4, the site was efficiently modified, since all polypeptides were now glycosylated either once or twice, demonstrating that they had translocated either the spacer sequence (one glycosylation) or the COOH-terminal domain (two glycosylations). However, in comparison to V40A , the fraction of twice glycosylated proteins dropped significantly. In a protease protection assay, it was confirmed that the efficiency of glycosylation in the translocated COOH terminus was not reduced (data not shown). Therefore, the ratio between the two topologies was significantly altered by addition of the glycosylation site in favor of polypeptides with a translocated spacer sequence. The fraction of products with a translocated COOH terminus decreased from ∼75 to ∼30% . A similar effect was seen for the longer spacer of 60 residues, where insertion of the glycosylation site reduced this fraction from ∼50% in V60A to <20% in V60(NIT)A . This showed that topogenesis is affected by glycosylation, a lumenal modification, which clearly takes place after targeting. To exclude the possibility that the mutation exerted its effect by altering the conformational properties of the spacer sequence rather than by glycosylation itself, the sequence NIT was mutated to NIN, which is not modified by oligosaccharyl transferase : the effect on the topology was largely reverted. A different consensus sequence, NMT, which was also efficiently glycosylated, showed the same increase of polypeptides with translocated spacer sequences as NIT . The sequence NGS, in contrast, did not affect topology, but also produced hardly any polypeptides with a single glycan . This confirms that a consensus sequence N-X-S/T is not sufficient for the topogenic effect, but that efficient glycosylation is required. . The effect of glycosylation on topology was also reproduced with an entirely different spacer sequence of 45 amino acids corresponding to a segment of the exoplasmic portion of H1 with a natural glycosylation site NFT. Mutation to QFT caused the fraction of COOH-terminally translocated polypeptides to increase from ∼30% (V45[NFT]A) to ∼50% (V45[QFT]A) . As expected, the topogenic influence of glycosylation was also observed with hemagglutinin as the NH 2 -terminal signal sequence (H40[NIT]A vs. H40A) . Glycosylation affects topogenesis and thus takes place before the topology of the polypeptides is determined, most likely by trapping the spacer sequence on the lumenal side, preventing its return to the cytosolic side of the membrane . The insertion of multispanning membrane proteins is a complex process that is still poorly understood. To test how successive topogenic determinants in a polypeptide chain are decoded by the targeting and translocation machinery of the mammalian ER, we expressed a series of chimeric proteins containing two conflicting signal sequences in COS cells. Only when the two signals are sufficiently separated from each other can the results be explained by a simple linear insertion process, in which the most NH 2 -terminal signal sequence determines its own orientation as well as that of a subsequent transmembrane domain. The required spacer length depends on the characteristics (the strength) of the two signals. The weakest NH 2 -terminal signal tested, that of vasopressin, completely forced the internal signal-anchor of the ASGP receptor H1 into a stop-transfer orientation with a spacer of 80 residues or longer, whereas the H1 signal-anchor itself could do so with a spacer of only 40 amino acids. With shorter spacers, mixed topologies were observed, indicating that topogenic information in the second signal of the polypeptide codetermined the insertion process. Hence, insertion did not occur in a strictly linear manner from the NH 2 to the COOH terminus. The situation is thus comparable to that found in bacteria by Coleman et al. 1985 using proteins with two copies of the prolipoprotein signal separated by either 13 or 27 residues. The spacer was predominantly translocated with the longer spacer, and the COOH terminus with the shorter one. However, a point mutation that shifted the first signal from a cotranslational to a posttranslational mode of action caused COOH-terminal translocation also with the longer spacer, suggesting that targeting and insertion was now directed by the second signal. Similarly, if in our system the binding kinetics of SRP to the NH 2 -terminal signal are sufficiently slow, SRP binding and targeting by the second signal might explain our results. Experiments by Johnsson et al. provided evidence that in yeast many signal sequences are surprisingly slow in targeting to the ER membrane. The signal sequences tested were of different efficiencies, allowing for between ∼100 (invertase) and ∼300 residues (carboxypeptidase Y and Ste6) to be exposed to the cytosol for 50% of the polypeptides. However, most of the weak signals, such as that of caboxypeptidase Y, were later shown to function independently of SRP , and even invertase secretion was only slightly affected in an SRP-defective strain . The question of SRP-dependent targeting kinetics in mammalian cells, where only few proteins are known to be targeted independently of SRP , was still open. In this study, we analyzed the kinetics of SRP-dependent targeting in COS cells using the hemagglutinin and vasopressin signals followed by reporter polypeptides of increasing length. Targeting was clearly faster (relative to translation) than in all the examples tested in yeast. 60% of the chains with a hemagglutinin signal and >90% with a vasopressin signal had been targeted by the time 55 residues past the signal had been translated. The relative rate of targeting might reflect that pro-vasopressin–neurophysin II is a relatively short protein of 145 residues in comparison to hemagglutinin with 547 amino acids, and thus needs to be targeted more rapidly. From the sequence, it is not clear what makes the vasopressin signal more efficient for SRP recruitment. For our constructs with conflicting signal sequences, the targeting rates of the hemagglutinin and vasopressin signals do not provide an explanation for the observed topologies, since there is no correlation with COOH-terminal translocation. Although a fraction of some of the proteins, e.g., with a hemagglutinin signal and with the shortest spacer, may be targeted by the second signal, the topogenic influence of the second signal appears to be largely independent of how the protein was brought to the translocon. Glycosylation sites introduced into the spacer segment between the signal sequences provided a surprising insight into the dynamics of the orientation process in the translocon. N -glycosylation significantly affected the equilibrium of topologies in favor of that with a translocated spacer sequence and a cytosolic COOH terminus. This implies that with or without a glycosylation site, the spacer segment destined for cytosolic localization is transiently exposed to the ER lumen. With the glycosylation site, the spacer is modified by oligosaccharyl transferase when it appears in the lumen and is trapped there . The results indicate that the polypeptides dynamically reorient within the translocon, exploring the possible topologies favored by either of the two signals. Hydrophilic sequences of up to 60 residues between transmembrane domains move in and out of the ER before the topology is decided. A likely mechanism for this glycosylation effect is steric hindrance of the modified segment to return to the cytosolic side. It has been previously observed in vitro that a glycosylated segment of a model membrane protein was finally exposed to the cytosol . We did not recover any threefold glycosylated products indicative of a lumenal COOH terminus and a glycosylated spacer, suggesting either that in our system glycosylated sequences cannot flip back to the cytosolic side or, if they can, that they are rapidly deglycosylated in the cytosol . For the insertion process of natural multispanning membrane proteins, our results illustrate that successive membrane-spanning sequences insert in a coordinated manner, if sufficiently close within the polypeptide. What determines the topogenic strength of a signal is not obvious from the few examples analyzed. It does not simply correlate with the charge difference, since Δ(C–N) of the constructs with a spacer of 40 residues, for example, are −6.5 (P40A), −1.5 (H40A), and −5 (V40A), listed in the order of decreasing topogenic strength. Other criteria shown to influence the orientation of individual signals, such as hydrophobicity and the size of the NH 2 -terminal hydrophilic segment , are likely to contribute as well. In addition, longer and more hydrophobic signals, like that of H1 and, to a lesser extent, that of preprolactin, may exit the translocation pore more rapidly and might thereby limit the influence of the second signal. Spacer segments of 80 or more residues may be sterically unable to reorient themselves within the translocon. An additional mechanism that could limit an effect of the second topogenic sequence is the binding of lumenal chaperones, particularly BiP. The yeast homologue of BiP, Kar2p, has been shown to promote posttranslational translocation, but probably acts also in cotranslational transport . It is likely that spacer sequences that are long enough to engage with lumenal chaperones will be anchored by this interaction, thus determining the topology. In summary, topologenesis of membrane proteins appears to be a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences throughout the polypeptide is integrated. Enzymes associated with the translocation machinery and acting on the protein to be inserted, such as oligosaccharyl transferase and potentially signal peptidase and chaperones, can affect the process and thereby contribute to the efficient and uniform insertion of natural membrane proteins. | Study | biomedical | en | 0.999998 |
10525534 | tha4-ml was recovered in a screen of F2 families derived from a maize line with active Mutator ( Mu ) transposons. Numerous tha4-m1 /+ plants were propagated in parallel for several generations by crossing to inbred lines. Heterozygous plants were self-pollinated to recover homozygous mutant seedlings. tha4 mutant seedlings used for DNA and RNA extraction were identified initially by their subtle chlorophyll deficiency, and then confirmed by immunoblot analysis of leaf proteins. Plants used in these experiments were grown for 10–12 d at 26°C in 16 h of light (400 μE/m 2 /s) and 8 h of dark. Basal leaf tissue used for RNA extraction was obtained from the basal 0.5 cm of the second leaf of 10-d-old seedlings (inbred line B73) grown according to this regime. Etiolated leaf tissue used for RNA extraction was obtained from B73 seedlings that were grown in the complete absence of light for 9 d. tha4 was mapped to chromosome 1L by crossing tha4-m1 /+ plants with stocks harboring various B-A translocations . Total DNA was extracted from maize seedlings and analyzed by Southern hybridization with digoxigenin-labeled Mu probes, as described previously . Total leaf RNA was extracted with TRIzol Reagent (GIBCO BRL) according to the manufacturer's instructions. RNA gel blot hybridizations were performed as described previously . Because the amount of tha9 -specific cDNA sequence was insufficient to generate a sensitive gene-specific probe for RNA gel blots (∼40 nucleotides), RNase protection assays were used to distinguish the tha4 and tha9 mRNAs in different maize tissues. RNase protection assays were performed as described in Voelker et al. 1997 . The radioactive antisense probes were generated from near full-length tha4 and tha9 cDNA sequences. The tha4 probe was derived by in vitro transcription of the tha4 cDNA clone encoding the complete tha4 open reading frame in pGEM-3Z (see Isolation and Analysis of cDNA). The tha9 probe was generated by in vitro transcription of a derivative of the tha9 cDNA clone described below, which had been truncated to delete sequences corresponding to the polyA tail. To clone the 1.9-kb XhoI fragment containing the Mu1 insertion linked to tha4-m1 , DNA from a homozygous tha4-ml mutant was first digested with XhoI and fractionated in an agarose gel. DNA was extracted from a gel slice containing DNA fragments of 1.8–2 kb, by using QIAEX beads according to the manufacturer's instructions (Qiagen). The DNA was ligated into pBluescript SK+ (Stratagene) that had been treated with XhoI and calf intestine phosphatase and electroporated into XL1-Blue MRF′ cells (Stratagene). Colony lifts were probed with a radiolabeled Mu1 probe, leading to the identification of clone A . A genomic library derived from the maize inbred line B73 (a gift of Doug Rice, Pioneer Hi-Bred, Johnston, Iowa) was screened to obtain sequence information both upstream and downstream of clone A. A radiolabeled 190-bp fragment derived from gene-specific sequences in the 3′ untranslated region (UTR) of the tha4 cDNA (see next section for cDNA isolation) identified two overlapping genomic clones, which contained clone A sequences within 2.5- and 11-kb XbaI fragments, respectively. The 11-kb XbaI fragment was digested with SacI to yield a 2-kb fragment containing clone A sequences. The 2.5-kb XbaI and 2-kb SacI fragments were subcloned into a modified pBluescript SK+ vector and used as templates for sequencing. DNA sequences were analyzed by Yanling Wang in the Institute of Molecular Biology DNA Sequencing Facility (University of Oregon, Eugene, OR). Primers used for PCR analysis of the tha4 locus in the revertant sector were as follows: primer M, 5′ CGAAATGGCACCGTGTTACAC 3′; primer N, 5′ GGGAACCACCACGGGTATC 3′; and Mu primer, 5′ AGAGAAGCCAACGCCAWCGCCTCYATTT 3′. To isolate a tha4 cDNA, a maize leaf cDNA library was screened by PCR using primers designed to amplify the 3′ ends of tha4 cDNAs. For this purpose, tha4 gene primers were chosen that mapped to sequences in clone A encoding amino acids just downstream of the predicted transmembrane domain. The initial PCR used a tha4 gene primer (5′ CCAAGCAGCTCCCCGAGATC 3′) and a vector primer (5′ AGGGTTTTCCCAGTCACGAC 3′) according to the following profile: 94°C/4 min, followed by 30 cycles of 94°C/1 min, 60°C/45 s, 72°C/2 min, and a final extension at 72°C/5 min. A second round of PCR was performed with a nested tha4 gene primer (5′ ATCGGCAAGACCGTCAAGAGC 3′) and an EcoRI-oligo dT primer (5′CGGAATTC(T) 17 ) according to the profile: 94°C/4 min, followed by an initial two cycles of 94°C/1 min, 37°C/45 s, 72°C/2 min, followed by 30 cycles of 94°C/1 min, 60°C/45 s, 72°C/2 min, and a final extension at 72°C/5 min. Amplifications were performed in 50-μl reactions containing 50 mM KCl, 10 mM Tris-HCl, pH 9, 1% Triton X-100, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 5 μl cDNA library stock or 1 μl of initial PCR, and 250 ng of each primer plus Taq DNA polymerase. The PCR product was cloned into pBluescript SK+ and its DNA sequence was determined. The cDNA library was screened using this PCR clone as a probe, yielding two types of cDNA encoding highly similar proteins but with distinct 3′ UTR sequences. This revealed the presence of a closely related gene in the maize genome, which we named tha9 . Authentic tha4 cDNAs were distinguished from tha9 cDNAs by four criteria: (1) their 3′ UTR detected an mRNA that accumulated to reduced levels in tha4 mutant seedlings ; (2) their 3′ UTR detected only authentic tha4 genomic clones (i.e., that matched clone A) when used to screen a genomic library of B73 DNA; (3) a nearly full-length, spliced tha4 cDNA obtained by reverse transcriptase-PCR (see below) was identical throughout a 382 bp region of overlap to genomic clone A; and (4) partial unspliced tha4 cDNAs recovered from the cDNA library contained intron sequence that matched intron sequence in the tha4 clone obtained from the B73 genomic library. Because library screens failed to yield full-length tha4 cDNAs, a cDNA containing the entire coding region of the tha4 mRNA was obtained by reverse transcription–PCR amplification of poly (A) + seedling leaf RNA from the inbred maize line B73 (Pioneer Hi-Bred). cDNA synthesis was catalyzed by M-MuLV reverse transcriptase (Promega) and primed with a tha4 3′ UTR gene-specific primer (5′ CTTCAATACGTAGAAGCTC 3′). The cDNA was amplified in a PCR reaction containing a gene primer spanning the start codon (5′ AGCAGGCATGGGGATAC 3′) and a nested 3′UTR primer (5′ GGATATGAACTGCTAACTCG3′), by using the profile: 94°C/4 min, followed by 30 cycles of 94°C/1 min, 60°C/45 s, 72°C/1 min, and a final extension at 72°C/5 min. Amplification buffer included 10% glycerol. The PCR product was cloned into pGEM-3Z (Promega). The tha4 cDNA sequence has been entered in GenBank/EMBL/DDBJ under accession number AF145755. The tha9 cDNA sequence has been entered in GenBank/EMBL/DDBJ under accession number AF145756. Sequences of THA4 , THA9 , HCF106 , TatA , and TatB were aligned using ClustalW 1.7 and BoxShade (Bioinformatics Group, ISREC). Methods for the extraction of leaf protein, SDS-PAGE, and immunoblot analysis are described in Barkan 1998 . Chloroplasts were isolated as described in Voelker and Barkan 1995 and fractionated as follows. Unless otherwise noted, all buffers included protease inhibitors at the following concentrations: 2 μg/ml aprotinin, 5 mM EDTA, 2 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM PMSF. Chloroplasts were lysed by resuspension in ice-cold 10 mM Hepes-KOH, pH 8, at a chlorophyll concentration of 0.3 mg/ml for 10 min. Thylakoid membranes were separated from the stroma by centrifugation for 5 min at 12,000 g , and resuspended to a chlorophyll concentration of 0.3 mg/ml in thylakoid resuspension buffer (10 mM Hepes-KOH, pH 8, 100 mM sucrose). For the salt-washed membranes, the membrane fraction was resuspended to a chlorophyll concentration of 0.2 mg/ml chlorophyll in either 150 mM sodium carbonate or 2 M sodium bromide and incubated on ice for 30 min, followed by centrifugation at 12,000 g for 5 min. The pellets were rinsed in HS (50 mM Hepes-KOH, pH 8, 330 mM sorbitol), centrifuged at 12,000 g for 5 min, and resuspended in HS. Proteins from the salt-washed supernatants were precipitated by the addition of BSA to 0.5 mg/ml and TCA to 10% (wt/vol), incubation on ice for 30 min, and centrifugation at 12,000 g for 20 min. The pellet was resuspended in HS. For protease treatments, aliquots of unwashed thylakoid membranes at 0.2 mg/ml chlorophyll in thylakoid resuspension buffer (without protease inhibitors) were treated with either 0.2 mg/ml thermolysin or 0.03 mg/ml proteinase K on ice for 30 min. Thermolysin was inactivated by the addition of 1/20 vol of 0.5 M EDTA and 3 vol of thermolysin stop buffer (HS plus 20 mM EDTA). Proteinase K was inactivated by the addition of 1/20 vol of 40 mM PMSF, 1/10 vol of 0.1 M EGTA, and 3 vol of proteinase K stop buffer (HS plus 10 mM EGTA). Samples were centrifuged at 12,000 g for 5 min, and the membrane pellets were resuspended in thermolysin stop buffer or proteinase K stop buffer, as appropriate. In control experiments, to test the protease accessibility of lumenal proteins in the absence of intact thylakoid membranes, Triton X-100 (0.1%) was added before protease addition. The near identity of THA4 and THA9 prevented the development of a THA4-specific antiserum . An antigen was generated by ligating a fragment of the tha9 cDNA encoding the COOH-terminal 63 amino acids (amino acids 106–169) into the pET28-C(+) vector (Novagen), to yield an in-frame fusion with sequences encoding an NH 2 -terminal 6× histidine tag. The fusion protein was purified on a nickel column and injected into rabbits for the production of polyclonal antiserum. The antiserum obtained does not recognize HCF106 (data not shown). A recombinant HCF106 fragment was generated by subcloning a 0.8-kb SacI-HindIII fragment from an hcf106 cDNA clone into the pET28-C(+) vector (Novagen), to yield an in-frame fusion with sequences encoding an NH 2 -terminal 6× histidine tag. This fusion protein included amino acids 75–243 of the predicted HCF106 gene product. The fusion protein was purified on a nickel column and injected into rabbits for the production of polyclonal antiserum. The anti-HCF106 antiserum does not detect THA4/THA9 on immunoblots (data not shown). Antisera to the 33-, 23-, and 16-kD subunits of the oxygen-evolving complex (OE33, OE23, and OE16, respectively) and to plastocyanin (PC) were described previously . The reference allele of tha4, tha4-m1 :: Mu1 (hereafter referred to as tha4-m1 ) was detected in a seedling screen of the F2 progeny of maize plants with active Mu transposons. Homozygous mutant seedlings were subtly chlorophyll-deficient and died after the development of three to four leaves. In these ways, tha4 mutants resembled many previously described maize mutants that lack subsets of thylakoid membrane proteins . Immunoblot data presented previously showed that tha4-m1 mutants accumulate only 20% of the normal levels of the core subunits of photosystems I and II and the cytochrome b 6 f complex. However, the thylakoid ATP synthase and the major light harvesting chlorophyll a/b binding protein accumulate normally. The pigmentation and thylakoid protein deficiencies of tha4-m1 mutants were similar to those of hcf106 and tha1 mutants, which have defects in the translocation of proteins across the thylakoid membrane . To explore the possibility that tha4-m1 likewise disrupts thylakoid protein targeting, the abundance and processing of proteins found in the thylakoid lumen were assessed with immunoblots. Two cpSecA substrates, PC and OE33, accumulated to normal levels in tha4-m1 mutants . In contrast, OE23 and OE16, which are translocated via the Δ pH pathway, accumulated to only 10–20% of wild-type levels. Furthermore, antibodies to OE23 and OE16 detected higher molecular weight proteins in tha4-m1 mutants with the sizes predicted for precursors that have retained their lumenal targeting sequences. Thus, the tha4-m1 phenotype strongly resembles that of hcf106 mutants, in which the Δ pH pathway is disrupted . Increased accumulation of incompletely processed OE23 and OE16 could result from either a defect in the lumenal processing protease or in the movement of the proteins across the thylakoid membrane. To distinguish between these possibilities, the intrachloroplast location of the precursors was determined by fractionating tha4-m1 mutant chloroplasts to separate the stroma from the thylakoid membrane vesicles. Precursors of both OE23 and OE16 were enriched in the stromal fraction, whereas the mature forms were found predominantly in the thylakoid membrane fraction . A small proportion of each precursor remained bound to the thylakoid membrane. To determine whether these bound precursors had been translocated across the membrane, the stromal face of the thylakoid vesicles was treated with carbonate, NaBr, or proteases. The carbonate and NaBr treatments caused some disruption of the vesicles, as revealed by the recovery of a small fraction of mature OE23 and OE16 in the supernatant; wild-type thylakoid membranes treated in the same fashion behaved similarly . Nonetheless, it is clear that the membrane-bound precursors were extrinsic proteins on the stromal face of the membrane: they were removed by treatment with carbonate or NaBr, and were selectively degraded by the proteases thermolysin or proteinase K . These results provide strong evidence that the accumulation of incompletely processed OE23 and OE16 in tha4-m1 mutants results from a defect in their translocation to the thylakoid lumen. Taken together, these results strongly suggest that tha4 , like hcf106 , functions in the Δ pH–dependent system for translocating proteins across the thylakoid membrane. The tha4 gene was mapped to chromosome 1L by crossing with a series of B-A translocation stocks . In contrast, hcf106 maps to chromosome 2, indicating that tha4-m1 defines a new gene. To address the possibility that tha4-m1 disrupts the Δ pH pathway by interfering with the accumulation of membrane bound HCF106, the abundance and location of HCF106 were monitored in tha4-m1 mutants. HCF106 accumulates to normal levels in tha4-m1 mutants . The same tha4-m1 chloroplast fractions used in Fig. 2 were probed with anti–HCF106 antibody , revealing that HCF106 is tightly associated with the thylakoid membrane in tha4-m1 mutants. Furthermore, HCF106 is highly susceptible to proteases in tha4-m1 thylakoid membrane vesicles . This fractionation behavior is the same as that described for HCF106 in wild-type chloroplasts . Therefore, tha4-m1 does not alter the accumulation of HCF106 or its insertion into the thylakoid membrane. tha4-m1 arose in a maize line with active Mu transposons and was somatically unstable, suggesting that it was caused by the insertion of a Mu transposon. To identify Mu insertions that were genetically linked to tha4 , DNA from mutants derived from diverse branches of the tha4-m1 pedigree were analyzed by Southern hybridization, using probes corresponding to each member of the Mu family . A Mu1 probe detected a 1.9-kb XhoI fragment in all mutants that was absent in closely related homozygous wild-type plants (data not shown). Probes corresponding to the other members of the Mu family failed to detect a genetically linked insertion. The 1.9-kb XhoI fragment was cloned from a size-enriched genomic library of tha4-m1 mutant DNA . Southern blots of wild-type and tha4-m1 mutant DNAs were probed with the genomic sequence flanking the cloned Mu1 insertion, revealing that all mutant and no wild-type plants were homozygous for the cloned insertion (data not shown). Longer clones corresponding to this region were isolated from a genomic library of wild-type DNA. To test whether clone A contained the insertion that is the cause of the tha4 - m1 mutant phenotype, the structure of the corresponding genomic region was monitored in a revertant sector that appeared on a tha4-m1 mutant leaf. DNA extracted from a dark green revertant sector and from the flanking, slightly paler mutant tissue was analyzed with PCR using primer pairs designed to selectively amplify either the mutant or wild-type allele . Control reactions first established that the predicted amplification products were obtained with homozygous mutant and wild-type DNA samples. As expected, DNA from homozygous wild-type tissue (WT) gave no amplification products when the Mu primer was used in conjunction with either of the gene-specific primers, M or N. Primers M and N together, however, gave rise to an amplification product of 210 bp with a WT template DNA, the size predicted for the wild-type allele. Amplification of DNA from the mutant tissue on the sectored leaf with the Mu primer in conjunction with primers M or N resulted in the predicted DNA fragments of 223 and 136 bp, respectively. As expected, the tha4 mutant DNA did not yield an abundant product when the gene-specific primers M and N were paired because PCR fails to amplify across intact Mu elements. Revertant sectors are expected to be heterozygous and should, therefore, give rise to the products representing both alleles. With revertant DNA as a template, primer M or N paired with the Mu primer gave rise to the 223- or 136-bp fragments expected for the mutant allele . The key finding was that the revertant DNA also contained an allele lacking the cloned Mu insertion: primer M paired with primer N yielded a robust amplification product. This product was slightly smaller than that resulting from a wild-type DNA template, indicating that it did not result from contamination with wild-type DNA. These results suggest that imprecise excision of Mu1 caused a small deletion of flanking genomic sequences, and that this excision, nonetheless, restored tha4 gene function. As described below, the Mu insertion in tha4-m1 disrupts the untranslated sequence in the 5′ portion of the tha4 gene, such that excision accompanied by a small deletion could well restore gene function. These results indicate that excision of the Mu1 insertion represented by clone A correlates with reversion to a wild-type phenotype, providing strong evidence that the clone contains a portion of the tha4 gene. The genomic sequence flanking the cloned Mu1 insertion was used to obtain tha4 cDNAs. The tha4 cDNA encoded a continuous open reading frame of 170 amino acids. The Mu1 insertion disrupted sequences mapping 35 bp upstream of those encoding the predicted start codon . A probe prepared from the unique 3′ UTR sequences of the tha4 cDNA detected a leaf mRNA of ∼900 nucleotides that accumulated normally in hcf106 mutants, but was barely detectable in tha4 - m1 mutants . The longest tha4 cDNA obtained began at the predicted start codon and included 740 nucleotides between the start codon and the beginning of the poly(A) tail. Given that the RNA gel blot analysis indicated a length of ∼900 nucleotides for the polyadenylated mRNA and that poly(A) tails commonly contain >100 residues in plants, we predict that the 5′ UTR of the tha4 mRNA contains 50–100 nucleotides. No in-frame ATGs are found in 300 bp of genomic sequence upstream of those encoding the predicted start codon. The Mu1 insertion, mapping 35 bp upstream of the predicted start codon, therefore, likely disrupts the 5′ UTR of the tha4 gene. The deduced tha4 gene product (THA4) is 170 amino acids in length and has a single predicted membrane spanning domain. The ChloroP algorithm predicts that THA4 is a chloroplast-localized protein. THA4 is related to maize HCF106 and to the products of the bacterial tatA and tatB genes . TatA and TatB are bacterial proteins implicated in Sec-independent protein export and hcf106 functions in the thylakoid Δ pH mechanism . The four proteins are closely related in their membrane-spanning domains and in the adjacent amphipathic helical domain . The predicted mature form of THA4 is similar in size to TatA, whereas the predicted mature form of HCF106 is similar in size to TatB. THA4 and TatA lack the extended COOH-terminal acidic region found in both HCF106 and TatB. Previously, it was proposed that hcf106 is more closely related to tatA than to tatB . However, these results suggest that hcf106 is more closely related to tatB and that tha4 is more closely related to tatA . In addition, an Arabidopsis cDNA sequence proposed previously to represent an hcf106 homologue is, in fact, much more similar to tha4 (data not shown) and likely represents a tha4 ortholog. cDNA library screens yielded two classes of cDNA, representing tha4 and a closely related gene, which we named tha9 (see Materials and Methods). Both genes were represented as cDNAs in a seedling leaf cDNA library, indicating that both are transcribed in seedling leaf tissue. The tha9 cDNA encodes a protein that is very closely related to THA4 , and that is predicted by the ChloroP algorithm to be chloroplast-localized. The predicted mature form of THA9 is nearly identical to that of THA4 ; even the predicted transit peptides diverge to only a small degree. This degree of identity strongly suggests that these two proteins are localized similarly in the cell and that they have similar or identical functions. The near identity of the mature regions of THA4 and THA9 precluded the generation of a THA4-specific antiserum. A polyclonal antiserum that would detect both THA4 and THA9 was generated to the COOH-terminal region of THA9 . The antiserum detected a protein in wild-type leaf tissue that migrated during SDS-PAGE with an apparent mass of 16 kD and accumulated to much reduced levels in tha4-m1 mutant leaf tissue . The residual protein in the mutant sample could result from incomplete disruption of tha4 gene expression by the Mu1 insertion and/or could be the product of the tha9 gene. In either case, these findings strongly suggest that the tha9 gene contributes no more than a small percentage of the immunoreactive protein in wild-type seedling leaves. However, it is also possible that the tha9 and tha4 gene products interact in such a way that THA9 is destabilized in the absence of THA4. These antibodies can be used to localize THA4 in the cell because the bulk of the immunoreactive signal in wild-type leaves most likely represents THA4 . The near identity of THA9 and THA4 suggests that the two proteins are, in any case, localized similarly. For simplicity, the immunoreactive protein in leaf tissue will be referred to as THA4. A substantial proportion of THA4 copurified with intact chloroplasts during sedimentation through Percoll gradients . THA4 was largely recovered in the thylakoid membrane fraction of chloroplasts . THA4 is tightly associated with the membrane, as it was not removed by carbonate or NaBr washes. Treatment of thylakoid membrane vesicles with the proteases thermolysin and proteinase K eliminated immunologically reactive material. Because the antiserum was raised to the COOH terminus of the protein and because the THA4 amino acid sequence predicts a short hydrophilic NH 2 -terminal domain but a long hydrophilic COOH-terminal domain, these results suggest that the COOH terminus of THA4 is exposed to the stroma. The fractionation behavior of HCF106 was similar to that of THA4 , consistent with the previous report that HCF106 is tightly associated with the membrane and is susceptible to proteases applied to the stromal face . Thus, THA4 and HCF106 are likely to be oriented similarly in the membrane, with their COOH-terminal acidic tails in the stroma. Duplicate gene pairs analogous to tha4/tha9 are common in maize as a consequence of the tetraploidy of the ancestral maize genome . The near identity of the THA4 and THA9 amino acid sequences suggests that the two proteins have retained similar or identical biochemical functions. Nonetheless, tha9 function cannot fully compensate for the absence of tha4 since mutations in tha4 disrupt the Δ pH–dependent translocation mechanism in seedling leaves. To address the possibility that the two genes have acquired different patterns of regulation, their mRNAs were quantified in green seedling leaf tissue, etiolated seedling leaf tissue, basal leaf tissue, roots, endosperm, and immature ears . These tissues differ with regard to their plastid populations: green seedling leaf contains mature chloroplasts; basal leaf is enriched in proplastids, the chloroplast progenitors found in undifferentiated cells; etiolated leaf contains etioplasts, which differentiate into chloroplasts upon exposure to light; and root, endosperm, and immature ear contain a variety of nonphotosynthetic plastid forms. RNase protection assays were used to distinguish the tha4 and tha9 mRNAs. The probe generated from the tha4 cDNA was nearly completely protected by tha4 mRNA, as expected . The tha9 mRNA protected smaller but still substantial fragments of the tha4 probe, because of their high degree of sequence complementarity ; this permitted the simultaneous detection of both mRNAs in each lane. In addition, the tha9 mRNA was assayed with a probe generated from the tha9 cDNA . The results of these two tha9 mRNA assays were consistent and served to reinforce one another. The level of the tha4 mRNA was substantially reduced in tha4-m1 mutants , whereas the accumulation of tha9 mRNA was unaltered in tha4 mutants . The tha4 mRNA accumulated to similar levels in green seedling leaves, etiolated seedling leaves, basal leaf, and immature ear . Its level was somewhat lower in endosperm and it was barely detectable in the root . The profile of tha9 mRNA accumulation differed significantly from that of tha4 . The tha9 mRNA accumulated to the highest level in green leaf tissue, to slightly lower levels in etiolated leaf tissue, and to much lower levels in leaf base, endosperm, immature ear, and root. Most notably, the ratio of tha4 to tha9 mRNA was much higher in leaf base and in immature ear than in green leaf . The fact that tha4 mRNA is predominant in basal leaf tissue suggests that tha4 function is required early in the proplastid to chloroplast transition, when the elaboration of thylakoid membranes is initiated. The tha9 gene may then provide supplemental protein in young and mature chloroplasts to maintain optimal function of the Δ pH–dependent translocation machinery. We have presented evidence that the maize tha4 gene functions in the Δ pH–dependent mechanism for the translocation of proteins across the chloroplast thylakoid membrane. THA4 is the second plant protein to be identified that participates in this mechanism, the first being the related protein HCF106 . Both THA4 and HCF106 are tightly associated with the thylakoid membrane; sequence analysis and protease sensitivity studies suggest they both have a single transmembrane domain and an acidic COOH-terminal tail on the stromal side of the membrane. However, the stromal domain of HCF106 is considerably longer and more acidic than that of THA4. THA4 and HCF106 are related to the tatA and tatB genes found in all sequenced eubacterial genomes. tatA and tatB were recently implicated in a novel sec-independent mechanism for the export of periplasmic proteins that bind redox cofactors . Alignment of the most conserved regions of these proteins (the transmembrane and amphipathic helical domains) revealed a comparable degree of similarity between all protein pairs. However, these proteins fall into two classes based upon the length of their COOH-terminal hydrophilic tails: HCF106 and TatB have a long tail of nearly identical length, and THA4 and TatA have a much shorter tail, also of nearly identical length . On this basis, we propose that tha4 is more closely related to tatA and hcf106 is more closely related to tatB . The similarity between the THA4/TatA and HCF106/TatB sequences raises questions about the functional relationship between these genes. As is true of tha4 and hcf106 , mutations in either tatA or tatB disrupt protein export , indicating that these genes have at least some distinct functions. The situation is still more complex because many bacterial genomes include a second tatA -like gene, termed tatE in E . coli , and mutations in tatE also disrupt export to some extent . In maize, both tha4 and hcf106 are themselves members of duplicate gene pairs: from a maize leaf cDNA library, we recovered cDNAs encoding a protein with >90% identity to HCF106 (Barkan, A., unpublished results) as well as the tha9 cDNA described here, which is very closely related to tha4 . That mutant phenotypes result from disruption of either hcf106 or tha4 is consistent with the notion that the members of each gene pair may not be completely redundant in their biochemical function. However, the near identity of the proteins in each pair leads us to favor the notion that the members of each pair play identical biochemical roles, but are subject to different patterns of regulation. This is supported by our finding that the tha4 and tha9 mRNAs accumulate differentially in different plant tissues. In any case, a picture is emerging of a group of related proteins involved in this novel protein translocation mechanism. The biochemical role of these proteins is not known, although it has been proposed based upon their orientation in the membrane that they function as receptors . It will be fascinating to learn how these proteins are organized in the membrane and how they relate in a functional sense to one another. Mutant phenotypes originally implicated THA4 and HCF106 in the Δ pH–dependent translocation mechanism. Recently, conclusive evidence that these proteins function directly in translocation was obtained from in vitro import assays performed in the presence of anti–THA4 or anti–HCF106 antibodies . Thus far, THA4 and HCF106 are the only proteins known to participate in the Δ pH–dependent mechanism in chloroplasts. However, by comparison to the bacterial system, it seems likely that a plant homologue of the bacterial TatC protein is also involved. TatC is predicted to be a polytopic membrane protein and is encoded in the same operon as TatA and TatB in E . coli . A tatC deletion mutant has a severe defect in the export of tat substrates . Genes with similarity to tatC are found in the chloroplast genomes of certain algae and plant cDNAs encoding tatC homologues have recently appeared in the databases. However, direct evidence for the role of these tatC homologues has not been reported. We recently recovered a maize mutant with a more severe defect in the Δ pH–dependent pathway than either hcf106 or tha4 mutants, and we have established that the mutation defines a third gene involved in this process (Pedersen, R., M. Walker, and A. Barkan, unpublished results). Whether this new mutation disrupts a tatC homologue or defines a novel component of this interesting protein translocation mechanism remains to be determined. | Study | biomedical | en | 0.999997 |
10525535 | The cDNAs coding for human APP695, the London mutant, and human APP695ΔCT, lacking the region encoding the carboxy-terminal cytoplasmic tail of APP, were cloned blunt end in the SmaI site of pSFV-1 as described previously . APP695/KK was generated by exchanging the two amino acids at position 3 and 4 of APP695 to two lysines by site-directed mutagenesis . The chimera APP695/LDLR was constructed by exchanging the carboxy-terminal cytoplasmic domain of APP with that of the LDL receptor (cDNA provided by Walter Hunziker). In all sorting mutants, an amino-terminal c-myc epitope (EQKLISEEDL) was inserted into the KpnI site of the 5′-terminus of APP695 . All cDNAs were verified by sequencing. pSFV1/APP constructs were linearized with SpeI, and run-off transcription using SP6 polymerase was performed to produce mRNA. The transcribed mix of APP and pSFV-helper were cotransfected into BHK cells by electroporation to yield recombinant SFV . BHK cells were grown in DME/F-12 supplemented with 5% FCS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. 24 h after transfection, the culture supernatant containing infective recombinant SFV was collected. Aliquots were snap-frozen in liquid nitrogen and stored at –70°C until use. Transgenic FVB×C57 black mice expressing wild-type human PS1 or the clinical mutants Met146Leu (PS1 M146L ) or Leu286Val (PS1 L286V ) under the control of the prion promoter were used . The transgene proteins were efficiently cleaved and their subcellular localization in hippocampal neurons at different developmental stages was virtually indistinguishable from PS1 wild-type . Heterozygous transgenic mice were crossed with wild-type FVB mice, and pregnant females were killed 17–18 d postcoitum. Hippocampal neuron cultures were prepared from E17–E18-d-old fetal mice according to Goslin and Banker 1991 . Single cell suspensions obtained from the hippocampi of individual embryos were plated on poly- l -lysine–coated plastic dishes (Nunc) or on poly- l -lysine–coated coverslips in minimal essential medium (MEM) supplemented with 10% horse serum. After 3–4 h, culture medium was replaced by serum-free neurobasal medium with B27 supplement (GIBCO BRL). Cytosine arabinoside (5 μM) was added 24 h after plating to prevent nonneuronal (glial) cell proliferation. Hippocampal neurons on plastic dishes were used at day 3 after plating. Hippocampal neurons on coverslips were maintained in culture for 14–16 d in the presence of a glial feeder layer to allow for full differentiation and polarization. Brain cortices of 1-wk-old control and transgenic PS1 wild-type mice were used as starting material. Since the transgene is under the control of the prion promoter, the human PS1 is mainly expressed in neurons. Analysis of the subcellular fractions obtained with this brain material using a human-specific antibody, therefore, reflects the distribution of PS1 in neurons in vivo. This allowed us to directly compare the fractionation data with the findings obtained in the cultured hippocampal neurons. The preparation of nuclei and nuclear envelopes was based on the method described by Otto et al. 1992 except that RNase 1 and PMSF were added before centrifugation of the nuclear envelopes. For the isolation of the intermediate compartment (IC) fractions, the protocol of Schweizer et al. 1991 was used with modifications . Gradient fractions were collected using a Büchler Auto-Densi Flow apparatus. Sucrose concentrations were calculated from the refractive index (Refractometer RF 490; Euromex) by linear regression. Highly purified intact lysosomes were prepared according to Maguire and Luzio 1985 . The protein content of each fraction was measured spectrophotometrically using a protein assay (Bio-Rad Laboratories) according to the manufacturer's instructions. Unless stated otherwise, equal amounts of protein (30 mg) were dissolved in Laemmli sample buffer, incubated for 10 min at room temperature, and resolved on 13% polyacrylamide gels (Mini-Protean II; Bio-Rad Laboratories). After SDS-PAGE and transfer of the proteins to nitrocellulose , blots were blocked in TBS containing 5% lowfat milk and 0.1% Tween 20 and incubated overnight (4°C) with the appropriate primary antibodies. Detection was done with HRP-conjugated secondary antibodies (Bio-Rad Laboratories) followed by enhanced chemiluminescence (ECL; Amersham). 12-d-old hippocampal neurons were fixed in 4% paraformaldehyde and 4% sucrose in 120 mM sodium phosphate buffer, pH 7.3, for 30 min at room temperature. Fixed neurons were rinsed twice in Dulbecco's PBS and transferred to ice-cold methanol (−20°C, for 4 min) followed by ice-cold acetone (−20°C, for 2 min). Coverslips were transferred to a humid chamber, washed three times with PBS, and blocked for 1 h at room temperature or overnight at 4°C (blocking solution: PBS containing 2% BSA, 2% FCS, 0.2% fish gelatin, and 4% donkey serum). Neurons were subsequently incubated with primary antibodies in blocking solution (for 2 h at room temperature). After four washes with PBS, secondary antibodies were applied (for 2 h at room temperature). CY2- and CY3- or Lissamine rhodamine-conjugated donkey anti–mouse and donkey anti–rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) were used for detection. Coverslips were washed three times with PBS, once in distilled water, and finally mounted in Mowiol (Calbiochem-Novabiochem). Specimens were viewed through a Nikon Diaphot 300 (PlanApo 60/1.40 oil) connected to the MRC1024 confocal microscope (Bio-Rad Laboratories). Images were captured by LaserSharp (version 2.0) on a Compaq Prosignia 300 workstation and finally processed using Adobe Photoshop 5.0. The following antibodies were used to detect PS1 : polyclonal antibody B17.2 recognizes both human and mouse PS1-CTF (peptide–antigen residues 300–315, EGDPEAQRRVSKNSKY of the human PS1 sequence); and mAb 5.2 is specific for the human CTF-PS1 (peptide–antigen residues 307–331, RRVSKNSKYNAESTERESQDTVAEN). Polyclonal antibody B14.5 detects the human PS1-NTF (peptide–antigen residues 30–44, NDNRERQEHNDRRSL); B19.2 and B32.1 are directed against the murine PS1-NTF (peptide–antigen residues 32–46, SQERQQQHGRQRLDN) and PS1-CTF (peptide–antigen residues 310–330, PKNPKYNTQRAERETQDSGSG), respectively. All polyclonal antibodies were affinity-purified against the peptide immobilized on NHS-activated Sepharose 4b (Pharmacia) according to the manufacturer's instructions. PS1 knockout brain tissue was used to confirm the specificity of the PS1 affinity-purified polyclonal antibodies. HoloAPP, α- and/or β-cleaved carboxy-terminal fragments of APP and amyloid peptides were immunoprecipitated from cell extracts and media using polyclonal antibodies raised against the 20 carboxy-terminal amino acids of APP (B10/4 and B11/8) or against the synthetic human 1-40 βA4 peptide (B7/7) . mAbs against lamin B1, a marker for nuclear envelopes, or against p58/Golgi, a marker for Golgi membranes, were obtained from Zymed or Sigma Chemical Co., respectively. mAbs to the transferrin receptor or to synaptobrevin II (clone 69.1) were provided by Ian Trowbridge (Salk Institute, San Diego, CA), and Reinhard Jahn (MPI-Biophysical Chemistry, Göttingen, Germany), respectively. Monoclonal anti–c-myc antibody was used for the detection of the carboxy-terminal–inserted c-myc epitope. Polyclonal antibodies to rSEC61αp, calnexin, BAP31, p58/ERGIC-53, GM130, SEC23p, and TGN38 were provided by Enno Hartmann (Georg-August Universität, Göttingen), Ari Helenius (Yale University, New Haven, CT), Michael Reth (MPI-Immunologie, Freibourg, Germany), Jaakko Saraste (University of Bergen, Norway), Graham Warren (Yale University, New Haven, CT), Jean-Pierre Paccaud, and George Banting (Bristol, UK), respectively. Recombinant SFV was diluted 10-fold in conditioned culture medium and added to the cells (1 ml/dish). Cultures were incubated for 1 h at 37°C to allow entry of the virus, followed by incubation in conditioned medium in the absence of virus (for 2 h). Metabolic labeling was performed using methionine-free N2 medium containing 100 μCi [ 35 S]methionine/ml (NEN). After 4 h, the conditioned medium was collected and the neuronal cell layer was washed once in PBS, and then scraped in extraction buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). Both the media and cell extracts were centrifuged (14,000 rpm for 10 min) to remove detached cells and aggregates. Polyclonal antibody B11/8 (1/500) was added to cell extracts and B7/7 (1/100) to the media, together with protein G–Sepharose (Pharmacia) and incubated overnight (at 4°C). The immunoprecipitates were washed five times in extraction buffer and once in TBS. To check the glycosylation status of APPwt and the APP/KK mutant (see below), the washed precipitate was treated overnight with endoH (20 mU; Boehringer) in phosphate buffer (at 37°C). Immunoprecipitated proteins were solubilized with Tricine-SDS sample buffer . Samples were boiled (for 5 min) and electrophoresed on 10–20% Tris-Tricine gels (Novex). Radiolabeled bands were detected by a PhosphorImager (Molecular Dynamics, Inc.) and analyzed (ImageQuaNT 4.1). Levels of α- and β-cleaved carboxy-terminal stubs and secreted total βA4 were normalized to the level of expression of APP holoprotein precipitated from the cell extracts. Sandwich ELISA assays to measure βA4(1-40) and βA4(1-42) used the combination of antibodies G2-10/W02 and G2-11/W02, respectively . Nunc MaxiSorb immunoassay plates were coated overnight at 4°C with 0.4 mg/well G2-10 in 100 mM NaHCO 3 , pH 9.5, or with 1 mg/well G2-11 in 100 mM Tris-HCl, pH 7.4. Subsequently, the antibody solution was removed and the wells were incubated overnight at 4°C with 5% BSA in TBS. Wells were washed with TBS plus 0.05% Tween 20 (TTBS) and were stored at 4°C (maximum 6 mo). Conditioned media were diluted with 10% BSA to yield a final concentration of 2% BSA and 100 ml of diluted media was added to each well along with 40 ng/well biotinylated W02. Biotinylation of W02 was performed with the EZ-Link TM Sulfo-NHS-LC biotinylation kit (Pierce Chemical Co.) according to the manufacturer's instructions. The plate was incubated at 4°C with gentle shaking for 12 h (for βA4(1-40) measurements) or for 24 h (for βA4(1-42) measurements). Plates were washed five times with TTBS. 100 ml of 0.5 mg/ml HRP-conjugated NeutrAvidin (Pierce Chemical Co.) was added to each well and plates were further incubated at room temperature for 1 h. The color reaction was performed with the TMB-H 2 O 2 system (Kirkegaard & Perry Laboratories, Inc.) according to the manufacturer's instructions and absorption at 450 nm was measured. For the assessment of intracellular βA4, the cells of one 60-mm culture dish were solubilized with 450 ml buffer containing 20 mM Tris-HCl, pH 7, 2 mM EDTA, 0.2% SDS, 1% Triton X-100, 1% NP-40, and protease inhibitors (0.7 mg/ml pepstatin A, 23 mg/ml PMSF, and 11 mg/ml TPCK). 80 ml of the cell lysates was used for every measurement. The sensitivity of the assays is ∼50–100 pg/ml and values ranged between 60–800 pg/ml for intracellular βA4 and 100–3,500 pg/ml for secreted β4A. This large variation is mainly a consequence of variations in the number of cells per dish. Mouse brain and hippocampal neurons derived from wild-type mouse brain were used to localize endogenously expressed PS1 using subcellular fractionation and confocal immunofluorescence microscopy. In addition, transgenic mice expressing human PS1 under the control of the prion promoter, which drives expression of the transgene in neurons, were used for confocal microscopy and to obtain fractionation data on the distribution of PS1 in neurons in situ. Because of their relatively high buoyant density, nuclei can be easily purified by differential centrifugation in high sucrose media. As is clear from Fig. 1 , only a little PS1 staining is observed in purified nuclei, which contain mainly histones in the protein fraction (not shown). However, additional DNA digestion combined with ultracentrifugation yields a fraction of highly purified nuclear envelopes, as demonstrated by the abundant lamin B1 immunoreactivity . Interestingly, this final purification step results in a strong enrichment for PS1-CTF and -NTF immunoreactive bands , indicating that the little amount of PS1 present in the nuclei is almost exclusively associated with the nuclear envelope. It should be noticed that this fraction also stains with calnexin antibodies, in agreement with the continuity between nuclear envelope and RER. Remarkably, in this nuclear envelope fraction, a prominent immunoreactive band representing full-length PS1 is detected with both NTF- and CTF-specific antibodies (see below). The presence of presenilin in the nuclear envelope was confirmed in fixed polarized hippocampal neurons using confocal microscopy. PS1 immunoreactivity colocalized with lamin B1 in confocal sections through the middle of the nucleus . Together, these results indicate that PS1 fragments and PS1 holoprotein are present in the nuclear envelope. Next, the distribution of PS1 was investigated in pre-Golgi compartments of hippocampal neurons derived from human PS1wt transgenic mice . rSEC61αp is an essential component of the mammalian ER protein translocation complex and is tightly associated with membrane-bound ribosomes in RER . PS1 immunoreactivity was found to codistribute only partially with the reticular staining pattern observed for rSEC61αp . In contrast, a considerable fraction of PS1 staining, especially obvious in the perinuclear region, did not localize with rSEC61αp . Antibodies to calnexin and BAP31 , two proteins that are abundantly distributed throughout the ER, displayed a reticular staining that overlapped very well with most of the immunolabeling for PS1-CTF. However, it should be noticed that several distinct PS1-positive spots did not colocalize with calnexin immunoreactivity. Transport from the ER to the IC and Golgi apparatus occurs in specific subdomains of the ER where transported proteins are concentrated in budding transport vesicles coated by COPII coat proteins . SEC23p is one of the cytosolic components of this COPII coat complex. As shown in Fig. 2j–l , this COPII protein displayed an overall colocalization with PS1-CTF . At a higher magnification, all PS1-CTF–positive patches were also immunolabeled for SEC23p . These export sites are often found juxtaposed to vesiculotubular clusters (VTCs) of the IC . Indeed, immunolabeling of ERGIC-53/p58, so far the best characterized IC resident protein , showed a close colocalization with PS1-CTF . Therefore, these results indicate an abundant localization of presenilin in the ER. Especially the close overlap with SEC23p and ERGIC-53 prompted us to further document the interesting localization of PS1 in the transit zone between ER and Golgi. We applied a cell fractionation scheme described by Schweizer et al. 1991 that allows one to obtain highly enriched fractions of the IC in VERO cells. This method starts with a Percoll gradient centrifugation step, resulting in a rough separation of RER and Golgi membranes. When applied on the postnuclear fraction of mouse brain, most of the PS1 immunoreactivity was detected in two peaks . One small peak (15.1 ± 0.5% of total PS1-CTF or 19.4 ± 3.3%, n = 3, of total PS1–NTF immunoreactivity, respectively) was associated with the bottom fractions of the gradient coinciding with the distribution of the RER marker protein rSEC61αp , confirming that a relative low amount of PS1 is associated with the RER compartment. A second, major peak (pool II: 42.7 ± 2.4% for PS1-CTF and 48.1 ± 0.5%, n = 3, for PS1–NTF) was located in the low density region coinciding with the density of Golgi membranes . Typically, IC membranes equilibrate as a broad peak in the midportion of this self-formed gradient. Fractions 5–14, containing 61.6 ± 7.3% ( n = 3) of the total ERGIC-53/p58 immunoreactivity contained 25.1 ± 2.1% of PS1-CTF and 17.9 ± 1.5% of PS1-NTF ( n = 3). This fraction was pooled and further purified and concentrated by an additional centrifugation step in a discontinuous Nycodenz gradient . Three interfaces, F1, F2, and F3, are recovered. The interface F3 at 18.5–27% Nycodenz is highly enriched in IC membranes, as demonstrated by the strong immunoreactivity for p58/ERGIC-53 . Both PS1-NTF and -CTF coenrich to a similar extent in this final F3 fraction. Unfortunately, and in contrast to the original method , immunoblotting for rSEC61αp and TRAPα, a protein associated with the translocon site , revealed contamination of F3 with RER membranes . This discrepancy is probably a consequence of the difference in starting material, i.e., VERO cells versus mouse brain used here. Therefore, we purified the F3 fraction in an additional continuous sucrose gradient (0.8–1.6 M sucrose). Whereas the marker proteins calnexin and ERGIC-53/p58 displayed together with PS1 a bimodal distribution , the rSEC61αp and TRAPα immunoreactivity was solely recovered in the high density region of the gradient. Thus, the PS1 immunoreactivity in the low density region (fractions 4 and 5) of the gradient cannot be explained by contaminating RER membranes. Furthermore, we quantified all marker proteins by densitometric scanning. When the results were expressed as density units per milligram of protein , it became obvious that PS1 fragments specifically coenriched with ERGIC-53/p58 rather than with calnexin, suggesting their localization in the IC. Moreover, the enrichment factor for ERGIC-53/p58 obtained in the low density peak fraction was 35 compared with total homogenate, reaching a similar degree of enrichment as originally described for VERO cells . From the initial Percoll gradient , a major peak of PS1 immunoreactivity (Pool II) can be observed corresponding to less dense regions where Golgi-derived membranes tend to equilibrate . To analyze whether this reflected an association of PS1 with Golgi membranes, we further purified the pooled fractions 15–19 using discontinuous Nycodenz gradient centrifugation consisting of three layers of different Nycodenz concentrations. Most of the PS1 immunoreactivity was recovered in the 14–21% interface (IF3) together with marker proteins of the Golgi apparatus (such as GM130). This interface IF3 was loaded on a continuous sucrose gradient (0.3–1.7 M sucrose) and the distribution of PS1 was analyzed after ultracentrifugation . Established Golgi marker proteins such as GM130 and β-cop (not shown) equilibrated at high sucrose densities in fractions 4 and 5, whereas the bulk amount of PS1-CTF and -NTF immunoreactivity was recovered at a lower buoyant density with a peak in fractions 7 and 8. The striking similar distributions of the unglycosylated form of TGN38 and RAP with the PS1-CTF and -NTF indicate that the PS1 in this pool is mainly associated with early cis-Golgi membranes. Indeed, unglycosylated TGN38 is located in the early cis-Golgi, whereas RAP is a molecular chaperone assisting membrane proteins such as the LDL receptor in their passage through the early secretory pathway between the ER and Golgi . Since the IC resident protein p58/ERGIC-53 did not codistribute with fractions 7 and 8, these fractions may identify a membrane-bound intermediate between the IC and cis-Golgi. Mature (glycosylated) TGN38, which is a marker for the TGN tubules, is mainly found in the bottom fraction . Since this fraction is completely devoid of PS1 immunoreactivity, we can assume that PS1 does not reach the TGN in significant amounts. This observation was entirely corroborated by confocal laser microscopy of control hippocampal neurons. Antibodies against the peripherally associated 58-kD Golgi protein , detected Golgi staining restricted to the cell body and proximal dendrites of neurons in agreement with Krijnse-Locker et al. 1995 . At first glance , a high degree of overlap with the immunostaining for PS1-CTF seemed to be observed. However, when a more careful analysis was performed at a higher magnification , only a limited overlap could be demonstrated . This becomes even more clear in the vertical plane section of a proximal dendrite . PS1-CTF staining was concentrated in defined stacks often capped by or juxtaposed to staining for p58/Golgi . Both proteins colocalized only partially in discrete areas. This is in line with the fractionation data. α- and β-secretase cleavage of APP occurs in the late Golgi, in transport vesicles, at the cell surface, and in endocytic compartments , generating the α- and β-APP carboxy-terminal stubs associated with the membranes. These stubs are the direct substrates for γ-secretase. Given the restricted distribution of PS1 in the ER, IC, and early cis-Golgi compartments, the question arises how the γ-secretase cleavage of these APP stubs can be controlled by PS1. Therefore, we analyzed the distribution of endogenous APP in the same fractions analyzed before for presenilin immunoreactivity. As shown in Fig. 5 a, both the gradient distributions of the APP holoprotein and the APP carboxy-terminal fragments were bimodal, which is similar to what was found for PS1 . The larger amounts are found in the fractions of Pool II. Further purification resulted in the concomitant enrichment for the APP carboxy-terminal fragments in the F3 interface , where PS1 is also recovered. Remarkably, this enrichment was much less obvious for the APP holoprotein, suggesting a selective accumulation of the carboxy-terminal APP fragments in this fraction. Further analysis of the Pool II–associated APP immunoreactivity revealed the enrichment of the APP carboxy-terminal fragments in exactly the same fractions of the sucrose gradient where PS1 fragments (together with RAP) are recovered . Interestingly, therefore, the carboxy-terminal fragments of APP are apparently at least partially codistributing with PS1-NTF and -CTF in the same subcellular membrane compartments (see Discussion). Given the existing controversy whether or not PS1 can be found in compartments beyond the ER/IC and cis-Golgi, we extended our study to some well-defined post-Golgi compartments such as lysosomes, endosomes, synaptic vesicles, the cell surface, and clathrin-coated vesicles. Both PS1 fragments clearly purify away from mature and intact lysosomes , and were observed only after prolonged exposure times. The weak immunoreactivity in the pure lysosomal fractions is most likely a consequence of minor contamination of the isolated fraction. We also investigated the possible colocalization of CTF-PS1 with marker proteins of post-Golgi compartments such as the transferrin receptor and synaptobrevin II in hippocampal neurons of control, nontransgenic mice . In fully polarized neurons, the transferrin receptor is localized somatodendritically and, accordingly, numerous transferrin receptor–positive punctae are noticed in the dendritic arbor . Virtually no overlap is observed for PS1-CTF and transferrin receptor staining, indicating that PS1 is not present in recycling endosomes. Synaptobrevin II is often used as a marker for synaptic vesicles that cluster in specialized axonal regions of mature neurons referred to as synaptic boutons. Again no immunostaining for PS1 could be detected within these boutons , suggesting that no PS1 is associated with the membranes of synaptic vesicles. Further analysis of highly purified clathrin-coated vesicles from nerve terminals, or double immunostaining with specific cell surface marker such as ICAM-5, and surface biotinylation experiments (not shown) did not yield any conclusive evidence that PS1 could be associated with clathrin-coated vesicles or with the cell membrane. (data not shown). It is clear that the amounts of presenilin present in the membranous compartments beyond the ER–cis-Golgi in hippocampal neurons are very limited and, in any event, below the detection limits of our current available technology. In conclusion, the data obtained from both cell fractionation and immunofluorescence studies on brain and neurons, demonstrate the abundant association of PS1 with the ER, IC, and to a lesser extent, the early cis-Golgi. This prompted us to further investigate the relationship between APP processing and presenilin at a more functional level. Therefore, we generated a series of APP trafficking mutants that limit APP processing to specific subcellular compartments. A dilysine motif was added to the carboxy terminus of APP to retain APP/KK in pre-Golgi compartments, thereby emphasizing the βA4(1-42) production . Reinternalization of cell surface APP was blocked by deleting the cytoplasmic tail (APPΔCT), limiting APP trafficking to the biosynthetic pathway , and severely impairing βA4 secretion . A chimerical APP/LDLR was generated by replacing the cytoplasmic domain of APP by that of the LDL receptor. This mutation increases the recycling of APP in the endosomal compartments and promotes βA4(1-40) production (see below). We also investigated the effects of the clinical APP/London mutation in combination with the PS1 mutations, to see whether these mutations operate additively or not. The APP constructs were expressed in neurons using the SFV expression system. Similar expression levels for all constructs were obtained in hippocampal cultures, except for APPΔCT . For comparisons between the different experiments, the obtained phosphorimaging results were normalized to the expression levels of APP holoprotein in the cell extracts. Expression of APPΔCT led to decreased βA4 secretion concomitantly with the production of a short cell-associated β-stub. In the case of APP/KK infection, the decrease of secreted βA4 was even more pronounced (also see below). The APP/KK mutant is actively retrieved from the cis-Golgi to the ER as a consequence of the dilysine motif. This was confirmed by its complete endo-H glycosidase sensitivity (data not shown) and its codistribution with calnexin and endogenous PS1 . The striking colocalization of endogenous PS1 with virally expressed APP/KK should be noticed. As a control, SFV-APPwt is shown, which confirms the axonal delivery of this protein . We analyzed systematically the metabolism of the expressed APP mutants ( Table ). Quantitative immunoprecipitation and phosphorimaging determined the total amounts of cell-associated α- and β-stubs and secreted βA4. To standardize and to allow comparisons between different experiments, we normalized all values to cell-associated APP holoprotein. Similarly, normalization of the numbers obtained in the ELISA experiments was performed because of the relative large variations in the absolute amounts of peptide produced in separate independent experiments. Therefore, only ratios of secreted and intracellular βA4(1-42)/(1-40) are given. Except for APP/LDLR, all constructs were completely analyzed in the three types of neuronal culture, i.e., expressing human PS1 wt or the clinical mutants PS1 L286V or PS1 M146L . The results were compared with those obtained in cells derived from control and nontransgenic littermate embryos. Table summarizes the data obtained for APPwt (A), the London mutant (B), APPΔCT (C), and APP/KK (D). With respect to α- and β-cleaved carboxy-terminal fragments, no significant differences were noticed related to PS1 wild-type or clinical mutants except for the London mutant. The clinical mutations in PS1 do not affect α- and β-secretase activity, complementing the data obtained previously in PS1-deficient neurons . However, in the case of the London mutant, levels of α- and β-stubs were surprisingly decreased in the FAD mutant PS1 neurons. This reflects probably the increased turnover of both stubs by γ-secretase cleavage, in accordance with the strong additive effects of the London- and FAD-linked PS1 mutations (especially PS1 M146L ) on γ-secretase processing (see below). To evaluate γ-secretase activities, we measured the total pool of secreted βA4 by immune precipitation as well as the ratio of the individual peptides by ELISA. A modest increase in the total amount of secreted βA4 was observed for the PS1 M146L mutant. This effect was not very dramatic (except for the APP/KK, see below). In contrast, PS1 mutations caused dramatic increases in the βA4(1-42) to βA4(1-40) peptide ratio, and this effect was even more pronounced when the effect of the mild overexpression of PS1wt alone was directly compared with that of PS1 containing clinical mutations. The PS1 M146L mutation was overall more effective than the PS1 L286V mutation. The London mutation on its own caused a threefold increase (3.44±0.43; n = 6) in the ratio of secreted βA4(1-42)/(1-40) versus APP wild-type in control neurons (not shown). Interestingly, an additional rise in this ratio was observed in combination with PS1 mutations ( Table B). This rise ranged from 1.38±0.07 ( n = 8) for the PS1 L286V mutation to 2.97±0.65 ( n = 3) for the PS1 M146L mutation. When compared with APPwt expressed in wild-type neurons, the total raise ranged up to 12.3 ± 3.1 ( n = 3). The increase appears to be solely due to an increased secretion of the β4A(1-42) peptide and not a consequence of a decreased βA4(1-40) production, explaining the decrease in α- and β-carboxy-terminal fragments as mentioned above. APPΔCT in control neurons yielded low levels of secreted βA4. However, the ratio between the two peptides was slightly raised compared with SFV-APPwt (1.30±0.14, n = 8, versus SFV-APPwt). This ratio increased even further when this mutant was expressed in PS1 M146L neurons ( Table C). For APP/KK, the total secretion of βA4(1-40) was also lowered dramatically to 5.0±1.6% ( n = 6) when compared with APPwt . The βA4(1-42) secretion was not affected as strongly as the βA4(1-40) secretion and only dropped to 18.0±4.7% ( n = 6) when compared with APPwt. The introduction of the ER-retrieving dilysine motif on its own, therefore, caused an appreciable increase in the 42:40 ratio of the secreted peptides to 3.98±0.70 ( n = 6) times relative to APPwt. Upon expression in PS1 M146L neurons, an additional raise in this ratio of 2.83±0.57 ( n = 6, Table D) was observed. The increase could again solely be contributed to an increased secretion of the βA4(1-42) peptide. Interestingly, only for this construct, a sharp rise in total secreted βA4 was noticed in PS1 M146L neurons. It should be noticed that the main peptide produced by APP/KK is βA4(1-42). This observation indicates that some of the presenilin mutations do not only affect the ratio of the secreted peptides, but can also increase the total production of βA4(1-42). The results obtained with this ER-retained APP/KK mutant also indicate that the clinical PS mutations are operating in the ER. This conclusion was further corroborated in an experiment comparing the amyloid peptide production from APP/LDLR in control and PS1 M146L hippocampal neurons. As expected for a mutant trafficking mainly in the endocytic compartments, a relative increase in secreted βA4(1-40) of 2.36±0.18 ( n = 3, SEM) fold was observed, whereas secretion of βA4(1-42) remained largely unaffected. Interestingly, the PS1 M146L mutation did not cause a statistically significant effect on the ratio of secreted βA4 peptides in these neurons. Finally, we note that in our experimental system, and for all APP constructs studied, PS1 clinical mutations do not cause a significant increase in the βA4(1-42)/(1-40) ratio of intracellular peptides. In this study, we provide conclusive evidence that the bulk of endogenously expressed PS1 in neurons is localized in the early compartments of the secretory pathway, i.e., in the ER, the IC, and the early Golgi. Relatively limited levels of PS1 are furthermore detected in the nuclear envelope and the RER. The ER, in contrast to the Golgi apparatus, is widely distributed in the soma and the dendrites (including the most distal regions) of neurons . Therefore, our data explain the previously documented somatodendritic localization of the presenilins . Within these early compartments of the biosynthetic pathway, a clear gradient of increasing amounts of the supposedly functional PS1 fragments is observed. The PS1 immunoreactivity previously detected in the nuclear membrane is, therefore, a mixture of (predominantly) PS1 fragments and full-length PS1, indicating that cleavage of PS1 by the unknown presenilinase occurs very early after biosynthesis . Importantly, these results definitively resolve the controversy whether and where full-length presenilin can be detected, namely in nuclear envelope fractions. Interestingly, both the PS1-NTF and -CTF codistributed and coenriched in all fractionation schemes applied, confirming at the endogenous level of protein expression that both fragments remain in a 1:1 stoichiometric relationship along the whole early biosynthetic pathway . Our data not only settle the debate on the subcellular localization of presenilin, but also document for the first time that PS1 immunoreactivity in the ER is concentrated in discrete patches that are sec23p-positive. Sec23p is one out of the five cytosolic proteins that constitute the COPII coat complex required for vesicle budding from the ER . These COPII components bind specifically with cargo molecules destined for sorting into transport vesicles and separate them from the ER resident proteins . The formation of COPII transport vesicles occurs nonrandomly in the ER at tubular protrusions often juxtaposed to VTC, referred to as the IC . PS1 codistributes and coenriches with markers of this intriguing compartment such as p58/ERGIC-53 , suggesting that PS1 is operating in these compartments. A similar conclusion was recently made by Culvenor et al. 1997 who, based on temperature block experiments, suggested that PS1 is localized in the IC and cis-Golgi in stably transfected SY5Y and P19 cell lines. On the other hand, and in contrast with many earlier studies investigating overexpressed PS1 , we could not confirm that PS1 at the endogenous level of expression distributes to a significant extent into the Golgi apparatus. This limits the subcellular compartment where PS1 is operating to the early compartments of the biosynthetic pathway. The earlier reported abundant Golgi localization of the presenilins is probably a consequence of overexpression and missorting of the protein. As illustrated in Fig. 4 B, f and i, colocalization studies should indeed be carefully interpreted. As we suggested before , experiments based on overexpression of presenilins may lead to erroneous conclusions since normal proteolytic maturation and also normal subcellular ER localization is apparently significantly disturbed. This conclusion is further corroborated by recent findings that overexpression of presenilins results in their redistribution into aggresomes . These structures are located at the perinuclear microtubule organizing center, where also the Golgi apparatus is located and are the signature of abnormal biosynthetic stress in the cell. In view of our new data, it is conceivable that PS1 passes from the ER, via VTCs, to cis-Golgi cisternae. The close colocalization of PS1 with sec23p further indicates that at least part of this transport is associated with the trafficking of COPII vesicles. . Since PS1 fragments exist as a stable complex with a long half-life and no accumulation in the Golgi apparatus was observed, PS1 is probably also retrogradely transported. Electron microscopical observations demonstrating abundant PS1 immunoreactivity in vesiculotubular structures and coated vesicles in close vicinity or in continuity with Golgi cisternae support the view that presenilins are actively transported back and forward between the ER and cis-Golgi . The lack of PS1 immunoreactivity in TGN-enriched fractions suggests that only minimal amounts of PS1, if any, exit the Golgi area. Indeed, no significant amounts of PS1 were found in recycling endosomes, lysosomes, synaptic vesicles, or clathrin-coated vesicles . Finally, all published data that support the localization of PS1 at the plasma membrane imply a topology of PS1 with the amino-terminal domain at the luminal side of the ER. This is not in agreement with several studies that addressed this question more specifically, although it could be speculated that cell type–specific differences exist. In any event, our data in hippocampal neurons clearly indicate that the bulk of PS1 fragments are localized in compartments of the early biosynthetic pathway and that only limited amounts (if any at all) traffic beyond the Golgi apparatus, a finding that needs to be taken into account when considering the association of presenilin with γ- secretase activities. Accumulating evidence indicate that presenilins are either part of the catalytic γ-secretase activity or at least controlling its activity . Wolfe et al. 1999 presented evidence that presenilins are aspartate proteases that become inactivated by mutating one of the two aspartic acid residues in the sixth or seventh transmembrane region respectively. Overexpression of this mutant PS1 results in dominant negative effects on γ-secretase activity . The most simple explanation is that PS1 is γ-secretase itself. This interpretation is consistent with the observation that deletion of the PS1 gene also results in the inhibition of γ-secretase . In both experiments, secretion of βA4(1-40) as well as βA4(1-42) peptides was equally affected . The subcellular localization of PS1 in the ER as we demonstrate here, is, however, difficult to reconcile with current concepts that γ-secretase processing of APP mainly occurs in the endocytic limb of the subcellular trafficking pathways. There is clearly a spatial paradox. The first question to be addressed in this regard is whether the real substrates of γ-secretase, namely the α- and β-secretase cleaved APP carboxy-terminal stubs generally believed to be generated in the late biosynthetic compartments and the endocytic pathways, can indeed reach the subcellular compartments where PS1 resides. Unexpectedly, we could demonstrate the enrichment of the APP carboxy-terminal stubs in the ER/IC subcellular fractions where PS1 resides. From a cell biological point of view, it remains to be explained how this retrograde transport of the APP carboxy-terminal fragments to the ER/IC can occur and which sorting signals are involved, but this finding is at least consistent with the PS1–γ-secretase hypothesis . The second question is whether and how PS1 can affect the generation of amyloid peptide in the endocytic limb of the protein trafficking pathways in the cell. This problem was addressed via a functional approach by expressing a series of APP trafficking mutants in hippocampal neurons using the recombinant SFV system. In previous work, it has been extensively demonstrated that the processing and subcellular trafficking of APP expressed with this system does not differ from that observed with endogenously expressed APP . Therefore, and in contrast with presenilins, the results obtained after transfection of APP with SFV under the experimental conditions used, can be considered as biologically relevant. The results (see Table ) obtained with APP retained in the ER (APP/KK), with APP processed in the biosynthetic pathway (APPΔCT) or with APP retrieved in the endocytic pathway (APP/LDLR, not shown), corroborated the conclusion that presenilins operate in the ER/IC. Significant increases in βA4(1-42) secretion were observed with the APP/KK and to a lesser extent with the APPΔCT mutant in neurons expressing PS1 M146L. . Such an effect was almost undetectable with the APP/LDL mutant. The latter most likely reflects the inability of the APP/LDLR carboxy-terminal fragments generated by α- or β-secretase to be recycled to compartments where γ-secretase resides. The APP/KK mutant was particular relevant in this regard. The dilysine Golgi–ER retrieval signal is recognized by COPI subunits in proteins such as p58/ERGIC-53 and results in their retrograde retrieval from VTCs and cis-Golgi cisternae to the ER . The KK motif limits the trafficking of APP/KK to those compartments where also PS1 resides. Indeed, the subcellular distribution of APP/KK was strikingly similar to that of endogenous PS1 . As previously demonstrated, only very low amounts of amyloid peptide are recovered in the media with this construct (5% compared with wild-type APP). Since β-secretase is mainly operating beyond the ER/IC , it is not surprising that only very small amounts of the immediate precursor of βA4, i.e., the β-secretase cleaved carboxy-terminal APP fragment , are generated with APP/KK . In contrast to the interpretation of Chyung et al. 1997 , we do not consider the residual β-secretase activity as evidence for a novel, ER-linked β-secretase activity. Since >95% of the normal β-secretase activity is precluded with this construct, this indicates in fact that β-secretase is operating mainly beyond the cis-Golgi, and that APP/KK is only marginally processed by enzyme trailing in the cis-Golgi. This interpretation is certainly more in line with general concepts of β-secretase activity in other cell types since shown to depend on the activity of Rab1B and on O-glycosylation . In any event, the residual generation of β-secretase cleaved fragments obtained with APP/KK was sufficient to demonstrate that mainly 1-42 peptide is generated in ER, IC, and early cis-Golgi, where the APP/KK carboxy-terminal stub is recycling. More importantly, the presenilin mutants have a pronounced effect on this process, which is clearly consistent with their subcellular localization. On the other hand, γ 40 -secretase processing in the endocytic pathways is not at all influenced by the PS1 mutations as was most dramatically illustrated with the APP/LDLR mutant. The processing of this mutant, which selectively increases the production and secretion of the β4A(1-40) peptide (this study), was not altered by coexpression of the PS1 M146L . It is generally accepted that the bulk of the βA4(1-40) peptide is generated beyond the TGN and in the endocytic pathways compartments where no PS1 is found (this study). This explains why PS1 mutations have no major effects on the secretion of βA4(1-40). Further evidence for the selective effect of the presenilin mutations on γ-secretase cleavage at position 42 was obtained when coexpressing APP-London together with mutant PS1 . Both clinical mutations increase the βA4(1-42) secretion and act additively, which suggests independent or at least synergistic mechanisms operating with the two mutations. The experimental evidence presented here together with many reports in the literature suggests strongly an exclusive role for the presenilins in γ 42 -cleavage (as opposed to γ 40 cleavage). The effect of the inactivation of PS1 or the expression of dominant negative forms of PS1 on γ 40 -secretase activity must, therefore, be indirect. One possibility is a precursor–product relationship between βA4(1-42) and βA4(1-40). In this view, only one γ-secretase is needed that generates βA4(1-42), whereas in compartments distal to the ER, carboxypeptidases convert this peptide to βA4(1-40). This would explain the above-mentioned spatial paradox, but not completely the selective increased secretion of βA4(1-42) linked to PS1 clinical mutations. In a precursor–product relationship, one should also expect a rise in the secretion of βA4(1-40), and this has not been really observed yet. Along similar lines, the fact that the APP/LDLR or APPΔCT mutations that affect APP trafficking in the endocytic compartments have a direct effect on βA4(1-40) production and secretion, support the general importance of this pathway in APP processing, and imply the existence of a γ 40 -secretase activity in the endocytic limb. Therefore, it is clear that further investigations are needed to explain the relationship between PS1 and γ 40 -secretase activity. Unexpectedly, blocking the processing of APP in the endocytic pathway also decreases secretion of βA4(1-42) , whereas stimulating the endocytic recycling (APP/LDLR) selectively promotes βA4(1-40) secretion. A recent study provided evidence that the cytoplasmic tail of APP contains, in addition to endocytic signals, information that influences its metabolism in the exocytotic limb . Apart from the spatial paradox, it remains to be explained why mainly the immature, N -glycosylated form of APP holoprotein has been found to interact physically with PS1 . One would predict a preferential association with the carboxy-terminal APP fragments if PS1 was indeed γ-secretase. It could be speculated that PS1 is involved in a posttranslational modification of the newly synthesized APP, tagging it for later recognition by the γ-secretases, although any direct evidence supporting such a possibility is lacking. Other hypotheses have been proposed as well. PS1 might control trafficking of APP to its processing compartments, or alternatively, control trafficking of the γ-secretases to APP-bearing compartments. For instance, the subtle effects on the maturation of TrkB and on BDNF-inducible TrkB autophosphorylation caused by PS1 deficiency in neurons was interpreted to reflect such a function . Our findings that endogenous PS1 colocalizes with marker proteins of the IC and ER, would agree with a recruiting or activating function for PS1, either as a chaperone or an adaptor molecule bringing γ-secretase, APP carboxy-terminal fragments, and maybe other substrates together in the right subcellular microenvironment needed for the controlled and limited proteolytic cleavage of the transmembrane domain. In conclusion, our data indicate clearly a direct role for PS1 in γ 42 -secretase activity in the early compartments of the biosynthetic pathway. They are compatible with the hypothesis that PS1 is γ 42 -secretase, and imply that γ 42 -secretase exerts its activity in close association with transport processes between ER and the cis-Golgi. However, some caution with this straightforward interpretation remains indicated, since several other observations remain difficult to integrate with this concept, most notoriously the relationship between PS1 deficiency and γ 40 -secretase processing. Therefore, the alternative possibilities discussed above have to be further explored, and further research should clarify the exact molecular relationship between PS1 and γ 40 - and γ 42 -secretase activity. | Study | biomedical | en | 0.999998 |
10525536 | HeLa cells were cultured in DME and synchronized in G2 phase by a thymidine-aphidicolin block . PtK1 cells were cultured in Ham's F-12 medium (GIBCO BRL), 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, 1 mM Na pyruvate, 10% foetal bovine serum, and 1% (vol/vol) Fungizone (GIBCO BRL) at 37°C/6% CO 2 . Human p21 Cip1 (1–90) was generated by PCR from the p21 Cip1 cDNA (kind gift of Dr. J. Roberts, Fred Hutchison Cancer Center, Seattle, WA) using the following primers; 5′ GCCGGATCCCCATGTCAGAACCG, and 3′ GCCGGATCCCTATCCCAACTCATCCCGGCCTCG. After digestion with BamHI, the PCR product was subcloned into the BamHI site of pGEX3T (Pharmacia) to yield pGEX-p21N. For expression, pGEX-p21N was used to transform E . coli (BL21) and the GST-p21N fusion protein was purified by glutathione Sepharose chromatography after which it was >90% pure on a Coomassie blue R250–stained SDS–polyacrylamide gel. The concentration of purified GST-p21N was determined by Coomassie blue R250 staining using BSA standards. All constructs were confirmed by sequencing on an automatic sequencer (Department of Biochemistry, University of Cambridge). Cyclin A/CDK2, cyclin E-CDK2, and cyclin A/CDK2 K33R were expressed in and purified from baculovirus-infected Sf9 cells as previously described , as were cyclin B1-MmGFP and cyclin B1 . Proteins were >90% pure on silver-stained gels. Proteins were concentrated in injection buffer (200 mM NaCl, 12.5 mM Tris-Cl, pH 8.0, 2.5 mM DTT, 1 mM EDTA, and 1 mM glutathione) in a Vivaspin 5,000 MW cut-off microconcentrator (Vivascience) and ∼5% of the cell volume injected into cells using an Eppendorf semiautomatic microinjector (Eppendorf) attached to a Leica DMIRBE microscope (Leica). The cDNAs for Cdc25B and Cdc25C, mutant and wild-type (kind gifts of Dr. I. Hoffmann, DKFZ, Germany) were cloned into pCMX under the CMV promoter, and injected into cells as described . Histone H1 kinase assays with purified cyclin A/CDK2 or cyclin B1-cdc2 were performed as described . The amount of 33 P incorporated into histone H1 was quantified using a scintillation counter (Beckman). Cells were fixed in 50:50 vol/vol methanol/acetone and stained with affinity-purified anti–cyclin B1 serum at 1:200 dilution, and with an anti–β tubulin monoclonal antibody (Amersham) at 1:100 as described . As secondary antibodies, Cy5-labeled anti–rabbit and Cy2-labeled anti–mouse antibody were used at 1:200 and 1:500, respectively. Cells were plated and synchronized in ΔT 0.15-mm dishes (Bioptechs). For microinjection and observation, the culture medium was replaced with CO 2 -independent medium without phenol red (GIBCO BRL) and overlaid with mineral oil. Dishes were maintained at 37°C using the ΔT system (Bioptechs). Lambda 10-2 filter wheels (Sutter Instruments) with custom filters (Chroma Technology) were used to control the excitation and emission wavelengths (for cyclin B1-GFP imaging). The filter wheel on the camera port also had a polaroid filter to capture DIC images. Images were captured on a Leica DMIRBE with a PentaMax camera (Princeton Instruments), and a PowerWave computer (PowerComputing) or Macintosh 8600 (Apple) computer running IP Lab Spectrum imaging software (Scanalytics Inc.) as described . To quantify the amount of fluorescence in a cell over time, a region encompassing the cell was drawn manually and copied onto successive fluorescence images of a time series saved in IP Lab Spectrum format. The sum of pixel intensities (total fluorescence) and the number of pixels (area) of this region in successive images was quantified using IP Lab Spectrum. The mean pixel intensity outside the region of cell fluorescence (mean background) was measured and multiplied by the number of pixels (area) of the original cell region. This represented that amount of the total fluorescence in the cell region which was due to background rather than GFP fluorescence. This was subtracted from the total fluorescence of the cell to give a figure representing the total amount of GFP fluorescence, and thus, the amount of GFP-linked protein in the cell (total fluorescence minus background). Captured images were animated with Adobe Premiere and the time to reach mitosis recorded for each cell. Cells were scored as entering mitosis when they had rounded up and condensed their chromosomes. Time-lapse imaging showed that cells were still viable after premature mitosis because cells exited mitosis and flattened out. Images were exported to Adobe Photoshop for printing. To investigate the role of cyclin A in mitosis, we have used a time-lapse microscopy assay to determine the effect on entry into mitosis of inhibiting or enhancing cyclin A/CDK2 kinase activity. In this assay, cells were first synchronized in S or G2 phase by a thymidine-aphidicolin block and release regime, and then viewed by time-lapse DIC microscopy to assess when they entered, and subsequently exited, mitosis. In each experiment, control cells were simultaneously visualized in the same field as the experimental cells. Cells were demonstrated to be in S or G2 phase by flow cytometry and by the incorporation of BrdU. To increase the amount of active cyclin A/CDK2 in a specific phase of the cell cycle we microinjected cyclin A/CDK2 purified from baculovirus-infected Sf9 cells into synchronized HeLa cells. This complex was an active histone H1 kinase and >90% pure on a silver-stained SDS-PAGE gel, as previously described . We found that injecting a final concentration of 2–4 μM cyclin A/CDK2, which represents ∼20–40-fold excess over the endogenous cyclin A, into early G2 cells (∼7 h after release from an aphidicolin block), caused the cells to begin to enter mitosis 30 min to 1 h after injection . This was at least 4 h before control. Uninjected cells began mitosis. This effect required CDK2 kinase activity, because cyclin A, in a complex with an inactive CDK2 K33R mutant, did not promote, indeed it slightly inhibited, entry into mitosis . The effect was specific to cyclin A/CDK2 because microinjecting the same amount of cyclin E-CDK2 did not cause G2 phase cells prematurely to enter mitosis . (We injected an equal amount of cyclin E/CDK2 rather than attempt to inject an equal amount of kinase activity, because no single substrate can be used to compare the physiological specific activities of different cyclin/CDKs. This is due to the substrate-targeting role of the cyclin which causes the apparent activity of a cyclin/CDK to depend on the substrate in the assay. For example, unlike cyclin A/CDK2, cyclin E/CDK2 only weakly phosphorylates histone H1, but it strongly phosphorylates NPAT .) The mitosis-promoting activity of cyclin A/CDK2 was also dose dependent, because injecting a final concentration of 0.4–0.8 μM cyclin A/CDK2 (a four- to eightfold excess over endogenous cyclin A/CDK2) caused early G2 phase cells to enter mitosis earlier than control cells, but later than cells injected with 2–4 μM cyclin A/CDK2 . Overall, these results suggested that cyclin A/CDK2 might be a rate-limiting component in G2 phase for the initiation of mitosis. We then asked whether cyclin A was simply able to accelerate cells through G2 phase, or whether it was also able to cause mitosis in cells with unreplicated DNA. Therefore, we injected cyclin A/CDK2 into S phase cells (2 h after release from an aphidicolin block). This caused <10% of the cells to enter mitosis and, furthermore, >90% of the cells had an apparently normal length G2 phase, suggesting either that the exogenous cyclin A/CDK2 had been inactivated, or that the appropriate mitotic substrates were not present in S phase . Although human cyclin A had been shown to accumulate through S and G2 phases until it was rapidly degraded in mitosis , it was possible that the exogenous cyclin A was inactivated in S phase through proteolysis. To test this, we injected a cyclin A–GFP fusion protein which acts as a proper marker for cyclin A; it binds and activates CDK2, localizes to the nucleus, and is rapidly degraded in mitosis (del Elzen, N., and J. Pines, manuscript in preparation). This fusion protein also has mitosis promoting activity in G2 phase cells. We were able to measure the rate of proteolysis of the protein by measuring the amount of GFP fluorescence . There was a low rate of degradation in S and in G2 phases, which increased with larger amounts of protein, but this rate was insufficient to account for the inactivation of cyclin A. Furthermore, the rate was the same in S or G2 phase cells . Given that cyclin A was equally stable whether injected into cells in S phase or G2 phase, this suggested that S phase cells do not inactivate exogenous cyclin A by proteolysis. Cells that are forced to enter mitosis without completing DNA synthesis undergo premature chromosome condensation/mitotic catastrophe and die after arresting in mitosis. However, this was not the case for the G2 cells that were forced to enter mitosis prematurely by the injection of cyclin A/CDK2. In many of these cells, mitosis was considerably prolonged, from ∼1 h to between 3 and 4 h. Prophase and prometaphase/metaphase were the most prolonged; anaphase and telophase were of relatively normal duration. In cells injected in mid to late G2 phase the chromosomes clearly condensed and aligned on the metaphase plate, and these cells subsequently divided and remained separate . However, in cells injected in early G2 phase, and in the small minority of S phase cells that prematurely entered mitosis when we injected cyclin A/CDK2, condensed chromosomes were never visible and in these cases the sister cells fused soon after cytokinesis . This phenotype is, therefore, likely to reflect premature condensation of incompletely replicated DNA. We wished to determine whether the M phase–promoting activity of cyclin A/CDK2 was acting through known regulators of cyclin B1/CDK1. Therefore, we injected cells with expression constructs that encoded inactive mutants of Cdc25B or Cdc25C, which act as dominant negative mutants and arrest cells in G2 phase. A dominant negative mutant of Cdc25B was able to block the effect of injecting cyclin A/CDK2 , as did a dominant negative Cdc25C, but to a lesser extent than the Cdc25B mutant (data not shown). Cells injected with cyclin A/CDK2 plus the dominant negative forms of Cdc25B or Cdc25C did round up, and their chromosomes partially condensed, but their nuclear envelopes remained intact and they did not divide (data not shown). These results suggested that cyclin A/CDK2 was acting upstream of the activation of cyclin B/CDK1, and that cyclin B1/CDK1 activity was required for nuclear envelope breakdown but not for the initial stages of chromosome condensation (see below). Our results suggested that cyclin A was able to promote mitosis once cells had replicated their DNA, and, therefore, we wished to determine the period in G2 phase for which cyclin A was required to initiate mitosis. To answer this question, we designed a means to inhibit cyclin A/CDK2 activity at specific points in G2 and M phase. To inhibit cyclin A/CDK2 kinase activity, we chose to microinject the first 90 amino acids of human p21 Waf1/Cip1/Sdi1 (hereafter referred to as p21N) purified as a GST-fusion protein from E . coli . This construct contained both a cyclin binding motif (Cy1) and a CDK-inhibitory motif , and efficiently inhibited cyclin A/CDK2 more than cyclin B/CDK1 in vitro . The effect of injecting GST-p21N was specific to the p21N moiety because injecting either buffer or GST had no effect on the dynamics of entry into mitosis . Furthermore, coinjection with an equal amount of cyclin A/CDK2 negated the effect of p21N . We did not use full-length p21 Cip1/Waf1/Sic1 because we found that this activated a G2 checkpoint (data not shown) related to that activated by overexpressing PCNA, as previously reported . We found that injecting ∼2 μM p21N, which represents a ∼40-fold excess over the endogenous cyclin A (data not shown), at 10 h after release from an aphidicolin block caused cells to arrest in G2 phase . (We injected excess p21N to ensure that we completely blocked cyclin A/CDK2 activity.) Previous studies in which full-length p21 was overexpressed have found that cyclin B1 accumulated in the nucleus . Therefore, we stained cells blocked in G2 phase by p21N with anti–cyclin B1 antibodies and found that cyclin B1 remained cytoplasmic . Thus, full-length p21 Cip1/Waf1/Sic1 does not cause cyclin B1 to accumulate in the nucleus by inhibiting cyclin A/CDK kinase activity. Our previous results showed that dominant negative mutants of Cdc25B and Cdc25C blocked the mitosis-promoting activity of cyclin A/CDK2. Therefore, we assayed whether overexpressing Cdc25B and C, or microinjecting active cyclin B1/CDK1, could promote mitosis when cyclin A/CDK2 was inhibited with p21N. Injecting the cDNAs for Cdc25B or Cdc25C , into cells blocked in G2 phase by injection of p21N caused cells to enter mitosis, although passage through mitosis was delayed . We were also able to force cells into mitosis with active cyclin B1/CDK1 purified from baculovirus-infected Sf9 cells . However, this differed from the effect of cyclin A/CDK2 because the time to entry into mitosis did not depend on the dose of the cyclin B1/CDK1 kinase, and the nuclear envelope broke down as soon as the cells rounded up. Thus, it appeared that the function of cyclin A/CDK2 to promote mitosis was bypassed by directly activating cyclin B1/CDK1, suggesting that cyclin A/CDK2 acted upstream of cyclin B/CDK1. These results also showed that p21N did not inhibit cyclin B/CDK1 in vivo. To define the period of the cell cycle for which cyclin A/CDK2 kinase was required for the initiation of mitosis, we injected p21N into late G2 phase and mitotic cells. As expected, we found that late G2 phase cells remained in G2 phase. Prometaphase cells that had undergone nuclear envelope breakdown continued to progress through mitosis (10/10 cells), and this was further evidence that p21N did not block cyclin B1/CDK1. However, when we injected p21N into prophase HeLa cells that had not yet reached NEBD, these cells did not continue through mitosis but decondensed their chromosomes, flattened out and arrested, apparently in interphase again (6/6 cells, and see below). Control prophase cells injected with GST completed mitosis (data not shown). After 2–3 h, cells injected with p21N began to re-enter mitosis and this time completed cell division. This observation was reminiscent of the prophase checkpoint that had recently been identified by Rieder and Cole 1998 , who found that PtK 1 cells in early prophase, >30 min before NEBD, would return to interphase after nuclear irradiation. Therefore, we microinjected p21N into prophase PtK 1 cells. We found that prophase PtK 1 cells returned to interphase up to the time when the nucleoli disappeared in mid prophase . p21N could not prevent cells later in prophase from progressing through mitosis; these cells underwent NEBD ∼10–20 min after microinjection (8/8 cells). Thus, microinjecting p21N mimicked the effect of the prophase checkpoint. From our studies, and from those of Rieder and Cole 1998 , there appeared to be a point in mid to late prophase after which the cells are committed to NEBD. From our results, this point correlated with the time when the nucleoli disappeared. One event that occurs at approximately this time is the translocation of cyclin B1/CDK1 into the nucleus, therefore, we reasoned that the translocation of cyclin B1/CDK1 might mark the point of no return for mitosis. We have previously shown that a cyclin B1-GFP fusion protein acts as a proper marker for the endogenous cyclin B1 . Therefore, we correlated the behavior of a cyclin B1-GFP chimaera in PtK 1 cells with the morphology of prophase nuclei under DIC microscopy. We found an excellent agreement between the time at which the nucleoli disappeared, which marks the point when p21N is unable to return the cells to interphase, and the time when cyclin B1-GFP translocated into the nucleus . In this paper we have used time-lapse DIC microscopy to study the requirement for cyclin A/CDK2 at mitosis. In our experiments we used cyclin A/CDK2 because this is the major form of cyclin A/CDK activity in G2 phase in HeLa cells . We have demonstrated that cyclin A/CDK2 activity appears to be rate limiting for the initiation of mitosis in G2 phase because purified, active cyclin A/CDK2 kinase will initiate mitosis within 30 min of microinjection if cells have finished DNA replication. This role for CDK2 kinase in the initiation of mitosis is consistent with experiments using Xenopus extracts, where CDK2 was shown to be a positive regulator of cyclin B/CDK1 . However, in these embryonic extracts CDK2 was partnered with cyclin E, whereas we found that cyclin E/CDK2 was unable to promote mitosis. Thus, it appears that the role of the CDK2 kinase in promoting mitosis is conserved in adult, mammalian cells but that the cyclin partner has altered to cyclin A. We have also used the amino terminus of p21 as a specific inhibitor of cyclin A/CDK2 to demonstrate that cyclin A/CDK2 activity is not only required for cells to enter mitosis, but also to progress through mitosis until the middle of prophase. If cyclin A/CDK2 is inhibited in early or mid prophase this causes the cell to return to interphase. However, cyclin A/CDK2 becomes dispensable once cyclin B has translocated into the nucleus. Active cyclin A/CDK2 is able to accelerate entry into mitosis by at least 4 h, effectively eliminating G2 phase. However, if cyclin A/CDK2 is injected into cells that are still replicating their DNA, this has no effect on the timing of mitosis in >90% of the injected cells. This may be because the appropriate mitotic substrates are not present, or are inaccessible, in S phase cells. Alternatively, S phase cells may be able to inactivate the mitosis-promoting activity of excess cyclin A/CDK2. Thus, in addition to the inhibition of Cdc25 by Cds1 and Chk1 , the negative regulation of cyclin A/CDK2 may underlie the observation that S phase nuclei are able to delay the entry of G2 cells into mitosis . We have shown that any inactivation is not through degrading cyclin A, therefore, it could be through cyclin A/CDK being bound by an excess of CDK inhibitors such as p21 Cip1 , or p27 Kip1 or p57 Kip2 . However, recent results have shown that there is little free p21 Cip1 after S phase begins and p27 Kip1 levels fall substantially through ubiquitin-dependent proteolysis in late G1 phase . By analogy with the inactivation of cyclin B-CDK1 after DNA damage, cyclin A/CDK2 might be inactivated by phosphorylating CDK2 on threonine 14 (T14) and/or tyrosine 15 (Y15). Interestingly, when we microinjected cyclin A/CDK2 into cells that were arrested in G2 phase by UV irradiation, we found that the cells rounded up but then flattened out again without breaking down their nuclear envelopes (data not shown). Our interpretation of this is that cyclin A/CDK2 was not inactivated, but it could not drive cells further than prophase because cyclin B1/CDK1 was inhibited by the UV treatment. Our results may be relevant to the observations of Bunz et al. 1998 , who showed that in addition to phosphorylating and inactivating cyclin B/CDK1, cells require p21 Cip1 to maintain a G2 arrest after DNA damage. We would suggest that p21 Cip1 is required to inhibit the M-phase–promoting activity of cyclin A/CDK in G2 phase. Interestingly, when the p53 target gene GADD45 is overexpressed in primary human fibroblasts, they arrest as rounded up cells with condensed DNA and stain strongly for MPM-2 epitopes, but still have intact nuclear envelopes . This arrest point corresponds to that of cells with active cyclin A/CDK2 but inactive cyclin B1/CDK1. Thus, GADD45 appears to inhibit the step(s) between the initiation of prophase by cyclin A/CDK, and the activation of cyclin B1/CDK1 to initiate nuclear envelope breakdown. Indeed, GADD45 has been shown to bind and inactivate cyclin B/CDK complexes . In most cases, we observed that the early entry into mitosis promoted by excess cyclin A/CDK2 was accompanied by a delayed metaphase, but eventually the cells did divide properly and remained separate. In other cases, mostly in early G2 phase cells and in the small minority of S phase cells that could be forced into mitosis by cyclin A/CDK2, the chromosomes never fully condensed, probably because DNA replication was not complete. These cells had a prolonged mitosis and the daughter cells fused after division. The cell fusion may have been caused by incompletely replicated chromosomes, leading to the presence of chromatin in the region of the cytokinetic furrow, which has been shown to cause the furrow to regress and the daughter cells to fuse . Cyclin A/CDK2 appears to act upstream of cyclin B1/CDK1 activation. We were able to block the mitosis-promoting effects of cyclin A/CDK2 by expressing a phosphatase-inactive mutant of Cdc25B or, less effectively, by a catalytically inactive form of Cdc25C. (The partial block by the phosphatase-dead form of Cdc25C is likely to be because it is less effective at competing against the pool of the endogenous protein compared with the mutant Cdc25B, given that Cdc25C is a more stable protein than Cdc25B.) The dominant negative Cdc25s blocked cells in prophase, before nuclear envelope breakdown. Consistent with these results, overexpressing either form of Cdc25, or injecting active cyclin B1/CDK1, was able to overcome the block to mitosis imposed by inhibiting cyclin A/CDK2 with p21N. These results suggest that cyclin A/CDK activity is able to drive cells through most of prophase, but that cyclin B/CDK activity is required at the end of prophase for nuclear envelope breakdown. Our experiments have revealed that cyclin A/CDK activity, rather than cyclin B1/CDK1 activity, is likely to be responsible, directly or indirectly, for a number of changes in the cell as it enters mitosis. Cells that return to interphase when cyclin A/CDK is inactivated by p21N have already begun to condense their chromosomes, to disassemble their nucleoli and to round up. Rieder and Cole 1998 showed that a number of mitosis-specific MPM-2 antigens and phosphorylated histone H3 are also present in cells at this point in mitosis. However, once cyclin B1/CDK1 has translocated into the nucleus, inhibiting cyclin A cannot force cells back into interphase, and very soon after this (∼10 min later) the cells undergo NEBD. Moreover, NEBD marks the point at which cyclin A begins to be degraded (den Elzen, N., and J. Pines, manuscript in preparation). Thus, we can view the entry into (mammalian) mitosis as a two stage event; cyclin A–associated kinase is required for cells to progress into and through prophase, i.e., to condense chromosomes, for the disassembly of the nucleoli, and for the activation and or translocation of cyclin B/CDK1. Once cyclin B1/CDK1 has been activated it is required for progress into metaphase, i.e., for completing chromosome condensation, for nuclear envelope breakdown and completing formation of the mitotic spindle. We note that the centrosomes begin to separate just before cyclin B1 enters the nucleus, making it possible for either cyclin A or cyclin B1 to initiate centrosome separation, although our observation that p21N does not block cells from completing mitosis after the nucleolus disappears suggests that cyclin A/CDK activity is not required to form the spindle itself. Nevertheless, determining the precise time at which cyclin B1/CDK1 is activated, i.e., before or after translocation to the nucleus, is critical for a proper understanding of the irreversible commitment to mitosis. In this respect, studies on maturing starfish oocytes have shown that cyclin B-associated histone H1 kinase activity appears ∼10 min before cyclin B1 enters the nucleus at meiosis I . It will also be interesting to determine whether there is another cyclin, perhaps a mammalian cyclin B3, that is subsequently required for some of the events in anaphase and/or telophase. Our studies also suggest that cyclin A/CDK could be the target of the DNA damage checkpoint in prophase. Rieder and Cole 1998 noted that prophase PtK 1 cells would return to interphase if they were damaged >30 min before NEBD. In our experiments, p21N causes PtK 1 cells to return to mitosis if they were >10–20 min before NEBD. The difference in timing may reflect the time needed to transduce the signal from damaged DNA in Rieder and Cole's studies, compared with our experiments in which we directly introduce an effector molecule. Should a CKI inhibitor be the natural effector molecule we would predict that p21 Cip1−/− (or p21 Cip1−/− /p27 Kip1−/− ) cells will have a defective prophase checkpoint. | Study | biomedical | en | 0.999995 |
10525537 | Dynactin isolated from bovine brain and purified to 95% homogeneity as previously described was used in all experiments. 20 μg of dynactin was combined with 144 μg of recombinant human dynamitin in 0.5 ml disruption buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA plus protease inhibitors) and incubated for 30 min on ice. Control dynactin samples that did not contain added dynamitin were incubated in parallel. Samples were diluted to 1.5 ml and the proteins were sedimented into 5–20% sucrose gradients at 35,000 rpm in an SW41 rotor for ∼17 h. 1-ml fractions were collected and the proteins were chloroform/methanol precipitated and subjected to SDS-PAGE followed by Coomassie blue staining (80% of the sample) or immunoblotting (20% of the sample). For quantitation of the stoichiometry of subunits released by dynamitin, 200 μg of dynactin was disrupted with 1.4 mg dynamitin as described above. Sucrose gradient fractions 6 and 7, which contained the peak of p150 Glued and p24, were combined and resedimented into a second 5–20% sucrose gradient. Fractions 6 and 7 from the second gradient were combined, precipitated, and subjected to SDS-PAGE followed by Coomassie blue staining. Shoulder/sidearm subunit stoichiometry was determined by densitometry (see below). 5 mg of bovine dynactin was disrupted by adding freshly prepared 6 M KI in H 2 O, to yield a final concentration of 0.7 M. The sample was incubated on ice for 25 min, and then chromatographed on a Superose12 (Pharmacia LKB Biotechnology Inc.) gel filtration column preequilibrated in column buffer (2 mM Tris-Cl, pH 8, 0.2 mM ATP, 0.2 mM CaCl 2 , 0.5 mM DTT) containing 0.7 M KI. Individual column fractions (0.5 ml) were aliquoted and analyzed by SDS-PAGE to identify fractions of interest. The column corresponds to a 500-μl sample run at 0.5 ml/min, but dynactin subcomplexes were no better resolved when the column was run at closer to ideal conditions (e.g., 50-μl sample load run at 0.2 ml/min). To further purify shoulder/sidearm, shoulder, and pointed-end complexes, the relevant protein fractions were pooled, and then dialyzed briefly (30–60 min × 2,000 vol) in column buffer without KI. 1 ml of the different dialyzed pools was then loaded onto separate 11-ml sucrose gradients (5–20% sucrose in column buffer) and centrifuged at 34,000 rpm in an SW-41 rotor for 13.5 h. Sedimentation standards were run in parallel. 1-ml fractions were collected from the bottom of each tube and analyzed by SDS-PAGE. The pellet was resuspended in 1 ml of 1× sample buffer just before analysis. In some cases, fractions containing the peak of the protein of interest were reanalyzed by Superose12 chromatography in column buffer without KI or pooled and further fractionated by MonoS chromatography (pointed-end complex). The MonoS column was eluted with a linear gradient of 0–500 mM KCl over 20-column volumes. Efforts to improve the resolution of pointed-end complex subunits were unsuccessful; these proteins eluted as a very broad peak under all conditions. Samples (2.5 μg protein in 0.5 ml column buffer) were adsorbed to freshly cleaved mica, rapidly pressure frozen, freeze fractured, and deep etched as previously described . Images of fields of molecules were made on conventional EM negatives; then molecules of interest were captured from projections of the negatives using a digital camera. Final galleries were assembled using Adobe Photoshop ® , but the images were not digitally image processed except to add pseudocolor. Monoclonal antibodies to p150 Glued , Arp1, p62, and actin were previously described (anti–p150 Glued : mAb 150B ; anti–Arp1: mAb 45A; anti–p62: mAb 62B ; anti–actin mAb: C4 . PAGE was carried out as described . Immunoblotting was performed as described ; appropriate alkaline phosphatase-conjugated secondary antibodies were detected by chemiluminescence (Tropix). For densitometry, gels were stained with Coomassie brilliant blue R-250, scanned on a flat-bed scanner (ScanMaker III; Microtek) and compared with a BSA standard dilution series using National Institutes of Health Image software. Molar ratios were determined based on cloned subunit M r s. Peptide fingerprints were determined by in-gel proteolysis of gel-purified proteins, as described . Dynactin subunits were separated by SDS-PAGE (12.5% acrylamide) and Coomassie stained at room temperature. For sequencing at EMBL (p62 and p27), stained gels were sent intact. For sequencing at Harvard University Microchemistry Facility (p25 and Arp11), gels were briefly destained in 10% acetic acid and individual bands were excised, washed twice in 50% acetonitrile, and frozen at −80°C. At both facilities, the proteins were subjected to trypsin digestion. p62 and p27 tryptic fragments were isolated by HPLC and their NH 2 -terminal sequences determined by Edman degradation. Arp11 and p25 sequences were obtained by in-line, tandem liquid chromatography and mass spectrometry, yielding multiple peptide sequences from a single digest. A λ ZAP II rat brain library (Stratagene Inc.) was screened using mAb 62B . Immunopositive lambda ZAP clones were plaque purified and cDNAs were recovered in the pBluescript KS II+ vector by induction with the R408 helper phage (Stratagene Inc.). Rapid amplification of cDNA ends (RACE)–PCR was used to obtain the putative ATG start codon of the cDNA. A gene-specific primer, 62.05: 5′-ATGCACCAGCTCGTGGTCGCTGAAGCC-3′ was used to prime the synthesis of first-strand cDNA using poly A+ mRNA from adult rat brains. The reverse transcription was done at 42°C with Superscript reverse transcriptase (Life Technologies, Inc.). After dA tailing, the PCR reactions were performed essentially as described . A primary PCR reaction was performed with the gene-specific primer, 62.03; 5′-TCCACTACATGAATAGTGTGTTGC-3′, and a primer matching the synthesized dA tail; 5′ AMP, 5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3′. The PCR products from the primary reaction were reamplified with a nested gene-specific primer, 62.06; 5′-CAAAGGCATGTAATTTCTTCGC-3′ and 5′ AMP. All PCR reactions were 30 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 2 min using AmpliTaq (Perkin-Elmer Cetus Corp.). All PCR products were gel purified and ligated into the pCRII TA cloning vector (Invitrogen Corp.). The deduced amino acid sequence was verified by comparison with sequences obtained from tryptic peptides prepared from bovine brain p62 . cDNAs were obtained by searching the EST database (NCBI) using peptide sequences obtained from the native bovine proteins. Full-length mouse EST sequences containing the required in-frame stops upstream of the initiator methionines and polyadenlylation sites were obtained from the American Type Culture Collection. Mouse p25 and p27 were encoded by single ESTs . pEGFP-p25 was constructed from AA869597 BamHI/HindIII and pEGFP-C2 BglII/HindIII. pEGFP-p27 was assembled from an EcoRI fragment containing the rat p27 open reading frame (clone 27.004) and pEGFP-C3 EcoRI. The Arp11 open reading frame was assembled from nonredundant fragments of three overlapping ESTs in three separate constructs (C, B, and A). Construct A contains the EcoRI/HindIII fragment of AA475054 in pEGFP-C2 HindIII/EcoRI. The EcoRI fragment of AA272131 was cloned into the EcoRI site of construct A to yield construct B (correct orientation was confirmed by restriction digest). Construct C (Arp11 ORF reverse) was finally assembled by ligating construct B digested with XhoI and BstXI with the SalI/BstXI fragment of AA109989. Bacterial strains were obtained from Genome Systems and large-scale plasmid preparations were CsCl-gradient purified before digests and subcloning. After cloning into the pEGFP vectors, plasmid inserts were completely sequenced from the EGFP-C primer (Clontech) and internal primers designed by the Johns Hopkins DNA Facility (Baltimore, MD). Sequences were visually confirmed from Editview 1.0 tracings of the sequencing gel output. The sequences of all three ESTs reported in the database were found to be correct . Overexpression of the dynactin subunit, dynamitin (p50), in cultured cells induces release of p150 Glued , but not p62, from the dynactin Arp1 minifilament . This effect can be mimicked in vitro by adding an excess of recombinant dynamitin to a Xenopus egg cell extract or partially purified dynactin preparation . Because all these studies were performed in complex protein mixtures, it was not clear whether dynamitin alone was sufficient to disrupt dynactin or whether additional cellular factors were required. To explore this question further, and to characterize in more detail the dynactin components that were released by dynamitin, we mixed highly purified bovine brain dynactin with a 30-fold excess of purified, recombinant human dynamitin . Sedimentation of the mixture into a sucrose density gradient revealed two major peaks of protein, one at ≈17S and one at ≈9S . The subunit compositions of both pools were evaluated by SDS-PAGE staining and immunoblot analysis with Abs to p150 Glued and conventional actin . The 19S pool contained p62 and Arp1, as previously described , as well as conventional actin, the barbed-end actin-capping protein, capZ, and dynamitin, all in concentrations similar to the undisrupted control sample. That p62, Arp1, actin, and capZ subunits remained tightly associated in a complex that sedimented ≈19S suggested that excess dynamitin did not significantly perturb the structure of the Arp1 minifilament. Quantitative densitometry revealed the Arp1: dynamitin ratio to be similar in intact dynactin and the ≈19S complex produced by dynamitin treatment (1.97 ± 0.14:1 in intact dynactin vs. 1.80 ± 0.16:1 in the 19S complex). This finding suggests that dynamitin may contribute to the stability of the Arp1 minifilament. Dynamitin treatment of dynactin released a 9S protein complex containing p150 Glued and p24 , as expected from previous studies . This material also appeared to contain dynamitin. However, the fractions most enriched in p150 Glued and p24 were not well resolved from the bulk of free recombinant dynamitin , making it impossible to estimate relative stoichiometries. To further characterize the sidearm structure released by dynamitin, 9S fractions were pooled, dialyzed, and sedimented into a second 5–20% sucrose gradient. p150 Glued , p24, and some dynamitin cosedimented at ≈9S, while the residual free dynamitin sedimented at ≈7S . Quantitative densitometry revealed the subunit stoichiometry of the 9S pool to be 2:4:2 p150 Glued :dynamitin:p24, which is the same as in intact dynactin . This result suggests that exogenous dynamitin effects disruption by introducing four additional dynamitin monomers at the Arp1/p150 Glued junction. This generates an Arp1 minifilament and a complex of p150 Glued and p24 subunits, both of which have four associated dynamitin subunits. Dynactin's Arp1 minifilament can be depolymerized by treatment with the chaotropic salt, KI . Gel filtration chromatography in the presence of 0.7 M KI yields primarily monomeric Arp1 and actin , but other dynactin subunits elute earlier from the sizing column, suggesting they might be organized into higher order complexes . Although the elution profile was complex, five overlapping peaks of protein were obvious. In highly concentrated samples, small amounts of Arp1 could be seen across the entire column profile, suggesting that some Arp1 might still be assembled into small multimers or associated with other dynactin subunits. Peak A contained p150 Glued , dynamitin, and p24 and eluted with an approximate Stokes' radius of 10.7 nm. Peak B, which contained dynamitin and p24, but little or no p150 Glued , eluted broadly from the column (mean Stokes' radius ≈6 nm). Peak C was enriched for the p62 and p27 subunits and also contained a doublet of proteins at ≈43 kD, the larger of which comigrated with Arp1, and a protein of ≈25 kD that electrophoresed slightly slower than p24. Peak C eluted from the column with a Stokes' radius of 4.7 nm. Peak D was highly enriched for capZ. Arp1 was most concentrated in peak E, which eluted with the expected Stokes' radius of monomers. The complex of p150 Glued , dynamitin, and p24 that was liberated from dynactin by KI appeared similar in composition to the p150/dynamitin/p24 complex released by excess soluble dynamitin. However, KI treatment also appeared to yield a second, distinct subcomplex containing dynamitin and p24 subunits (peak B). To gain a better understanding of the relationship between these three protein complexes, peaks A and B were dialyzed to remove KI, and then subjected to velocity sedimentation into a sucrose gradient. The three polypeptides present in peak A (p150 Glued , dynamitin, and p24) cosedimented at ≈9S , much like the p150/dynamitin/p24 complex released by dynamitin. The small amount of Arp1 that was sometimes present in peak A did not sediment at 9S, but instead spread between 11S and >20S, demonstrating that Arp1 is not an integral component or necessary for the stability of this assembly. Densitometric analysis of Coomassie blue–stained gels suggested that each 9S complex contained p150 Glued , dynamitin, and p24 in a ratio of 2:2:1 ( Table ). This stoichiometry, together with the predicted molecular weight of each subunit from the cloned cDNAs, predicts a subcomplex of 401.5 kD. In good agreement with this estimate, a subcomplex of 396.6 kD was predicted from the Svedberg equation solved with sedimentation coefficient and Stokes' radius values ( Table ). A parallel analysis of peak B revealed that dynamitin and p24 cosedimented at ≈5S. As was seen for peak A, the Arp1 in peak B sedimented cleanly away from the other polypeptides. The stoichiometric ratio of dynamitin to p24 in the 5S pool was estimated by densitometry to be 2:1 ( Table ). Subunit molecular weights deduced from cloned cDNAs predict a complex mass of 110.9 kD, in good agreement with the 110.8 kD mass predicted from hydrodynamic measurements. Together, the complexes in peaks A and B sum to yield p150 Glued , dynamitin, and p24 subunits in a ratio of 2:4:2, which is the same as in native dynactin. The biochemical composition and hydrodynamic properties of the p150 Glued /dynamitin/p24 assembly isolated by KI treatment strongly suggested that it corresponded to the flexible p150 Glued sidearm that projects from the dynactin Arp1 filament . To explore this possibility further, platinum replicas of quick-frozen, deep-etched 9S complex were visualized by EM . The majority of molecules were elongated (>20 nm) and many contained a pair of globular heads similar in size and shape to those seen at the distal end of the dynactin sidearm. The structures were kinked in the middle to varying degrees, suggesting that, like the dynactin sidearm, this structure is flexible. Because the p150 Glued /dynamitin/p24 assembly is biochemically and ultrastructurally similar to the projecting sidearm and shoulder domains of the intact dynactin molecule, we will refer to this protein complex as the dynactin shoulder/sidearm. The remarkably similar ultrastructures of the projecting sidearm seen for intact dynactin and the isolated shoulder/sidearm subcomplex raises the question of where the excess dynamitin/p24 present in peak B might be accommodated within the dynactin structure. Mass arguments, plus the fact that free dynamitin can dissociate the p150 Glued sidearm from the Arp1 filament, suggest that dynamitin is contained within the highly flexible, elastic shoulder that tethers the filamentous p150 Glued sidearm to the Arp1 minifilament. Previous attempts to label intact dynactin with antibodies to dynamitin were unsuccessful (Schafer, D.A., J.E. Heuser, and T.A. Schroer, unpublished data) and appropriate p24 antibodies are not available, so we could not address this question by direct labeling methods. Instead, we imaged isolated dynamitin/p24 heterotrimers as described above . These molecules appeared as flexible, elongated structures that ranged in length from 11 to 30 nm and were occasionally seen to associate with each other end-to-end. The flexibility and apparent elasticity of these structures is consistent with a location within the dynactin shoulder. We will refer to the dynamitin/p24 heterotrimer as shoulder. KI disruption yielded two other protein peaks whose components behaved as protein complexes. Fig. 2 C, peak D, was most enriched in the capZ α/β heterodimer and was not pursued further. Peak C, in contrast, was enriched in p62, p27, and two previously unidentified dynactin subunits of 43 and 25 kD. This material eluted from the sizing column with an apparent Stokes' radius of 4.7 nm ( Table ). These behaviors predict a globular protein of M r 135,000 D. However, peak C also contained some capZ, so we were unsure as to whether the four predominant components (p62, p27, plus 43- and 25-kD species) comprised a single protein complex or they might be organized into multiple, independent protein assemblies. To explore this possibility further, the proteins in peak C were sedimented into a sucrose gradient . CapZ sedimented at ≈5S, consistent with previous work , while p62, p27, and the 43- and 25-kD proteins cosedimented at ≈7S. To further purify the 7S complex, it was subjected to ion exchange chromatography on a MonoS column . p62 and the three associated polypeptides bound and coeluted in a broad peak, while other dynactin subunits (capZ, dynamitin, and p24) flowed through the column. The approximate stoichiometry of the p62, p27, and 43- and 25-kD polypeptides was 1:1:1:1 ( Table ), as estimated by densitometry of Coomassie blue–stained gels. The sample was then subjected to ultrastructural analysis . Round particles were observed with an approximate diameter of 8 nm, in agreement with our gel filtration size estimates ( Table ). The particles appeared to have a depression in the center and were reminiscent of the Arp2/3 complex , an important regulator of actin assembly that binds F-actin pointed ends . Our previous ultrastructural analysis suggested that p62 is associated with the pointed end of the Arp1 filament . This implies that the p62, p27, and the 43- and 25-kD assemblies are associated with the Arp1 filament pointed end. We will refer to this heterotetrameric protein complex as pointed-end complex. Though several dynactin subunits have been characterized at the amino acid sequence level , the four components of the pointed-end complex have not. To complete the molecular characterization of dynactin, we cloned all four remaining subunits. A full-length p62 clone was isolated from a rat brain expression library using mAb 62B . The predicted protein has an M r of 53,086 and pI of 7.4. Inspection of the sequence revealed a RING-finger–like motif and a series of predicted α helices with alternating acidic and basic character. Database searches indicate that p62 is homologous along its length with RO-2 (ropy-2), a Neurospora gene whose protein produce is also predicted to contain a metal binding motif . Analysis of ropy mutants reveals defects in dynein-dependent nuclear movement into growing hyphae. Since other RO genes encode subunits of dynein or dynactin , it seems likely that p62 is the vertebrate RO-2 homologue. In addition to p62, the pointed-end complex contains subunits of ≈25 and ≈43 kD that had not been observed previously. However, close inspection revealed a minor polypeptide migrating just above p24 in undisrupted bovine brain dynactin . Immunoblotting showed the ≈25-kD species to be immunologically distinct from p24 (data not shown). In intact dynactin, the novel 43-kD species is obscured by Arp1, the major component of dynactin (one third by mass). The 43-kD polypeptide in purified pointed-end complex was determined to be distinct from both Arp1 and actin by peptide fingerprinting and immunoblotting (data not shown). The identification of the ≈25- and 43-kD species as novel dynactin components was verified by peptide microsequencing and cDNA cloning (see below). To obtain clones for the 25- and 43-kD polypeptides, for which antibodies were not available, we screened genomic databases with tryptic peptide sequences from each protein. This yielded multiple EST clones that were assembled, as needed, to yield full-length sequences . Human p27 was previously identified by the Japanese genome sequencing project as a ubiquitously expressed gene. The mouse p27 gene encodes a protein of 20.6 kD with an isoelectric point of 6.0. p27 orthologs from fly and worm were identified using the predicted rat p27 amino acid sequence. Though the p27 gene appears to be evolutionarily conserved , no novel features are revealed in an alignment of the sequences. The 25-kD subunit is another novel protein with a predicted M r of 20.1 kD and pI of 7.9. Orthologs were identified in fly and worm; both had basic isoelectric points. Although we did not detect chick embryo brain dynactin p25 on two-dimensional (2-D) gels , presumably due to its alkaline pI, a spot corresponding to bovine p25 can be seen at a pI of ≈8.0 by silver staining (data not shown). p25 is predicted to fold into a β structure. The sequence of the 43-kD protein revealed it to be a novel Arp. A phylogenetic tree including representative actin and Arp sequences shows this new Arp to be less similar to actin than most other Arps. In keeping with existing nomenclature guidelines , we suggest the name Arp11. Alignment of the Arp11 sequence with actin reveals overall conservation of the “actin fold,” a primordial core structure that contains nucleotide and metal binding elements . As in other actin-related proteins, sequence differences, when they are observed, are largely limited to surface residues. In addition to scattered amino acid substitutions, the alignment reveals several changes in surface loops that play key roles in actin–actin and actin–protein interactions . The Arp11 barbed-end face is generally well conserved and is not predicted to contain large insertions that would prevent interactions with the pointed end of an Arp1 (or actin) filament. The NH 2 and COOH termini (found at the barbed-end face of subdomain 1) are only slightly longer (5–20 amino acids) than in actin, which should interfere only slightly, if at all, with subunit–subunit interactions. In contrast, the pointed-end face of Arp11 is predicted to be dramatically different from that of actin . The most conspicuous difference is a large deletion (actin residues 38–57) that eliminates an entire surface loop from subdomain 2. Subdomains 3 and 4 are predicted to contain insertions, some of which map within two important actin–actin interfaces inferred from the F-actin x-ray crystal structure . The predicted structure of Arp11 suggests it will not form filaments by itself and will only interact with filaments of Arp1, or conventional actin, at their pointed ends. The work presented here provides significant new insight into dynactin composition and subunit organization, and suggests novel mechanisms for Arp1 filament length regulation and dynactin-membrane binding. The availability of purified dynactin shoulder/sidearm, shoulder, and pointed-end complexes will facilitate biochemical and biophysical analysis of dynein–dynactin and dynactin–cargo interactions. The four new dynactin subunit cDNAs we have isolated will be useful tools in studies of the assembly pathway of dynactin in vitro and should permit a comprehensive analysis of dynactin function in cells. We are reasonably confident that the present effort represents the complete molecular characterization of dynactin subunits. 2-D gel analysis of isolated bovine brain dynactin reveals all of the proteins discovered thus far, including the novel subunits p25 and Arp11 (data not shown). Dynactin's predicted mass, based on the stoichiometries of its 11 different gene products and their predicted molecular weights, is 1.03 MD. This value agrees well with values (1.10 and 1.11 MD) predicted by hydrodynamic measurements (this study) and previous STEM analysis . Dynactin's complicated, highly asymmetric structure suggests a multiplicity of functions. To serve its many roles in cells, dynactin must be able to interact with dynein, endomembranes, and other cytoplasmic particles, centrosomes, chromosomes, and the plasma membrane. While the present analysis does not provide any new information about these binding interactions specifically, it does allow us to better define the dynactin subdomains thought to underlie them. A large body of evidence indicates that dynein binds dynactin via the projecting p150 Glued sidearm. p150 Glued was first implicated as a dynein-binding subunit in blot overlay and affinity chromatography studies using recombinant proteins . We find that isolated dynactin shoulder/sidearm inhibits dynein-based microtubule aster formation in vitro, suggesting that this domain can act as a competitive inhibitor of the dynein–dynactin interaction. Moreover, overexpression of the dynein-binding domain of p150 Glued profoundly interferes with dynein-based motility in cells , reinforcing the notion that this subunit interacts directly with dynein in vivo. Recombinant or overexpressed p150 Glued binds cellular microtubules via an NH 2 -terminal microtubule binding site that maps to the globular heads at the distal end of the dynactin sidearm. Native dynactin and isolated shoulder/sidearm subcomplex also bind microtubules in vitro, although the binding is transient. The dynamic binding of dynactin to microtubules allows dynein to remain associated with the microtubule track throughout the dynein ATPase cycle, which is believed to enhance motor processivity (King, S.J., and T.A. Schroer, manuscript submitted for publication). Together, dynactin's shoulder/sidearm and shoulder complexes form a flexible, elastic structure that can bind the Arp1 minifilament on one end, the microtubule substrate at the other, and dynein in between. The flexibility of the junction with the minifilament allows p150 Glued to undergo a wide range of movement relative to the Arp1 backbone. This may facilitate passage of bulky cargoes through cytoplasm. The flexibility of this structure may also facilitate intercalation by exogenous dynamitin. Isolated shoulder/sidearm and shoulder complexes both exhibit a range of morphologies, much like the pleomorphic shoulder in intact dynactin. Two dynamitins in isolated shoulder complex are bound to one subunit of p24, suggesting that this may be a fundamental assembly unit of the dynactin molecule. However, dynamitin disruption leaves four dynamitin subunits on the Arp1 filament and four subunits on the p150 Glued /p24 assembly, suggesting that dynamitin may be most stable in tetramer form. That KI dissociates the dynamitin tetramer into two dimers suggests a “fault line” at the dimer–dimer interface. Our disruption studies have also provided new insights into subunit–subunit interactions in the Arp1 minifilament. Dynamitin treatment appears to leave this structure intact. CapZ remains associated, indicating that the barbed end is still capped. The pointed-end complex is also apparently retained. p62 is a component of the 19S complex and Arp11, p27, and p25 can be discerned by 2-D gel analysis (K.A. Melkonian, unpublished observations). The 19S structure also contains dynamitin, suggesting that this protein may contribute to Arp1 minifilament stability. KI disruption, in contrast, depolymerizes the Arp1 polymer, as previously seen for conventional F-actin , but leaves the capZ α/β heterodimer intact, as expected . Our KI disruption studies provide no new information about the location of the single actin monomer within the Arp1 minifilament, as actin elutes as monomer under these conditions . However, association with the conventional actin-capping protein, capZ is a likely possibility. The predicted structure of Arp11, based on alignment with conventional actin and other Arps, suggests it has the capacity to bind Arp1 directly. Earlier attempts to demonstrate binding between p62 and Arp1 were unsuccessful (S.R. Gill, unpublished observations), as would be expected if this interaction were mediated by Arp11. The positions of insertions and deletions (relative to actin) within the Arp11 sequence predict it will associate with the Arp1 filament exclusively at its pointed end. Dynactin Arp1 minifilaments are uniform in length and they do not readily depolymerize, self-associate, or nucleate assembly of conventional actin (J.B. Bingham, unpublished observations), suggesting that they are capped at both ends. In contrast, the filaments formed by purified Arp1 in vitro are heterogeneous in length (mean length, 52 nm) and can anneal end-to-end . The conventional actin-capping protein α/β 2 heterodimer associated with dynactin is expected to prevent Arp1 growth and annealing at the Arp1 barbed end. Pointed-end complex may play a similar role at the opposite end of the filament and may also help template Arp1 assembly to ensure the filaments grow to a uniform size. The association of p27 and p25 with p62 and Arp11 in the pointed-end complex appears to be somewhat labile. During KI disruption, p62 and Arp11 always copurify, but p27 and p25 sometimes copurify with monomeric Arp1 and actin (J.B. Bingham, Ph.D. thesis). When the resulting Arp1/actin/p27/p25 sample is further fractionated by MonoQ chromatography , p25 and p27 again copurify (J.B. Bingham, Ph.D. thesis), suggesting they are tightly associated with one another. Thus, within the pointed-end complex, p62 and Arp11 appear to be binding partners, as do p27 and p25. The primary sequences of the remaining three pointed-end complex subunits provide fertile ground for speculation about the assembly and function of the Arp1 minifilament, dynactin's proposed cargo-binding domain. The mechanism of dynactin-membrane binding remains obscure, though recent work suggests that Arp1 may interact with Golgi-associated spectrin . However, the predicted biochemical properties of pointed-end complex subunits suggest that this structure may also participate in membrane binding. The conserved alkaline pIs of p62, Arp11, and p25 suggest that any or all may interact electrostatically with negatively charged membrane lipids or other acidic cargoes such as lipid droplets or viral nucleocapsids. p25 is predicted to fold into a β structure that may contain a lipid-binding, plekstrin homology domain. Thioacylation of p25 and/or p27 (both of which are cysteine rich) might further stabilize interactions between these subunits and membranes. Homologies of pointed-end complex subunits have been found in organisms as diverse as fly (all four subunits), worm (p62, p27, and p25), and Neurospora (p62 and Arp11), indicating that this structure is evolutionarily conserved. However, budding yeast, an organism that does not require cytoplasmic microtubules for endomembrane traffic, does not appear to contain pointed-end complex homologues. This suggests that yeast dynactin may not perform whatever function(s) this structure provides in other species and further supports a role in membrane interactions. But regardless of how dynactin binds membranes, it is important to keep in mind that dynein may have mechanisms for binding membranes that are independent of dynactin. Purified cytoplasmic dynein associates peripherally with purified synaptic vesicles and can bind purified phospholipid vesicles with similar affinity . The dynein light chain, TctexI, binds directly to the transmembrane protein rhodopsin . Thus the interaction of dynein and dynactin is likely to be complex and may involve multiple subunits of each protein. Previous work indicates that all dynactin subunits except conventional actin and actin-capping protein are found in dynactin only . We do not yet know if the same is true for Arp11, p27, or p25. It is intriguing that pointed-end complex ultrastructure so closely resembles the Arp 2/3 complex , a structure that plays an important role in events at conventional actin filament pointed ends . It will be interesting to determine whether pointed-end complex, or Arp11 alone, contributes to the dynamics of the conventional actin cytoskeleton. That Neurospora RO-2 mutants show defects in actin cytoskeletal organization suggests that this may indeed be the case. Dynactin is just one of a family of proteins proposed to mediate the interaction of membranes with microtubules. It shares a number of superficial similarities with CLIP-170, another protein in this class. CLIP-170 binds to and tracks with growing microtubule plus ends , where it may mediate static interactions between endosomes and microtubules . As in the case of the shoulder/sidearm subunit p150 Glued , microtubule binding occurs at the NH 2 terminus via a conserved binding site whose activity appears to be subject to complex physiological regulation. The CLIP-170 COOH terminus contains predicted metal-binding motifs that are proposed to participate in cargo binding. p62's predicted metal-binding, RING-finger–like motif may serve a similar function. In both dynactin and CLIP-170, microtubule and cargo binding activities are positioned at opposite ends of the molecule. Like CLIP-170, dynactin accumulates at microtubule plus ends . This association appears to depend on CLIP-170 , suggesting that CLIP-170 may recruit dynactin to microtubule plus ends to initiate dynein-based movement. How this process is coordinated and what other factors may contribute to cargo docking and motility will be an area of significant future work. | Study | biomedical | en | 0.999995 |
10525538 | Mitotic asters were assembled in HeLa cell lysates as previously described . In brief, synchronized cells were homogenized and a postnuclear supernatant was prepared. Endogenous microtubules were stabilized by addition of taxol. Purified shoulder/sidearm (see below) or intact dynactin was added to the extract at a concentration approximately equal to the endogenous dynactin concentration, as estimated from immunoblots for p150 Glued (D.A. Compton, unpublished observations). Purified bovine brain dynactin was prepared as described and shoulder/sidearm isolated as described . In brief, 10 mg of dynactin was dissociated by adding 0.7 M potassium iodide, incubated on ice for 30 min, and then dynactin subcomplexes and subunits were separated by gel filtration chromatography on a Superose12 column (Pharmacia LKB Biotechnology, Inc.). Fractions of interest were dialyzed, and then sedimented into a 5–20% sucrose gradient. Shoulder/sidearm complex purified by this method was cryoprotected by addition of 1.25 M sucrose, snap frozen in small aliquots, and stored at −80°C for later use. A full-length chicken p150 Glued cDNA was obtained by screening a λgt10 library (gift of B. Ranscht, Scripps Laboratories Inc.) with the original p150 Glued clone, p150A . The insert was subcloned into the EcoRI site of pGW1-CMV . Constructs encoding the predicted coiled-coil regions of p150 Glued were engineered using PCR from p150A . CC1 (amino acids 217–548) was made using the primers CGTGCCATGGAGGAAGAAAATCTGCGTTCC (upstream) and CCGGGATCCTTACTGCTGCTGCTTCTCTGC (downstream). CC2 was made using primers CGTGCCATGGCCGAGCTGCGGGCAGCTGC (upstream) and CCGGGATCCTTACCCCTCGATGGTCCGCTTGG (downstream). Both PCR products were ligated into pTA (Invitrogen Corp.), subcloned into the NcoI and BamHI sites of pET-3c (Novagen, Inc.), subcloned again into pVEX using XbaI and EcoRI, and then finally into pGW1-CMV using NdeI and BamHI. The mouse p24 gene was characterized by sequencing EST AA002440 completely on both strands. It contained a single conservative amino acid substitution (E 131 –Q 131 ) when compared with a previously published mouse p24 gene . p24-green fluorescent protein (GFP) 1 was engineered by subcloning the entire p24 cDNA into the EcoRI site of pEGFP-C2 (Clontech). Orientation was determined by diagnostic digests and the fusion open reading frame was confirmed by sequencing. Dynamitin-HA in pCB6 was a gift from C. Valetti . Dynamitin-GFP in pcDNA3 was a gift from E. Vaisberg (University of Colorado, Boulder, CO). In fixed cells, GFP-tagged proteins were detected by their intrinsic fluorescence; Abs were used on blots. p150 Glued : mAb 150.1 , mAb 150B , pAb UP502 (gift from E.L.F. Holzbaur, University of Pennsylvania, State College, PA). Arp1: mAb 45A , rabbit antibody to recombinant human Arp1 (gift from J. Lees-Miller, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). p62: mAb 62B . p24: affinity-purified rabbit antibody R5700 . Tubulin: α tubulin mAb DM1A (Sigma Chemical Co.), rabbit antibody white-wall Tyr , affinity-purified rabbit antibody against peptide KVEGEGEEEGEEY (gift from E. Karsenti, EMBL). γ Tubulin: mAb GTU 88 (Sigma Chemical Co.), rabbit antiserum pAb (Sigma Chemical Co.) against peptide EEFATEGTDRKDVFFYK. Pericentrin: rabbit antibody pAb 4b . Mannosidase II: rabbit antibody from K. Moremen (University of Georgia, Athens, GA). HA: anti–HA epitope mAb . β Galactosidase: mAb from Promega. GFP: pAb from Molecular Probes, Inc. FITC- and Texas red–conjugated horse anti–mouse and –rabbit (Vector Laboratories, Inc.) and Cy5-conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories Inc.) were used as secondary antibodies. Cos-7 and L cells were grown in DMEM (GIBCO-BRL, Life Technologies, Inc.), supplemented with 10% FCS (Summit Technologies). For transient transfections, cells were grown to 70–90% confluency, harvested with 0.05% trypsin-EDTA, and then 1–2 × 10 7 cells were resuspended in 0.5 ml OPTI-MEM (GIBCO-BRL) and electroporated with 10 μg DNA at 230–240 V using an electro cell manipulator 600 (BTX). Cells were seeded on 22-mm 2 coverslips (2 × 10 5 cells/coverslip) in six-well dishes and grown for 14–24 h before being processed for immunofluorescence. Transfection efficiencies of 60–80% (Cos7) or 20–50% (L) were routinely obtained. Cells were rinsed with D-PBS and then fixed in −20°C MeOH for 10 min. Coverslips were then blocked in TTBS (TBS, 0.1% Tween-20, and 2% BSA) incubated for 30 min in primary antibody, washed in TTBS (3 × 5 min), and incubated in secondary antibody for 15 min, all at room temperature. Samples were washed again and mounted on slides in 3:1 Mowiol 4–88 (Calbiochem Corp.): n -propyl gallate (Sigma Chemical Co.) in PBS plus 50% glycerol. For each overexpressed protein, at least 200 overexpressing cells on multiple coverslips were analyzed in two or more independent experiments. Overexpressed p150 Glued and CC1 were detected using mAb 150.1, which recognizes an epitope within CC1 and not the COOH terminus as reported earlier . mAb 150.1 does not react with mammalian p150 Glued . Overexpressed CC2 was detected using mAb 150B. Endogenous p150 Glued was detected with rabbit antibody UP502. Arp1 was detected with a pAb against human Arp1. Immunofluorescence microscopy was performed using an Axiovert 35 microscope (Carl Zeiss Inc.). Images were recorded on TMAX-400 film (Eastman-Kodak Co.), and digitized using a ScanMaker III scanner (Microtek). Additional images were recorded on a DeltaVision deconvolving microscope system (Applied Precision, Inc.). All images were imported into Adobe Photoshop ® v3.0 (Adobe Systems, Inc.) for contrast manipulation and figure assembly. Cells were transfected, seeded on coverslips, and grown 14–24 h as described above. Microtubules were depolymerized in 33 μM nocodazole (Sigma Chemical Co.) in DMEM for 30 min on ice, and then washed three times with room temperature DMEM and incubated at room temperature to allow regrowth. Coverslips were fixed at timed intervals in −20°C MeOH and processed for immunofluorescence as described above. Transfected cells were harvested, lysed, and sedimented as described in Echeverri et al. 1996 , except that 4 × 10 cm 2 dishes were used. Sucrose gradients (SW-50 rotor) were fractionated (400-μl fractions) and analyzed by immunoblotting on Immobilon-P membrane (Millipore Corp.). Blots were incubated with antibodies to dynactin subunits and the overexpressed protein, and then with alkaline phosphatase–conjugated goat anti–rabbit or –mouse IgG for detection using the Western-Light system (Tropix). Endogenous p150 Glued and Arp1 were detected with mAbs 150B and 45A. Overexpressed p150 Glued and CC1 were detected with mAb 150.1; CC2 was detected with mAb 150B. Cells overexpressing the dynactin subunit, dynamitin, show a wide variety of motility defects , all of which are thought to be due to the decoupling of dynactin's dynein- and cargo-binding functions. In these cells, the dynein-binding p150 Glued subunit released by excess dynamitin is assumed to continue to bind dynein. To explore this possibility, we used an assay for mitotic aster assembly to determine the effects of purified dynactin shoulder/sidearm on dynein activity in vitro. Aster formation requires dynein and dynactin function; asters do not form in extracts immunodepleted of either protein, and activity can be restored by readdition of purified dynein or dynactin. Dynactin, and a small amount of dynein, is incorporated into the asters . The shoulder/sidearm of dynactin was added to mitotic HeLa cell extracts before or after aster formation. When added at a concentration approximately equal to endogenous dynactin, shoulder/sidearm inhibited aster formation . Once asters were formed, however, excess shoulder/sidearm had no effect . Addition of equimolar dynactin did not inhibit aster formation under either condition. These findings support the hypothesis that free dynactin shoulder/sidearm can interact with dynein and prevent it from performing its normal functions. It also appears that the dynactin that incorporates into asters during assembly is adequate to maintain aster integrity, suggesting a relatively stable association with the aster core. We then performed a series of experiments to determine how excess shoulder/sidearm subunits might affect microtubule organization in living cells. In all this work, protein overexpression was driven by the cytomegalovirus promoter. We only analyzed cells that contained evenly distributed (i.e., soluble) recombinant proteins, and not those that contained large protein aggregates (seen in some cells overexpressing p24 or p62). We first determined the effects of chicken dynamitin overexpression on the interphase microtubule array. In a previous study , dynamitin was reported to have no effect on interphase microtubule organization in HeLa cells, which are an epithelial cell line that contains a broad microtubule organizing zone rather than a single, well-defined focus. Cos7 fibroblasts overexpressing dynamitin, in contrast, were reported to contain microtubules that were less well-focused than normal. We extended this observation by evaluating microtubule organization in Cos7 cells using immunofluorescence microscopy . Determination of the percentage of cells that contained normal or abnormal microtubule arrays revealed that most dynamitin overexpressing cells contained large numbers of microtubules, but that these were no longer organized into a tightly focused, radial array. Dynamitin overexpression causes release of dynactin shoulder/sidearm subunits that are hypothesized to competitively inhibit dynein-cargo binding. We reasoned that overexpression of just the dynein-binding subunit, p150 Glued , might mimic the effects of dynamitin. As previously reported for rat p150 Glued , overexpressed chicken p150 Glued bound microtubules along their length and, in some cells, induced microtubule bundling (data not shown). In addition, the overall organization of the microtubule cytoskeleton was perturbed and microtubules no longer appeared to radiate from a single perinuclear focus. The microtubule binding and bundling seen with overexpressed p150 Glued made it difficult to draw any clear conclusions about its effects on microtubule organization . We therefore engineered two p150 Glued expression vectors that lacked the NH 2 -terminal microtubule binding domain. Coiled-coil 1 (CC1; amino acids 217–548) is a 39,021-D fragment that corresponds to the central predicted coiled coil. This part of the protein binds dynein intermediate chain in vitro and is thus thought to be dynactin's dynein-binding domain. Within the dynactin molecule, coiled-coil 2 is thought to lie near the Arp1 filament , where it may bind Arp1 directly . Circular dichroism analysis revealed CC1 and CC2 to be α helices (data not shown), as predicted from their sequences. When overexpressed, neither CC1 nor CC2 bound microtubules, but overexpressing cells had disorganized, unfocused microtubule arrays similar to those seen previously . This suggested that the microtubule disorganization seen in cells overexpressing full-length p150 Glued was not simply due to its microtubule binding activity. Finally, we examined microtubule organization in cells overexpressing p24, the third shoulder/sidearm subunit, tagged with green fluorescent protein. Again, we saw disorganized microtubules and, in some cells, p24-GFP appeared to accumulate at centrosomes . Myc-tagged p24 had similar effects (data not shown), suggesting that the GFP tag did not affect function. Several controls were performed to verify the significance of our results. Nearly all (95%) cells present on the same coverslip that were not overexpressing the protein of interest had radially focused microtubules. Normal microtubule organization was also seen in cells overexpressing a control protein, β galactosidase (β-Gal). Cells overexpressing p62, a component of dynactin's Arp1 backbone had a slightly higher incidence of microtubule disorganization than controls, but significantly fewer cells were affected than with shoulder/sidearm subunit overexpression. We conclude that overexpression of dynactin shoulder/sidearm subunits specifically induces microtubule disorganization. Because overexpression of p150 Glued , CC1, CC2, or p24 all had similar effects on microtubule organization to dynamitin, we determined whether interphase cells showed other perturbations characteristic of the “dynamitin effect.” Dynamitin overexpression disrupts dynactin structure , presumably because dynamitin is the linker that binds shoulder/sidearm subunits to the Arp1 minifilament backbone. The disruptive effects of other shoulder/sidearm subunits on microtubule organization led us to ask whether any of these proteins also disrupted dynactin structure. To address this question, we determined whether or not dynactin remained a single complex that sedimented at ≈20S . Cells transfected with the different expression constructs were treated with detergent and the cell lysates were sedimented into sucrose gradients. Gradient fractions were then analyzed on immunoblots to determine the distribution of endogenous p150 Glued , p62, Arp1, and p24, as well as the overexpressed proteins . In samples prepared from cells overexpressing dynamitin, we observed two overlapping pools of p150 Glued and p24, one at ≈17–18S and one at ≈9S, as expected from previous studies . No other overexpressed dynactin subunit had a detectable effect on dynactin's sedimentation behavior. Most of the overexpressed proteins sedimented between 4–11S, the expected position of monomers or dimers, but a small portion of overexpressed p150 Glued and p24-GFP cosedimented at 20S with other dynactin subunits, suggesting that they were able to incorporate into dynactin. Apparently, overexpression of shoulder/sidearm components can disrupt microtubule organization without detectably altering dynactin structure. This suggests that the free subunits are acting independently of the whole molecule. Dynamitin overexpression causes cells to arrest in pseudoprometaphase owing to a variety of spindle defects . We therefore determined whether other shoulder/sidearm subunits had the same effect. As seen for dynamitin, most mitotic cells overexpressing CC1 had uni- or multipolar spindles (data not shown). Cells overexpressing p24-GFP or p24-myc died 20–24 h after transfection and mitotic cells were never observed, so we could not assess spindle morphology or mitotic progression. However, cells overexpressing CC2 were seen in all stages of mitosis and their spindles appeared normal (data not shown), indicating that mitosis was not affected. Another hallmark of dynamitin overexpression is disruption of the Golgi complex into small stacks dispersed throughout the cytoplasm . We therefore determined the extent of Golgi complex fragmentation in cells overexpressing shoulder/sidearm components . Mouse L cells transfected with the different expression constructs were stained with antibodies to the medial Golgi enzyme mannosidase II. Most cells overexpressing either p150 Glued or CC1 contained fragmented Golgi complexes similar to those seen in dynamitin overexpressing cells, while most cells overexpressing CC2, p24-GFP, or the control protein β-Gal contained Golgi complexes with the typical juxtanuclear localization and ribbon-like morphology. An intermediate number of cells overexpressing the dynactin backbone subunit, p62, had disrupted Golgi complexes. Full-length p150 Glued and CC1 disrupted microtubule and Golgi complex organization, but did so without detectably altering dynactin structure . Biochemical studies indicate that the NH 2 -terminal half of p150 Glued can bind dynein directly , which may explain our results. In living cells, overexpression of p150 Glued or CC1 might interfere with dynein-based motility via the same basic mechanism as dynamitin. Excess dynamitin causes release of shoulder/sidearm that is thought to bind dynein, while free p150 Glued and CC1 may bind dynein directly and compete for its interactions with intact dynactin. In both cases, the net effect would be that dynein can no longer interact with cargo. All three proteins would thus be expected to have similar effects on dynein-based motility. What this model does not explain, however, is how overexpressed CC2 and p24 interfere with dynein function, as they perturb microtubule organization but do not appear to have an effect on Golgi complex structure. To learn more about the underlying basis of the microtubule perturbations we saw, we examined centrosome structure and function in cells overexpressing dynactin shoulder/sidearm subunits. Cells were stained with antibodies to the centrosomal proteins γ tubulin or pericentrin. In the vast majority of untransfected cells or control-transfected cells expressing β-gal or p62, γ tubulin and pericentrin both localized to a single focus or paired foci near the nucleus. Pericentrin staining was not affected by overexpression of any dynactin subunit ( Table ). In contrast, γ tubulin localization was altered in about half the cells overexpressing dynactin shoulder/sidearm subunits . Multiple γ tubulin foci were present , in addition to a single perinuclear focus that also stained for pericentrin . Two patterns of γ tubulin foci were seen: individual foci scattered throughout the cell and clusters of foci near the nucleus. Cells overexpressing shoulder/sidearm subunits commonly had four or more foci (in addition to the parent centrosome), while controls contained at most two foci that were always perinuclear. As many as nine widely spread foci could be detected per cell, while up to 12 foci were seen per cluster. Scattered foci were more common than clusters (≈3:1). All shoulder/sidearm subunits had similar effects on γ tubulin localization. Dynactin itself is associated with centrosomes, both in vivo and in vitro . However, centrosomal localization requires intact cytoplasmic microtubules , suggesting that dynactin is not a bona fide centrosomal protein. Because shoulder/sidearm subunit overexpression affected γ tubulin distribution, it seemed possible that centrosomal dynactin localization might also be altered. To test this hypothesis, control and dynactin subunit overexpressing cells were stained with antibodies to Arp1, the major component of the dynactin backbone . Most control cells contained a single bright spot of Arp1 that colocalized with γ tubulin (data not shown). The same result was seen in cells overexpressing p24-GFP, CC2, or p62. In contrast, most cells overexpressing dynamitin, p150 Glued , or CC1 did not contain a detectable Arp1 focus. These are the same subunits whose overexpression correlates with Golgi complex dispersion and mitotic arrest. Overexpression of all shoulder/sidearm subunits had an effect on microtubule organization and γ tubulin localization, suggesting that the loss of microtubule focus might be correlated with centrosome integrity. Consistent with this, centrosomes in cells overexpressing dynamitin, p150 Glued or CC1 also appeared to lack dynactin, as judged by Arp1 staining. However, if microtubule disorganization is due to massive disruption of pericentriolar material, the two phenomena should correlate directly. This is not what we observed, since centrosomes in cells overexpressing CC2 and p24 still appeared to contain Arp1. To better characterize the centrosome-associated dynactin pool in these cells, they were stained for the shoulder/sidearm component p150 Glued . Most control cells contained a single centrosomal focus of p150 Glued , similar to what was seen for Arp1. Overexpressed subunits that caused a loss of Arp1 from centrosomes (i.e., dynamitin and CC1) also caused a loss of p150 Glued . Most cells overexpressing CC2 or p24-GFP also did not have a perinuclear focus of p150 Glued . Double labeling for Arp1 and p150 Glued revealed that most cells overexpressing p24-GFP had perinuclear Arp1 foci that were not associated with p150 Glued . Thus, overexpression of p24-GFP appears to selectively release p150 Glued from Arp1 at centrosomes. This occurs in the absence of a detectable effect on the bulk dynactin pool . Since centrosome organization was clearly altered by shoulder/sidearm subunit overexpression, we next examined effects on centrosome function. Many overexpressing cells contained ectopic γ tubulin foci, suggesting that noncentrosomal microtubule nucleation might at least partially account for the altered microtubule array seen in most cells. To test this hypothesis, we determined the pattern of microtubule regrowth after cold and nocodazole-induced depolymerization . After increasing intervals of regrowth (0 min to 6 h), cells were fixed and stained for α and γ tubulin. In untransfected control cells, single microtubule asters were seen at 5 min regrowth and, by 30 min, a robust, radial array had developed. By 1 h, the microtubule distribution appeared the same as at steady state , and remained unchanged for the rest of the experiment. Similar results were obtained in cells overexpressing β-Gal or the dynactin p62 subunit. Immediately after microtubule depolymerization, cells overexpressing dynamitin, CC1, CC2, or p24-GFP contained a single detectable γ tubulin focus rather than multiple spots. The focus was near the nucleus, stained for pericentrin (data not shown), and colocalized with the site of microtubule aster formation. This suggested it was the centrosome. Although this perinuclear structure could nucleate microtubules, a more careful analysis revealed that microtubule regrowth was not completely normal. Little if any microtubule regrowth was detected at 5 min and, at 10 min, only small asters were observed, suggesting that microtubule nucleation was delayed. However, growth continued steadily and at the end of 1 h, each cell had a well-developed, single radial microtubule array . Although we saw only a single aster during this time, multiple γ tubulin foci became apparent. These were first detected at 20 min of regrowth and became more abundant with time . Peripheral γ tubulin foci and perinuclear clusters were observed, although the latter were more prevalent. Cells that contained multiple γ tubulin foci appeared to contain only a single microtubule aster, suggesting that nucleation was not occurring at ectopic foci. This implies that the aberrant microtubule arrays seen at steady state were not the result of noncentrosomal nucleation. Although Fig. 8a and Fig. C , shows only the behavior of cells overexpressing dynamitin-GFP, similar results were obtained in cells overexpressing p24-GFP. We were surprised to find that the pattern of microtubule regrowth was relatively normal in these cells since, at steady state, microtubule and centrosomal protein distributions were so clearly perturbed. This result suggested that cells containing overexpressed shoulder/sidearm components still nucleated microtubules at the centrosome, but that the newly assembled microtubules were no longer retained at this site. To test this hypothesis, we examined microtubule distribution in cells at later times of regrowth . Disorganized, unfocused microtubules were detected in some cells at 2 h, and by 6 h the cells had returned to the steady state condition (60–80% abnormal). Analysis of the distribution of γ tubulin in cells overexpressing dynamitin-GFP revealed that the number of noncentrosomal γ tubulin foci also increased with time. At 20 min, most cells with multiple foci contained six foci or fewer but, by 3 h, as many as 12 foci were detected in some cells (data not shown). At all time points, both perinuclear clusters and widely spread foci were seen , suggesting that the two arose in parallel. The present study extends significantly our understanding of dynein and dynactin function in interphase cells and provides new insight into mechanisms of microtubule anchoring at centrosomes. Our findings suggest that dynein and dynactin play key roles in microtubule organization, centrosome integrity, and centrosome assembly. The use of multiple dynactin subunits and subunit fragments has allowed us to selectively explore the function of the dynein- and microtubule-binding dynactin subunit, p150 Glued . Our results lend strong support to the idea that dynein function requires binding to dynactin via p150 Glued . Our data also indicate that dynactin provides a previously undescribed microtubule anchoring function at centrosomes. The overexpressed proteins used in this study can be grouped into three classes based on the severity of the phenotype they elicit when overexpressed in cultured fibroblasts ( Table ). Dynamitin (class A) has the broadest range of effects, perturbing dynactin structure and centrosome integrity, and interfering with endomembrane motility, microtubule organization, and mitosis. Dynamitin overexpression is thought to act by disassembling the entire cellular pool of dynactin and leaving, in its place, decoupled dynein- and cargo-binding elements. Neither piece can function independently, leading to an inhibition of all dynein-based motile events. In addition to the previously reported effects of dynamitin overexpression on mitotic progression and membrane localization , we find that microtubule focusing and localization of pericentriolar components to centrosomes are perturbed in interphase cells. Full length p150 Glued and CC1 (class B) also affect a variety of functions but, unlike dynamitin, they do so without having a detectable effect on dynactin structure or stability. Class B agents most likely act by providing the cell with an excess of free dynein-binding polypeptides that competitively inhibit the interaction of dynein with intact dynactin. This inhibits all dynein-based motility in cells that still contain normal concentrations of dynactin. Antibodies such as mAb 70.1 or 74.1 can also be considered class B agents, as they bind dynein intermediate chain and interfere sterically with the dynein–dynactin interaction. The dynactin p24 subunit and CC2 (class C) are significantly more selective in their effects than are class A or B. They do not interfere with dynactin structure or stability and do not disrupt the organization or localization of the Golgi complex. In contrast to cells overexpressing dynamitin in which movement is abolished , cells overexpressing p24-GFP also show normal levels and patterns of endosome motility (Schroer, T.A., and N.J. Quintyne, unpublished observations). In addition, CC2 has no obvious effect on mitotic events. These findings indicate that class C agents do not interfere with cytosolic dynein activity, yet they have profound effects on interphase microtubule and centrosome organization. This suggests they interfere with dynactin function in a way that does not directly relate to its interaction with dynein. Overexpression of the dynactin p62 subunit affects only some dynein-dependent phenomena, and always to a lesser extent than the other subunits tested. Microtubule organization was altered in only a small population of cells. Centrosomal p150 Glued , Arp1, and γ tubulin localization appeared completely normal. In contrast, nearly 40% of cells (as compared with 10% of controls) contained disrupted Golgi complexes. These results suggest that p62, and possibly other components of dynactin's tetrameric pointed-end complex , may contribute to the interactions of dynactin with membranes, but not with centrosomes. Our understanding of dynein's contributions to microtubule organization in interphase are strongly influenced by what has been learned from in vitro studies in mitotic and meiotic systems . Dynein is essential for the formation and stability of asters or spindle poles. Its primary role is to transport microtubule minus ends and pole components to a common site, thereby driving pole formation and focusing. Ongoing dynein activity is also required for pole maintenance , suggesting that it helps keep microtubules in place. It is not clear that dynactin is required for microtubule–microtubule sliding events, although it may facilitate dynein–microtubule interactions. However, dynactin itself is actively transported to the pole, perhaps in a complex with other matrix components such as NuMA . Dynactin that has accumulated at poles is not affected by exogenous free shoulder/sidearm , suggesting that it is incorporated into a relatively stable structure. Dynein, in contrast, does not appear to be stably associated with spindle poles . This makes sense, since dynein acting at the pole will cause microtubules that are not well anchored to be ejected . Microtubule retention is proposed to involve microtubule-binding activities such as NuMA, as well as an opposing BimC family motor . Dynactin may also provide a microtubule binding function via its p150 Glued subunit. The perturbations of microtubule organization that occur in interphase fibroblasts are highly reminiscent of what is seen when dynein or dynactin function is inhibited in vitro. In both cases, microtubules are not focused into radial arrays, and dynactin subunits do not accumulate at microtubule minus ends. Whether microtubules are formed artificially or nucleated from centrosomes, our results suggest that two principles underlie microtubule organization throughout the cell cycle. First, to maintain a uni- or bipolar radial array, microtubules that are released from centrosomes must be retrieved, most likely by dynein. Second, in both interphase and mitosis, dynein appears to transport pericentriolar components to the centrosome. These include dynactin, γ tubulin, and perhaps pericentrin during interphase, and dynactin and NuMA during mitosis. The interactions between dynein, dynactin, microtubules, and centrosome components are complex. Dynactin is required for dynein to bind cargo, yet in some cases is cargo itself. Pericentriolar material serves as a docking site for dynactin, but dynein and dynactin are required for it to be recruited to centrosomes. Despite these interwoven relationships, our data allow some simple conclusions to be drawn. Disorganization of the interphase microtubule array is tightly correlated with the loss of p150 Glued from centrosomes ( Table ), suggesting that this dynactin subunit contributes a key microtubule anchoring function. That unfocused microtubules are seen in cells that contain centrosomal foci of γ tubulin or Arp1 indicates that neither protein is sufficient to maintain the radial microtubule array. We propose that dynein-mediated transport is required for targeting and delivery of dynactin to centrosomes. Dynein may also translocate free shoulder/sidearm, p150 Glued , or CC1 toward the centrosome, but no accumulation is observed, suggesting that the Arp1 minifilament is required to bind dynactin to pericentriolar material. We propose that centrosomal p150 Glued binds microtubules tightly, countering outward-directed pulling or ejection forces. The link between p150 Glued and Arp1 is therefore under constant tension, which may render centrosomal dynactin susceptible to disassembly when excess p24 or CC2 is present. CC2 may displace shoulder/sidearm by binding Arp1 directly . According to this model, class C agents should induce dynactin disassembly whenever the p150 Glued -Arp1 link is under tension, which might be expected to occur whenever cargo is moved. Yet overexpression of p24 or CC2 does not correlate with membrane localization or motility defects, and the bulk pool of cytosolic dynactin is not affected . The forces exerted on dynactin at centrosomes are likely to be significant since they involve large numbers of motor molecules operating on the entire microtubule cytoskeleton. This may not be true for endomembranes, particularly discrete tubulovesicular structures such as late endosomes and ER-to-Golgi transport complexes. Moreover, dynein can bind membranes via multiple dynactin-independent mechanisms. Binding can be mediated by transmembrane protein “receptors,” such as rhodopsin , as well as membrane lipids themselves . Multiple attachment mechanisms would reduce the net tension on the p150 Glued sidearm and prevent dynactin disassembly. Finally, individual dynactin molecules that are acting as dynein cargo are not expected to be under significant tension and would therefore remain intact. We find that defects in microtubule organization are much more prevalent than γ tubulin dispersion, suggesting that the two phenomena arise independently. Noncentrosomal aggregates of γ tubulin may form in two ways. First, γ tubulin fragmentation could be linked in some way to microtubule disorganization, as suggested by the widely spread, peripheral aggregates seen in cells overexpressing class A, B, or C agents. Outward-directed forces acting on microtubules may cause entire pieces of pericentriolar material to be torn away from the centrosome in conjunction with microtubules. Second, the noncentrosomal γ tubulin foci we observe might correspond to newly synthesized proteins that have accumulated in the periphery, perhaps in association with microtubules, but cannot be transported inward in the absence of dynein activity. The results of our microtubule regrowth experiments suggest that the latter can occur. Multiple γ tubulin foci are seen in cells that appear to contain single microtubule asters, indicating that γ tubulin dispersion can precede microtubule disorganization. While it seems very likely that pericentriolar components are transported to centrosomes in a microtubule- and dynein-dependent manner , the possibility that some γ tubulin foci arise by centrosome fragmentation cannot be rigorously excluded. Cells overexpressing class C agents share a number of superficial similarities with cultured epithelia. Such cells nucleate microtubules from centrosomes, and then release them to yield an unfocused array , and some epithelia contain noncentrosomal pools of γ tubulin . Cultured epithelial cells contain a single, perinuclear Golgi complex and are mitotically active. The radial microtubule array present in fibroblasts is obviously not required for normal cellular function, leading to the question of why such an arrangement exists. It is possible that the organization of microtubules into a single, radial array facilitates movement of the microtubule cytoskeleton as a whole. Precedent is seen in mitotic epithelial and embryonic cells, where the entire spindle rotates in a process that is thought to involve cortically anchored dynein and dynactin . A similar mechanism may underlie the movement of the entire interphase microtubule array in amoebae, migrating fibroblasts, macrophages, and T cells. Whatever the underlying reason for a radial array, our results suggest that the different microtubule organizations seen in fibroblasts, epithelia, and neurons may be profoundly influenced by the activities and subcellular localizations of dynein and dynactin. | Study | biomedical | en | 0.999996 |
10525539 | The S . cerevisiae strains used in these experiments are derivatives of S288C and are listed in Table . The dyn1::HIS3 , dyn1::URA3 , kar3::LEU2 , kip2::URA3 , kip3::kan and kip3-14 , cik1::LEU2 , and vik1::HIS3 alleles were described previously. The unmarked dyn1-Δ10 allele was created by transforming a dyn1::URA3 strain with a DNA fragment of DYN1 that spanned the URA3 insertion but harbored an internal deletion. Ura − were selected on media containing 5-fluoro-orotic acid (5-FOA) 1 (US Biological). The same strategy was used to create the unmarked kip2-Δ10 allele. The smy1-Δ10 unmarked deletion was created by PCR-targeted gene disruption . The PCR template DNA was URA3 flanked by a directly repeated sequence. Following transformation and selection for smy1::URA3 , we were able to select, using 5-FOA, for segregants that had looped out URA3 by homologous recombination between the repeated sequence. Multiple motor gene deletion mutants were constructed by standard genetic techniques. Strains with reduced spindle motor function often displayed evidence of aneuploidy (i.e., non-Mendelian segregation of genetic markers). Therefore, we routinely verified all seven microtubule motor gene alleles by PCR. For DYN1 and KIP2 , we were able to use a primer set that flanked the genes and yielded distinct PCR products from both the wild-type and deletion alleles. For the remaining five microtubule motor genes, two PCR reactions were used to test each allelic form. Both reactions used an upstream primer with a sequence present in both wild-type and deletion mutant alleles. This was paired with downstream primers that were specific for either the wild-type allele DNA in one reaction or the deletion DNA in the other. Rich (yeast extract, peptone, dextrose [YPD]) and minimal (synthetic dextrose [SD]) media were as described . Benomyl (DuPont) was added to solid YPD medium from a 10 mg/ml stock in DMSO. Nocadazole (Sigma Chemical Co.) was added to liquid YPD medium from a 3.3 mg/ml stock in DMSO. For G1 synchronization, α-factor (Bachem Bioscience) was added to 4 μg/ml to log-phase cells in liquid YPD, pH 4.0, and incubated at 26°C until >80% of cells were unbudded. For arrest in S phase, hydroxyurea (Sigma Chemical Co.) was added to 0.1 M to log-phase cells in liquid YPD, pH 5.8, and incubated at 26°C until >70% of cells were large-budded. Temperature-sensitive alleles of KAR3 were generated by a mutagenic PCR procedure . The entire KAR3 gene, including 412 bp of upstream sequence and 560 bp of downstream sequence, was amplified under mutagenic PCR conditions. We used Taq polymerase according to the supplier's instructions (Stratagene), except that Mn 2+ and Mg 2+ were added to final concentrations of 0.25 and 4.5 mM, respectively. The PCR products were concentrated using QIAquick spin columns (Qiagen). pMA1428 was digested with NruI and StuI to create a gapped construct in which all but the first 210 bp of KAR3 coding sequence was removed. The gapped pMA1428 construct was gel-purified and cotransformed with the concentrated PCR products into strain MAY4619. This strain is unable to survive loss of a plasmid carrying KAR3 and URA3 , and is therefore rendered sensitive to 5-FOA. Approximately 6,600 His + transformants were selected at 26°C and then replica plated to 5-FOA–containing media at 26 and 35°C. Six colonies were selected that were viable on 5-FOA at 26 but not 35°C. Plasmids were isolated from the six temperature-sensitive transformants and retransformed into MAY4619. After growth on 5-FOA at 26°C, four of the six retransformed strains displayed reduced growth on YPD at 35°C. These four plasmids were designated pMA1428 kar3-61 through pMA1428 kar3-64 . Of these, pMA1428 kar3-64 caused the greatest reduction of growth for kar3Δ kip3Δ strains at 35°C, and was chosen for study in all subsequent experiments. pMA1428 kar3-64 did not alter the temperature resistance of either wild-type or kip3Δ , dyn1Δ , and kar3Δ single mutant strains. Therefore, kar3-64 is a recessive and nonepistatic mutant. To stain for DNA, cells were pelleted out of liquid media, resuspended in 70% ethanol, and stored on ice for 30 min. The ethanol-fixed cells were washed once with water and then resuspended in 0.3 μg/ml 4,6-diamidino-2-phenylindole (DAPI) containing 1 mg/ml p -phenylenediamine to prevent fading (Sigma Chemical Co.). To stain for chitin-containing bud scars , cells were fixed as above, washed once with water, resuspended in 1 mg/ml calcofluor solution (Sigma Chemical Co.), and stored at room temperature in the dark for 5 min. The cells were washed four times with water and then resuspended in 0.3 μg/ml DAPI plus 1 mg/ml p -phenylenediamine before viewing. Mother cell bodies were defined as those which possessed at least two calcofluor-stained chitin rings on their surface. For antitubulin immunofluorescence microscopy, cells were fixed by adding formaldehyde directly to the medium to a final concentration of 3.7%. Microtubules were visualized by a procedure described previously using the rat antitubulin antibody YOL1/34 (Harlan Bioproducts for Science) and rhodamine-conjugated goat anti–rat secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Stained cells were examined with an inverted microscope (Axiovert 135; Carl Zeiss, Inc.) equipped with epifluorescent optics using a 100× objective. Digital images were captured with a cooled, slow-scan CCD camera. In cells expressing the spindle pole body (SPB)-associated fusion protein Nuf2p–green fluorescent protein (GFP) , bipolar spindles were quantitated by counting the number of cells with two clearly separated GFP dots. Electron microscopic examination of thin sections was performed as described by Byers and Goetsch 1991 . To quantitate nuclear mislocalization in cells, two assays were used. In the first, cells were arrested at 26°C with α-factor as described above. Cells were then washed once with water to remove the α-factor and resuspended in YPD media prewarmed to 35°C. The cultures were incubated at 35°C and at regular intervals samples were removed, fixed with ethanol, and stained with DAPI. The percentage of total cells that were large-budded (bud diameter greater than three quarters the diameter of the mother cell) with the nucleus away from the neck was determined for each time point. “Nucleus away from the neck” cells were defined as those in which the closest distance between the nucleus and the neck was greater than one half the diameter of the entire nuclear DNA mass, as judged by eye. In the second assay, cells were arrested at 26°C with hydroxyurea as described above. Next, cultures were shifted to 35°C, and at regular intervals samples were removed, fixed, and either stained with DAPI alone, or processed for both antitubulin immunofluorescence and DAPI staining. The percentage of large-budded cells with the nucleus away from the neck was determined for each time point. In a previous study we obtained evidence of overlap for an essential function between Kar3p, Kip3p, and dynein. Although neither KAR3 , KIP3 , nor DYN1 (encoding the dynein heavy chain) are individually essential for cell viability, the double deletion mutant combinations are inviable, or in the case of dyn1Δ kip3Δ , grow very slowly . To investigate the nature of this overlap and the mitotic role of Kar3p, we generated a recessive temperature-sensitive KAR3 mutant allele (see Materials and Methods). A plasmid carrying this allele, kar3-64 , was identified in a strain deleted for KAR3 and KIP3 . The kar3Δ kip3Δ (p kar3-64 ) strain was inviable at 33°C and higher . kar3Δ dyn1Δ (p kar3-64 ) cells were also temperature-sensitive for growth, although the effect was not as extreme as for kip3Δ . p kar3-64 also failed to complement the slight temperature sensitivity caused by kar3Δ . In the absence of dynein, kinesin-related Kip2p acts antagonistically to Kar3p and Kip3p. Loss of Kip2p suppresses the growth defects of the dyn1Δ kip3Δ and dyn1Δ kar3Δ double mutants, but not that of kip3Δ kar3Δ . Accordingly, we found that the kar3Δ dyn1Δ kip2Δ (p kar3-64 ) strain showed moderately improved growth at 35°C compared with the kar3Δ dyn1Δ (p kar3-64 ) strain . However, the kar3Δ kip3Δ kip2Δ (p kar3-64 ) strain was just as temperature-sensitive as the corresponding strain possessing the wild-type KIP2 allele . Log-phase cultures of kar3Δ kip3Δ (p kar3-64 ), kar3Δ dyn1Δ (p kar3-64 ), and wild-type cells were shifted to 35°C for 2 h and examined by microscopy. The kar3Δ kip3Δ (p kar3-64 ) and kar3Δ dyn1Δ (p kar3-64 ) showed evidence of reduced proficiency to accomplish mitosis; 57 and 52%, respectively, of the mutant cells were large-budded and mononucleate, relative to 26% of the wild-type ( n = 200 cells for each). The kar3Δ dyn1Δ (p kar3-64 ) cells often displayed mislocalized nuclei indicative of a spindle positioning defect (see next section). Cells lacking KAR3 display a bilateral karyogamy defect; kar3Δ cells will form diploids with normal efficiency with KAR3 cells of the opposite mating type, but not with kar3Δ cells . In addition to its mitotic defects, kar3-64 caused a temperature-sensitive defect in diploid formation when mated to a kar3Δ strain, but not a KAR3 strain (data not shown). In a previous study, the spindle positioning role of Kip3p was clearly revealed in cells missing dynein. Loss of Kip3 function in dynein-deficient cells caused spindle (and nuclear) mispositioning. The lethality of kar3 dyn1 and kar3 kip3 cells suggested that Kar3p may also participate in spindle positioning. To investigate this possibility, we assessed the proficiency of kar3-64 cells at positioning the nucleus at the mother–bud neck (a consequence of proper spindle positioning). Two related assays were used. In the first, cells were arrested in G1 with α-factor at 26°C and released to media at 35°C. The ability of the cells to translocate their nuclei to its proper position at the neck between mother and bud cell bodies was then assessed. In the second, spindle positioning was allowed to first occur at permissive temperature (26°C) in the presence of the DNA synthesis inhibitor hydroxyurea. Yeast cells treated with this inhibitor are still able to accomplish bipolar spindle assembly and spindle positioning . A temperature shift to 35°C was then imposed while the hydroxyurea block was maintained, and the ability of the cells to maintain the nucleus at the neck was assessed. In both assays, the inability to move the spindle or to maintain it at the neck was manifested by the appearance of large-budded cells with the nucleus located away from the neck . Both assays yielded similar results . Loss of Kar3p function alone did not cause nuclear mislocalization. In both assays, the kar3Δ (p kar3-64 ) strain behaved similar to wild-type . This agrees with previous reports, in which the nuclear migration index (the shortest distance between the nucleus and the bud neck divided by the mother cell diameter) for a kar3Δ strain was found to be comparable to the nuclear migration index of a wild-type strain . Although the kar3Δ (p kar3-64 ) strain did not exhibit nuclear mislocalization, this strain did accumulate a large percentage (∼50%) of large-budded mononucleate cells at 35°C, as reported previously for strains lacking Kar3p . In contrast, the elimination of Kar3p function in the absence of dynein resulted in a gradual but significant mislocalization of the nucleus . The extent of this phenotype was always greater than loss of dynein ( kar3Δ dyn1Δ [p KAR3 ] strain) or Kar3p ( kar3Δ [p kar3-64 ] strain) alone. As was observed for loss of both Kip3p and dynein activities , loss of both Kar3p and dynein caused the appearance of long cytoplasmic microtubules that extended into the bud. Bud scar staining with calcofluor was used to identify the mother bodies of cells in which Kar3p and dynein were inactivated ( n = 200). This analysis revealed that all of the nuclear mislocalization events occurred exclusively in the mother cells, as was previously observed for kip3 dyn1 cells. Therefore, efficient spindle positioning in the absence of dynein requires Kar3p as well as Kip3p. Furthermore, loss of Kar3p function in dynein-deficient cells that had previously achieved nuclear positioning results in movement of the nucleus away from the neck toward the mother cell cortex. To ensure that the effects of loss of dynein and Kar3p function observed here were not an artifact of the cell cycle block used in the protocols, we also examined temperature-shifted log-phase cultures. After 2 h at 35°C, 28% of large-budded kar3Δ dyn1Δ (p kar3-64 ) cells exhibited mislocalized nuclei or two nuclei in one cell body, compared with 6, 8, and 0% of kar3Δ (p kar3-64 ), dyn1Δ , and wild-type, respectively ( n = 200 cells for each). The ability of the deletion of KIP2 to suppress the growth defect of kar3 dyn1 cells (see above) was also reflected in its effects on spindle positioning proficiency. In both assays, the kar3Δ dyn1Δ kip2Δ (p kar3-64 ) strain displayed a greatly attenuated nuclear positioning defect relative to the kar3Δ dyn1Δ (p kar3-64 ) strain . This indicates that the spindle mislocalization observed in the absence of Kar3p and dynein was caused by the action of Kip2p. kar3Δ dyn1Δ kip2Δ (p kar3-64 ) cells were found to have significantly shorter cytoplasmic microtubules compared with the kar3Δ dyn1Δ (p kar3-64 ) strain . This is consistent with the previous observation that loss of Kip2p causes a reduction in cytoplasmic microtubule length . The spindle positioning defect caused by the absence of Kar3p and dynein, and its suppression by kip2Δ , was very similar to the defect observed previously for cells missing Kip3p and dynein . Cells missing both Kar3p and Kip3p are inviable, possibly also due to a spindle positioning defect (i.e., eliminating any two of either dynein, Kar3p, or Kip3p would prevent spindle positioning). To test this possibility, we performed the two nuclear position assays on a kar3Δ kip3Δ (p kar3-64 ) strain. Both assays demonstrated that loss of Kar3p in the absence of Kip3p caused only relatively mild nuclear mislocalization . However, this effect was greater than those observed in the absence of either Kar3p or Kip3p alone. Similar findings were obtained in experiments using a temperature-sensitive KIP3 allele in a kar3Δ strain (Cottingham, F.R., and M.A. Hoyt, unpublished observations). The minor nuclear position defect observed in the absence of Kar3p and Kip3p did not appear to be affected by the presence or absence of Kip2p, i.e., the kar3Δ kip3Δ kip2Δ (p kar3-64 ) strain behaved similarly to the kar3Δ kip3Δ (p kar3-64 ) strain in both nuclear position assays. These findings indicate that Kar3p and Kip3p together make only a minor contribution to spindle positioning when dynein is present. However, when dynein is absent, the contributions of both Kip3p and Kar3p (this study) become apparent. The fact that loss of both Kar3p and Kip3p caused only a mild spindle positioning defect, but that lethality was not suppressed by kip2Δ , suggests that they overlap for a different essential process. The nature of this process is addressed in the following section. The findings presented here and elsewhere implicate four microtubule-based motors in the process of spindle and nuclear positioning: dynein, Kar3p, Kip3p, and Kip2p. To investigate the consequences of loss of all four of these motors, we performed the two nuclear position assays described above on a strain deleted for DYN1 , KAR3 , KIP3 , and KIP2 and carrying the kar3-64 allele on a plasmid . In both assays, the kar3Δ dyn1Δ kip3Δ kip2Δ (p kar3-64 ) strain exhibited a nuclear positioning defect at 35°C; it was not able to move the nucleus to the neck efficiently or maintain it there following hydroxyurea synchronization. The movement of nuclei away from the neck after hydroxyurea treatment was found to occur independent of microtubule function (Cottingham, F.R., and M.A. Hoyt, unpublished observations). Therefore, in this experiment, Kar3p was acting as a solitary and essential spindle positioning motor required to resist a microtubule-independent force that operates on the nucleus. In the last Results section, we demonstrate that either Kar3p or Kip3p alone is sufficient to perform spindle positioning. As demonstrated above, loss of function of both Kar3p and Kip3p caused only a mild spindle positioning defect. Therefore, the inviability of kar3 kip3 double mutant cells may be due to a defect in another essential cellular process. In the course of our spindle positioning experiments, microscopy revealed frequent abnormal spindle morphologies in kar3 cells, as has also been observed in kar3 cells by others . The abnormal spindle phenotype appeared more severe when both Kar3p and Kip3p were absent. To assess bipolar spindle assembly proficiency, cells of various genotypes were synchronized in G1 with α-factor at 26°C and released into media at 35°C. Fig. 4 shows examples of spindles fixed and stained 1 h after release from the α-factor block. Most wild-type cells possessed spindles that were clearly bipolar; a bright bar of spindle microtubules was visible in the nuclei (in some cases the bar was long, indicating anaphase had initiated). Fewer bar structures were found in kar3Δ (p kar3-64 ) cells and even fewer in kar3Δ kip3Δ (p kar3-64 ) cells. Instead of bars of microtubules, kar3Δ kip3Δ (p kar3-64 ) cells often had a bright small mass of nuclear microtubules from which the cytoplasmic microtubules radiated. As described previously for cells deficient for Kar3p and Dyn1p or Kip3p and Dyn1p, the cytoplasmic microtubules of cells missing the functions of both Kar3p and Kip3p grew to much longer lengths than wild-type. To quantitate the effects of loss of function of Kar3p and Kip3p on bipolar structure, we examined cells whose spindle poles were marked with the GFP. A construct encoding a GFP-tagged SPB protein, Nuf2p, was integrated into our motor mutant strains . At various times after release from the α-factor block into 35°C, live cells were observed under the microscope and the percentage of cells with two clearly separated GFP dots was determined . Relative to the wild-type, the kar3Δ kip3Δ (p kar3-64 ) strain was severely compromised for its ability to generate cells with two distinct GFP dots. The majority of kar3Δ kip3Δ (p kar3-64 ) cells were large-budded, with a single GFP dot located at the bud neck . Therefore, Kip3p makes an important contribution to spindle assembly that is revealed when Kar3p activity is compromised. However, the activity provided by Kip3p cannot completely compensate for loss of Kar3p, since kar3-64 and kar3Δ (not shown) cells exhibited an intermediate reduction in spindle assembly proficiency, even when Kip3p was present. Dynein, on the other hand, does not appear to overlap with Kar3p for establishing spindle structure, since the kar3Δ dyn1Δ (p kar3-64 ) strain was no more severely affected than the kar3Δ (p kar3-64 ). To assess the ability of bipolar spindles to maintain their integrity after motor loss, we arrested the mutants with hydroxyurea at 26°C (a condition that allows bipolar spindle assembly) and then shifted to 35°C. Observation of the Nuf2p-GFP dots as a function of time revealed that wild-type and kip3-14 (a temperature-sensitive allele) cells maintained bipolar spindle structure . In contrast, ∼80% of the kar3Δ (p kar3-64 ) cells displayed two Nuf2p-GFP dots before the temperature shift, but only ∼45% displayed two dots after 3 h at 35°C. The presence or absence of dynein in the kar3-64 cells made no apparent difference. Spindles also did not lose bipolarity when both Kip3p and dynein were inactivated . However, the absence of Kip3p significantly enhanced the loss of bipolarity phenotype exhibited by kar3-64 cells; after 3 h, only ∼25% of kar3Δ kip3Δ (p kar3-64 ) cells had two clearly separated dots. Therefore, Kar3p and Kip3p overlap for a function required to maintain bipolar spindle integrity. Thin-section electron microscopy was used to examine the spindle morphology of kar3Δ kip3Δ (p kar3-64 ) and wild-type cells treated with hydroxyurea and shifted to 35°C for 3 h . The wild-type spindles were short and bipolar, with parallel SPBs separated by ∼1.5 μm and joined by a bundle of microtubules. No normal-appearing spindles were found in the kar3Δ kip3Δ (p kar3-64 ) culture. Of the spindles in which two poles could be visualized in one section, nine had SPBs located adjacent to one another. Six spindles had SPBs that were separated from between 0.2 and 1.2 μm (average = 0.53 ± 0.37 μm, SD), but were clearly defective. The poles of these spindles were not parallel. These findings agree with those from the Nuf2p-GFP analysis. Loss of Kar3p and Kip3p function caused preformed bipolar spindles to break with SPBs frequently moving close together. Kar3p forms distinct complexes with two homologous accessory factors, Cik1p and Vik1p . cik1 and vik1 mutants display different phenotypes that each overlap in part with the phenotypes of kar3 cells. It is likely that these factors target Kar3p to different cellular roles, the precise nature of which have not been determined. We found that, similar to a kar3Δ mutant , cik1Δ is lethal in combination with dyn1Δ , and this lethality is suppressed by kip2Δ . Viable cik1Δ dyn1Δ cells could only be created when KIP2 was deleted. Fig. 7 demonstrates that introduction of KIP2 by transformation into a cik1Δ dyn1Δ kip2Δ triple mutant prevented colony formation, indicating that lethality is due to the activity of KIP2 . In this case, the similarity in genetic behavior of cik1Δ to kar3Δ suggests that it is the Cik1p–Kar3p complex that overlaps with dynein for an essential spindle positioning role. However, unlike kar3Δ , cik1Δ could be combined with kip3Δ yielding viable, healthy cells. Therefore, Cik1p–Kar3p complexes do not uniquely perform the function required for spindle integrity that overlaps with Kip3p. This supports the hypothesis that Cik1p is only required for a subset of Kar3p's roles . vik1Δ dyn1Δ and vik1Δ kip3Δ double mutant cells were also viable and healthy. Therefore, Vik1p–Kar3p complexes do not uniquely overlap for essential functions with either Dyn1p or Kip3p. However, cik1Δ vik1Δ kip3Δ triple mutants were inviable. Strain MAY6417 ( cik1Δ vik1 kip3Δ [p KIP3 URA3 ]) could not survive loss of the KIP3 URA3 plasmid, as evidenced by its inability to segregate cells capable of growth on 5-FOA. This may indicate that the essential spindle integrity function that overlaps with Kip3p is performed by both Cik1p–Kar3p and Vik1p–Kar3p complexes, and that either alone is sufficient. Alternatively, the lethality of the cik1Δ vik1Δ kip3Δ triple mutant may reflect a nonspecific additive effect of combining three deleterious mutations. Kar3p can act as a microtubule destabilizer in vitro , and kar3Δ vegetative growth defects can be suppressed by the presence of the microtubule-destabilizing reagent benomyl . We found that the temperature sensitivity of kar3Δ kip3Δ (p kar3-64 ) cells could be partially suppressed by the presence of 5–10 μg/ml benomyl in the media . The temperature sensitivity of kar3Δ kip3Δ (p kip3-14 ) cells could be suppressed by the same treatment as well , similar to the suppression of kip3-30 (temperature-sensitive) kar3Δ reported by others . This finding suggests that both Kar3p and Kip3p act to destabilize microtubules, and that this is an important aspect of the essential function for which they overlap (see Discussion). Indeed, all multiple motor mutant genotypes that are dependent upon either kar3-64 or kip3-14 for viability were suppressed for temperature-sensitive growth by benomyl . The S . cerevisiae genome encodes six kinesin-related motor genes and a single dynein heavy chain. In this and a previous study we described two viable triple mutant combinations. Cells deleted for DYN1 and either KAR3 or KIP3 were viable when the antagonistically acting KIP2 was deleted as well. Since a stripped-down or minimal system can help reveal how a process is accomplished, we attempted to reduce further the number of functional microtubule-based motor genes. In generating strains with reduced spindle motor function, evidence of aneuploidy was sometimes detected (i.e., non-Mendelian segregation of genetic markers). Therefore, we routinely checked all seven motor gene alleles by a PCR assay to verify that the genotypes constructed were correct (see Materials and Methods). We also note that for practical reasons, the minimal motor strains we created carried one of the active motor genes on a low-copy centromere plasmid. Centromere plasmids are usually present in one to two copies per haploid cell, but can increase slightly if selective pressure is applied . From the two viable triple mutants described above ( dyn1Δ kip3Δ kip2Δ and dyn1Δ kar3Δ kip2Δ ) we were able to delete an additional two genes encoding kinesin-related proteins, KIP1 and SMY1 . KIP1 encodes the BimC family motor that is phenotypically less important (the more important BimC motor is encoded by CIN8 ) . SMY1 probably has no mitotic spindle role (see Introduction) but was deleted on account of its sequence relationship to kinesins. The strains created expressed only two of seven microtubule-based motor genes; CIN8 with either KAR3 or KIP3 . Cells expressing only CIN8 and KAR3 were quite healthy relative to wild-type, as judged by doubling time and other cell cycle criteria ( Table ). Some difficulty in progression through mitosis was evident from the approximately twofold elevation of large-budded, mononucleate cells in log-phase cultures. Spindle positioning errors were also evident (by the appearance of bi and anucleate cell bodies), but at a level no higher than a dyn1 single mutant . Cells expressing only CIN8 and KIP3 were considerably less well off. Their doubling time was significantly increased, and spindle positioning errors were approximately twice that of the CIN8 KAR3 cells. The plating efficiency of the CIN8 KIP3 cells was roughly half that of wild-type, indicating many dead cells in the culture. However, this figure is close to that observed for kar3 single mutants . To determine if Cin8p Kar3p or Cin8p Kip3p represent the lowest possible motor complements, we created strains in which one of the two active motor genes was replaced with a temperature-sensitive allele. While cells surviving with only CIN8 KAR3 or CIN8 KIP3 were able to grow at 35°C, neither CIN8 kar3-64 , CIN8 kip3-14 , nor cin8-3 KAR3 cells grew at this elevated temperature . Therefore, under normal growth conditions, Cin8p–Kar3p or Cin8p–Kip3p were the minimal motor sets that supported cell viability (but see below). We examined the effects on spindle integrity and positioning caused by elimination of the function of one of the two active motors in the two-motor strains. Wild-type, CIN8 kar3-64 , CIN8 kip3-14 , and cin8-3 KAR3 cells were arrested with hydroxyurea at 26°C and then shifted to 35°C for 3 h, maintaining the hydroxyurea block. The motor mutant strains exhibited greatly reduced numbers of bipolar spindles as judged by antitubulin immunofluorescence microscopy (78% for wild-type; 17% for CIN8 kar3-64 ; 9% for CIN8 kip3-14 ; and 7% for cin8-3 KAR3 ). This was an expected finding, because a BimC motor (either Cin8p or Kip1p) plus either Kar3p or Kip3p is required for bipolar spindle integrity. However, examination of the nuclear positioning proficiency of these strains did reveal differences. After hydroxyurea synchronization, the shift to 35°C caused nuclei in CIN8 kar3-64 and CIN8 kip3-14 cells, but not cin8-3 KAR3 cells, to mislocalize . Therefore, it is reasonable to conclude that Kar3p or Kip3p are the only motors performing spindle positioning in their respective two-motor strains, and that Cin8p makes no detectable contribution to this process. Finally, we found further support for a microtubule-destabilizing role for Kar3p and Kip3p in vivo. Addition of benomyl to the media could suppress the temperature-sensitive growth of cells surviving with only CIN8 kar3-64 or CIN8 kip3-14 . The slow growth of the CIN8 KIP3 strain was exacerbated at elevated temperature, a phenotype that was also suppressed by benomyl. In contrast, the temperature sensitivity of the cin8-3 KAR3 strain was not relieved by benomyl. Therefore, although microtubule destabilization may be an important function of Kar3p and Kip3p, this does not appear to be a role for the BimC motor Cin8p. Our findings demonstrate that S . cerevisiae Kar3p and Kip3p motors perform overlapping roles contributing to both mitotic spindle positioning and spindle structural integrity. These two activities are primarily accomplished by distinct sets of microtubules, cytoplasmic and nuclear, respectively. Therefore, we conclude that Kar3p and Kip3p act on both sides of the nuclear envelope, a property that may be unique among the seven S . cerevisiae microtubule-based motors. We suggest that a major aspect of the nuclear and cytoplasmic functions of both of these motors is to destabilize microtubules. In the absence of dynein, both Kar3p and Kip3p are required for spindle positioning and vigorous cell growth. Similar to cells in which dynein and Kip3p were eliminated , we demonstrated here that elimination of dynein and Kar3p caused spindle mispositioning and the appearance of extremely long cytoplasmic microtubules. In both cases, the long microtubule phenotype and the spindle positioning defect were suppressed by the elimination of Kip2p. In cells missing both dynein and Kip2p, either Kar3p or Kip3p would now suffice for viability, probably becoming the sole spindle positioning motor in these cells. We demonstrated that in cells surviving with only Cin8p and Kar3p or Kip3p, all spindle positioning function appeared to be accomplished by Kar3p or Kip3p. It is not clear whether Kar3p and Kip3p normally act in distinct spindle positioning pathways or if they redundantly perform an activity required for the same pathway. In previous studies, Kar3p was visualized only upon microtubules in the nucleus of mitotic cells . However, our finding that Kar3p participates in spindle positioning indicates that it must be acting upon cytoplasmic microtubules as well. Two other findings indicate that Kar3p can act upon cytoplasmic microtubules: Kar3p influences the dynamics of cytoplasmic microtubules in mitotic cells , and acts upon cytoplasmic microtubules in karyogamy to draw together the nuclei of mated cells . Our studies also demonstrate a role for Kar3p and Kip3p in the nucleus, overlapping for a function essential for bipolar spindle assembly and structural integrity. Previous observations of spindle abnormalities in kar3Δ cells suggested a spindle integrity role for Kar3p . A nuclear role for Kip3p was suggested by its localization to nuclear (as well as cytoplasmic) microtubules and the extra long pre and postanaphase spindles observed in kip3Δ cells . We demonstrated here that the structural integrity of the nuclear bipolar spindle requires the actions of at least one of these motors. Since loss of both motors is lethal, but causes only modest effects on spindle positioning, it is likely that spindle integrity is the essential activity for which they overlap. When both motors were inactivated, bipolar spindle assembly and the structural integrity of preformed spindles were compromised. The collapse of preformed spindles was slow, requiring over an hour to reach the maximal effect . Spindle poles moved together, but many poles were still slightly separated. This contrasts with the rapid spindle collapse observed when the two BimC motors, Cin8p and Kip1p, were inactivated; all spindle poles were pulled to an adjacent position after as little as 5 min at the nonpermissive temperature . This indicates that Kar3p and Kip3p probably function differently from the BimC motors in maintaining bipolar spindle structure. Kar3p forms functionally distinct complexes with two homologous accessory factors, Cik1p and Vik1p . Cik1p, but not Vik1p, is required for karyogamy, and Kar3p and Cik1p are interdependent for localization to cytoplasmic microtubules in mating cells. We found that, similar to a kar3Δ mutant, cik1Δ is lethal in combination with dyn1Δ , and this lethality is suppressed by kip2Δ . This overlap in function between Cik1p and dynein suggests that Kar3p complexed to Cik1p performs the cytoplasmic spindle positioning function. Our genetic studies indicated that neither Cik1p–Kar3p nor Vik1p–Kar3p complexes uniquely perform the function essential for spindle integrity that overlaps with Kip3p. However, since cik1Δ vik1Δ kip3Δ triple mutants were inviable, it remains possible that these two complexes redundantly provide this activity. Our findings lend further support to the hypothesis that Cik1p and Vik1p target the Kar3p motor to distinct cellular functions . The molecular nature of the spindle positioning and structural defects caused by loss of Kar3p and Kip3p is likely to involve a defect in microtubule polymer length regulation. Recent studies have implicated microtubule motors as important regulators of microtubule dynamics. Notably, members of the XKCM1/MCAK family of vertebrate kinesins have been demonstrated to cause microtubule instability by promoting catastrophe events . A microtubule-destabilizing activity for Kar3p has been detected in vitro . Findings reported here, combined with those of others, clearly demonstrate that Kar3p, Kip3p, and dynein either directly or indirectly act to shorten microtubules in vivo . These observations can be generally summarized as follows: mutants deficient for any one of these motors display elongated microtubules and/or phenotypes that are suppressible by the antimicrotubule compound benomyl. Double mutants are much more severely affected; they are either inviable or very sick and display extremely long microtubule structures. In the studies reported here, numerous multiple motor mutant genotypes were created that were missing Kar3p or Kip3p and relied upon a temperature-sensitive form of the other for viability. All of these strains were suppressed for temperature-sensitive growth by the addition of the antimicrotubule compound benomyl to the media. Although the mechanism of suppression could be indirect, it is probable that benomyl is substituting for an activity normally accomplished by Kar3p and Kip3p. Depending upon the genotype tested, benomyl suppressed either the lethal spindle structural defect, the lethal spindle positioning defect, or both (i.e., for cells surviving only with CIN8 and kar3-64 ). Therefore, both the spindle structural and positioning defects caused by kar3 and kip3 mutations involve effects on microtubule stability. It is not clear how increased microtubule stability or length caused the observed kar3 kip3 mutant spindle structural and positioning defects. The dynamic behavior of the ends of microtubules is probably important for functions such as interactions with kinetochores, antiparallel microtubules, and the cell cortex. Any of these processes may be disrupted by the inability to control the growth of microtubules. A less direct possibility for the kar3 kip3 spindle structural defect is that the longer or less dynamic spindle microtubules do not form proper interactions with the BimC motors, leading ultimately to spindle collapse. For the spindle positioning defect, it is possible that the extremely long cytoplasmic microtubules act to prohibit migration of the nucleus up to the cell neck or actually push the nucleus away. The functional overlap between Kar3p and Kip3p could not have been predicted from the amino acid sequence of these gene products, since each is distinct. Kar3p defines a unique subclass of kinesin-related proteins characterized by their COOH terminus–located motor domain and their minus end–directed force production on microtubules . The motor domain of Kip3p is located at its NH 2 terminus. The directionality of Kip3p has not been determined, but a related motor from Drosophila has been found to exhibit plus end–directed activity . The directionality of these motors may relate to activities for which they need not necessarily overlap. We predict that the only essential overlap between these motors is their ability to shorten both nuclear and cytoplasmic microtubules. In contrast to Kar3p, Kip3p, and dynein, the Kip2p motor acts to increase the length of cytoplasmic microtubules. Loss of function causes extremely short cytoplasmic microtubules , whereas overexpression causes an extra long microtubule phenotype . In addition to shortening the microtubules of kar3 dyn1 and kip3 dyn1 cells, kip2Δ also suppresses their lethal spindle positioning defect . The shortened microtubules may be the direct cause of the suppression, allowing spindles now to migrate unimpeded to the neck region. However, the possibility that other activities of Kip2p may antagonize spindle positioning in the absence of dynein cannot be ruled out. In contrast, the elimination of Kip2p did not suppress the lethal spindle structural defect of kar3 kip3 cells. This is consistent with its localization to cytoplasmic microtubules only , as well as our failure as yet to detect any nuclear role for this motor. Cytological descriptions of mitotic spindle function have revealed a complex series of motility events that affect the segregation of replicated chromatids. These events include bipolar spindle assembly, kinetochore capture and congression, and two chromosome-separating movements, anaphase A (movement of kinetochores towards spindle poles) and anaphase B (separation of spindle poles). In considering the minimal microtubule-based motor set required for S . cerevisiae viability, it first must be recognized that unlike higher eukaryotic cells, this yeast requires microtubules for only one essential role, mitotic spindle function. Nonetheless, our finding that S . cerevisiae can survive with only two microtubule-based motors is unexpected in light of the observed complexities of spindle motile behavior. We were able to create viable strains expressing only two of seven microtubule-based motors; the BimC-type motor Cin8p combined with either Kar3p or Kip3p. Attempts to reduce motor function further caused expected deleterious phenotypic consequences and revealed that these represented the minimal sets required for viability. Removing the function of Kar3p (from Cin8p Kar3p-ts cells) or Kip3p (from Cin8p Kip3-ts cells) caused spindles to both collapse and misposition. Removing the function of Cin8p caused spindle collapse, an expected consequence since these cells were missing the other BimC motor, Kip1p . However, elimination of Cin8p function caused no detectable spindle positioning defect. Therefore, it seems likely that in these two-motor strains, all nuclear microtubule motor functions were accomplished by the combination of Cin8p and either Kar3p or Kip3p, and cytoplasmic microtubule motor functions accomplished by either Kar3p or Kip3p. A few caveats must be considered regarding the conclusion that only two motors are required for viability. First, we cannot exclude the possibility that the observed vigorous growth of the two-motor cells could have been aided by suppressing mutations. The genotypes of created strains were verified with respect to the seven motor loci, so suppressors would necessarily affect genes that do not encode dyneins or kinesins. Second, we must strongly consider a contribution from microtubule-based motility mechanisms that do not include dyneins and kinesins. We are not aware of any force-producing microtubule-based ATPases outside of the dynein and kinesin families. However, an undiscovered class of motors cannot be excluded. All kinesins, dyneins, and myosins (as well as many other proteins) possess a domain with a nucleotide-binding sequence (G-X-X-X-X-G-K-T/S; and a distinct region of coiled α-helical coil sequence. An examination of the S . cerevisiae genome reveals gene products of unknown function that contain both of these sequence motifs (i.e., the gene products specified by YLR106C and YPL217C). Another possibility is a motility mechanism that uses a different form of force generation. For example, a protein that is able to couple the dynamic behavior at the ends of microtubules to force production . Such a coupler need not necessarily be a nucleotidease. Therefore, the proper conclusion for our findings is that of the six kinesins plus one dynein encoded in the genome, only two kinesins are required for S . cerevisiae viability. The presence of benomyl in the media partially suppressed the temperature-sensitive growth defects of the Cin8p Kar3p-ts and Cin8p Kip3p ts two-motor strains. However, we have been unable to find a condition allowing vigorous growth in the complete absence of Kar3p and Kip3p (cells surviving on Cin8p alone). Therefore, the temperature-sensitive forms of these motors may retain some activity at the elevated temperature that is required in addition to the microtubule-destabilizing activity of benomyl. Nonetheless, these findings further indicate that microtubule-destabilization is an important activity of Kar3p and Kip3p. In summary, S . cerevisiae cells only require two microtubule-based motors to accomplish mitosis. The BimC motor Cin8p is required to assemble and elongate the bipolar spindle, probably by virtue of its ability to cross-link and slide microtubules. The second motor, either Kar3p or Kip3p, acts to promote microtubule shortening both within the nucleus and in the cytoplasm. Until the possible discovery of other spindle motility-producing mechanisms, we must entertain models in which all essential spindle motile activities can be accomplished by this limited set of motor proteins. | Study | biomedical | en | 0.999998 |
10525540 | The human HeLa cell line and the monkey CV1 cell line were maintained in DME containing 10% FCS, 2 mM glutamine, 100 iU/ml penicillin, and 0.1 mg/ml streptomycin. The human CF-PAC1 cell line was maintained in Iscoves modified DME containing 10% FCS, 2 mM glutamine, 100 iU/ml penicillin, and 0.1 mg/ml streptomycin. Cells were grown at 37°C in a humidified incubator with a 5% CO 2 atmosphere. The HSET-specific antibodies were prepared by immunizing rabbits with recombinant HSET protein expressed in bacteria. A 1365-bp EcoRI fragment from the HSET cDNA ps55 was ligated into pGEX-5X-3 at the unique EcoRI site in the multicloning site. This construct results in the fusion of the open reading frames for GST and the COOH-terminal 377 amino acids of HSET. The orientation of the HSET sequence was verified by multiple combinatorial restriction digests and the construct transformed into Escherichia coli BL21 (Stratagene). Expression of the GST–HSET fusion protein was induced by addition of 1 mM IPTG to a liquid culture. Cells were harvested after 6 h, pelleted by centrifugation at 7,000 rpm at 4°C, resuspended in 10 ml PBS containing protease inhibitors (5 mg/ml chymostatin, leupeptin, antipain, pepstatin, and 100 mg/ml phenylmethylsulphonyl fluoride) and sonicated on ice. The lysed cells were then incubated on ice for 30 min with 1% Triton X-100 and the insoluble debris removed by centrifugation at 11,000 rpm for 15 min at 4°C. The soluble fraction was collected and passed over a column of packed glutathione Sepharose-4B (Pharmacia Biotechnology Inc.). The column was washed twice with PBS to remove any nonbound protein, after which the bound GST–HSET protein was eluted by three successive washes with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. GST–HSET was further purified from the eluate by SDS-PAGE, where the GST–HSET containing band was excised from a polyacrylamide gel, electroeluted from the gel, dialyzed against water, lyophilized, and resuspended in PBS. This pure GST–HSET fraction was used to immunize two rabbits, which produced two similar HSET specific antibodies, HSET-1 and HSET-2. The remaining antibodies used in these experiments were as follows. αCTP-2, raised against the COOH-terminal tail of XCTK2 , was generously donated by Claire Walczak (University of Indiana, Bloomington, IN). NuMA was detected with the rabbit polyclonal antibody . Tubulin was detected using the mAb DM1α (Sigma Chemical Co.). Eg5 was detected using a rabbit polyclonal antibody raised against the central rod domain, expressed as clone M4F . Cytoplasmic dynein was detected using a mAb specific for IC74 intermediate chain . Finally, γ-tubulin was detected using a mouse mAb (Sigma Chemical Co.). Indirect immunofluorescence microscopy was performed on cultured cells by immersion in microtubule stabilization buffer (MTSB; 4 M glycerol, 100 mM Pipes, pH 6.9, 1 mM EGTA, and 5 mM MgCl 2 ) for 1 min at room temperature, extraction in MTSB + 0.5% Triton X-100 for 2 min, followed by MTSB for 2 min. Cells were then fixed in −20°C methanol for 10 min. Indirect immunofluorescence microscopy on mitotic asters assembled in the cell free mitotic extract was performed by dilution of 5 μl of the extract into 25 μl of KHM buffer . The diluted sample was then spotted onto a poly- l -lysine–coated glass coverslip and fixed by immersion in −20°C methanol. Both the fixed cells and mitotic asters were rehydrated in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1% albumin and all antibody incubations and washes were performed in TBS + 1% albumin. Each primary antibody was incubated on the coverslip for 30 min, followed by 5 min washes in TBS + 1% albumin, and the bound antibodies were detected using either fluorescein- or Texas red-conjugated species-specific secondary antibodies at dilutions of 1:500 (Vector Labs, Inc.). The DNA was detected using DAPI (4′,6-diamidino-2-phenylindole) at 0.4 μg/ml (Sigma Chemical Co.). After a final wash, the coverslips were mounted in Vectashield FITC-guard mounting medium (Vector Labs, Inc.) and observed on a Nikon Optiphot microscope equipped for epifluorescence. Mouse oocytes were permeabilized, fixed, and processed for the immunocytochemical detection of spindle components as described previously . Cells were labeled with a fluorescein-conjugated secondary antibody to identify the injected antibody, antitubulin, followed by a rhodamine-conjugated secondary antibody and 5 μg/ml Hoechst 33342 for fluorescence DNA localization. Epifluorescent microscopy and photography were performed on a Zeiss Axiophot equipped with appropriate filters for all three fluorochromes. Cultured cells or proteins from the mitotic extracts were solubilized directly with SDS-PAGE sample buffer. The proteins were then separated by size using SDS-PAGE , and transferred to PVDF membrane (Millipore Corp.). The membranes were blocked in TBS containing 5% nonfat milk for 30 min at room temperature, and the primary antibody incubated for 6 h at room temperature in TBS containing 1% nonfat milk. Nonbound primary antibody was removed by washing five times for 3 min each in TBS, and the bound antibody was detected using either HRP-conjugated Protein A or HRP-conjugated goat anti–mouse (Bio-Rad Co.). The nonbound secondary reagent was removed by washing five times for 3 min each in TBS, and the signal detected using enhanced chemiluminescence (Nycomed Amersham Inc.). To localize HSET by immunogold EM on mitotic spindles in cultured CF-PAC1 cells, the cells were grown on photo-etched alphanumeric glass coverslips (Bellco Glass Co.). The position of mitotic cells was determined by phase-contrast microscopy and noted for subsequent selection for examination by EM. Cells were rinsed in MTSB for 1 min at room temperature, extracted in MTSB + 2% Triton X-100 for 5 min, followed by MTSB for 2 min. Cells on the coverslips were then fixed in 1% glutaraldehyde in 0.1 M Na-cacodylate buffer for 30 min. Detergent extraction of cells before fixation was necessary to remove soluble cytosolic components from cells which obscure visualization of HSET on the spindle. This extraction procedure was not deleterious to spindle structure as judged by the presence of interpolar, kinetochore, and astral microtubules. After fixation, the coverslips were washed twice for 15 min each in 0.1 M Na-cacodylate buffer, twice for 15 min each in PBS, three times for 15 min each in 0.5 mg/ml NaBH 4 , twice for 5 min each in PBS+1% BSA, and finally, once in TBS+1% BSA for 10 min. The anti-HSET rabbit polyclonal IgG was then added at a concentration of 0.13 mg/ml in TBS+1% BSA and incubated for 1 h. The coverslip was then washed with TBS+1% BSA, and incubated for 4 h with a 1/50 dilution of goat anti–rabbit FAb fragments conjugated with 3-nm gold particles (Nanoprobes Inc.) in TBS+1% BSA. The sample was then washed once with TBS+1% BSA, once in 0.1 M Na- cacodylate buffer, and fixed with 2% glutaraldehyde in 0.1 M Na cacodylate. After final fixation, the cells were rinsed in 0.1 M Na-cacodylate buffer, postfixed with 1% OsO 4 in 0.1 M Na-cacodylate buffer for 30 min at room temperature, and en-bloc stained in 2% aqueous uranyl acetate. Cells were dehydrated through a graded series of ethanols and propylene oxide, and flat-embedded in epon (LX112)/araldite (502). The glass coverslip was removed by etching in cold concentrated hydrofluoric acid as described by Moore 1975 and Rieder and Bowser 1987 . The area containing the mitotic cells that were previously selected by phase-contrast microscopy was identified with the help of a dissecting microscope, cut out of the flat-embedded rectangle, and remounted onto epoxy blanks. 120–150-nm sections were prepared and stained with 2% uranyl acetate for 45 min at 50°C. We specifically chose 3-nm gold-conjugated FAb fragments as the secondary reagent for all immuno-EM. These small gold particles are at or near the resolution limit for detection in EM, but were essential for optimal penetration of the dense microtubule structures, and to avoid silver enhancement techniques. This allowed us to fix the specimens with osmium tetroxide, which was important in revealing the electron dense material associated with the spindle microtubules. All electron micrographs were taken at 80 or 100 kV on a JEOL 100CX. Mitotic extracts from HeLa cells were prepared according to Gaglio et al. 1995 . HeLa cells were synchronized in the cell cycle by double block with 2 mM thymidine. After release from thymidine block, the cells were allowed to grow for 6 h and then nocodazole was added to a final concentration of 40 ng/ml. The mitotic cells that accumulated over the next 4 h were collected by mitotic shake-off and incubated for 30 min at 37°C with 20 μg/ml cytochalasin B. The cells were then collected by centrifugation at 1,500 rpm and washed twice with cold PBS containing 20 μg/ml cytochalasin B. Cells were washed one last time in cold KHM buffer containing 20 μg/ml cytochalasin B, and finally Dounce homogenized (tight pestle) at a concentration of ∼3 × 10 7 cells/ml in KHM buffer containing 20 μg/ml cytochalasin B, 20 μg/ml phenylmethylsulfonyl fluoride, and 1 μg/ml each of chymostatin, leupeptin, antipain, and pepstatin. The crude cell extract was then subjected to sedimentation at 100,000 g for 15 min at 4°C. The supernatant was recovered and supplemented with 2.5 mM ATP (prepared as Mg 2+ salts in KHM buffer) and 10 μM taxol, and the mitotic asters were stimulated to assemble by incubation at 30°C for 30 min. After incubation, the samples were processed for indirect immunofluorescence microscopy as described above, and the remainder of the extract containing the assembled mitotic asters was subjected to sedimentation at 10,000 g for 15 min at 4°C. The supernatant and pellet fractions were both recovered and solubilized in SDS-PAGE sample buffer for immunoblot analysis. In all experiments, HSET was perturbed by addition of the HSET-1 antibody at a final concentration of 0.1 mg/ml. Immunodepletions from the extract before aster assembly were carried out using 100 μg of anti-Eg5 affinity-purified rabbit polyclonal IgG, or mAb 70.1, which is specific for the IC74 intermediate chain of cytoplasmic dynein. Each antibody was adsorbed onto ∼25μl of either protein A-conjugated agarose or protein G-conjugated agarose (Boehringer Mannheim, Corp.). The 70.1 mAb against cytoplasmic dynein intermediate chain was coupled to protein A-conjugated agarose using goat anti-murine IgM-specific antibody (Vector Labs, Inc.). The antibody-coupled agarose was washed in KHM buffer and then packed by centrifugation to remove the excess fluid. Efficient depletion of the target protein was routinely achieved by sequential depletion reactions in which the total quantity of packed agarose did not exceed 40 μl per 100 μl of extract. First, half of the antibody-coupled agarose was resuspended with the mitotic extract and incubated with agitation for 1 h at 4°C. After this incubation, the agarose was removed from the extract by sedimentation at 15,000 g for 10 s and saved. Next, the extract was recovered and used to resuspend the other half of the antibody-coupled agarose and another incubation performed with agitation for 1 h at 4°C. After this incubation, the agarose was removed by sedimentation at 15,000 g for 10 s and pooled with the agarose pellet from the initial depletion reaction. In all cases, immunoblot analysis indicates that this depletion protocol results in ∼100% efficient depletion of the target protein in experiments both where only one protein was depleted and when more than one protein was depleted (see Results). The depleted extract was recovered and microtubule polymerization induced by the addition of taxol, ATP, and incubation at 30°C for 30 min. Each depletion experiment was performed at least three times and in all cases the data shown are representative of the microtubule structures we observed. CF-PAC1 or HeLa cells growing on photo-etched alphanumeric glass coverslips (Bellco Glass Co.) were microinjected following the procedures of Compton and Cleveland 1993 , and Capecchi 1980 . For the antibody microinjection experiments, interphase cells were microinjected in the cytoplasm with either a preimmune IgG or the immune IgG, and monitored by phase-contrast microscopy as they progressed into mitosis. αHSET-1, αEg5, and the rabbit preimmune IgG's were concentrated in 10 mM KPO 4 , 100 mM KCl, pH 7.0, at concentrations of 10 mg/ml (αHSET-1 and preimmune) and 1–2 mg/ml αEg5. After injection, cells were followed until they entered mitosis and then processed for immunofluorescence microscopy as detailed in the text. Mouse oocytes were obtained as described in Simerly et al. 1990 . Immature oocytes from outbred IRC mice (Sprague-Dawley) were collected from minced ovaries and the cumulus cells were removed by pipetting. Fully grown oocytes were maintained in a modified Tyrode's solution with 100 μg/ml dibutyryl cAMP (dbcAMP; Sigma Chemical Co.) to arrest spontaneous development . Meiotic maturation was initiated once the derivatized AMP was removed by rinsing in culture medium. Micropipettes were front-loaded with antibody from a small droplet under mineral oil juxtaposed to the culture medium containing the oocytes. Microinjection was performed by puncturing zona-intact oocytes with a 1-μm beveled micropipette (Sutter Instruments), sucking in a small amount of cytoplasm, and expelling the antibody and cytoplasm . Antibody concentrations used were as described, and ∼5% of the egg volume was microinjected with either preimmune or immune IgGs. To investigate the role of the minus end-directed kinesin-related protein, HSET, in mitotic spindle assembly in mammalian cells, we raised polyclonal antibodies against the COOH-terminal 377 amino acids of the protein. This segment of HSET was expressed as a GST fusion protein, purified by affinity chromatography as described in Materials and Methods, and used to immunize two rabbits. Both rabbits responded similarly to immunization and immunoblot analysis against total HeLa cell protein, showing that these antibodies, αHSET-1 and αHSET-2 (data not shown), specifically recognized two proteins with equal intensity at 80 and 75 kD. The 80-kD protein identified by our antibodies comigrated with the protein identified by αCTP-2, an antibody raised against the COOH-terminal 11 amino acids (CVIGTARANRK) of XCTK-2, the Xenopus laevis HSET homologue . Two lines of evidence indicate that the two proteins identified by our antibodies are different isoforms of HSET. First, we have immunoprecipitated sufficient quantities of each protein from a HeLa cell extract to obtain peptide sequence using mass spectrometry. We obtained 12 amino acid peptide sequences from both proteins that were 100% identical to the published HSET sequence. Second, searches of the EST database reveal two classes of HSET cDNAs. The segments of the HSET protein encoded by the two classes of HSET cDNAs are identical, except for the predicted COOH-terminal amino acids. The predicted protein sequence derived from one class of cDNA terminates in the sequence RLPPVSLVRTRGWL, whereas the predicted protein sequence from the other class of cDNA terminates in the sequence NQCVIGTAQANRK. This is consistent with the immunoblot showing that the αCTP-2 antibody is specific for only one of the two isoforms . The genomic organization of the HSET locus recently reported by Janitz et al. 1999 accounts for only one isoform that terminates in the sequence NQCVIGTAQANRK. Further inspection of the genomic sequence reveals that the COOH terminus of the other isoform is encoded on a distinct exon. We have labeled the 80-kD form HSET-H, and the 75-kD form HSET-L, and with a few specific exceptions as noted, we refer to these proteins collectively as HSET throughout this manuscript. We are currently investigating how the two isoforms are produced and if they differ in any specific functional properties. To localize HSET at high resolution within the mitotic spindle, we performed immunogold EM of human CF-PAC1 cells at metaphase. The protocol we have developed involves extraction with a microtubule stabilizing buffer, followed by fixation with glutaraldehyde. This process removes the soluble components of the cells, allowing good penetration of the antibodies, but preserves spindle structure including astral microtubules, centrosomes/centrioles, kinetochore fibers, and chromosomes . Immunofluorescence microscopy showed that HSET localization on the spindle was not detectably different between cells extracted before or after fixation, indicating that its localization was not altered by extraction (data not shown). Using this technique, we obtained good labeling within the metaphase spindle counting 768 gold particles in sections through the long (pole to pole) axis of the half spindle of four different mitotic cells. This labeling was specific because >90% of the gold particles were spindle associated ( Table ) and no gold labeling was observed when the αHSET-1 antibody was replaced with preimmune antibody (data not shown). We quantified the localization of the gold particles obtained by staining for HSET in two ways. First, we divided the half spindle into 1-μm sections perpendicular to the long axis of the spindle. We then counted the number of gold particles and microtubules in each of these sections . The average number of microtubules per section is relatively constant, although there are fewer microtubules nearest the centrosome, consistent with previous work . The average number of gold particles was also relatively constant, although there are fewer gold particles near the pole. These data indicate that HSET is concentrated within the main body of the half spindle and contrasts sharply with the concentration of NuMA at the spindle pole that we observed previously, using a similar technique . Second, we quantified the position of each individual gold particle relative to the microtubules ( Table ). More than 82% of the gold particles were localized within the main body of the spindle with <18% being either not spindle-associated or associated with the astral microtubules. Nearly half of all the gold particles were found to be located between adjacent microtubules . Individual gold particles can be seen in the high magnification images in Fig. 2 B and C, with HSET's association with spindle microtubules most clearly depicted in Fig. 2 C. This image shows an uninterrupted length of a pair of microtubules that have several gold particles in the intermicrotubule space. Many of the microtubules labeled for HSET terminate within a mass of chromatin or at a kinetochore . This indicated that the microtubule polymers in those specific images are oriented parallel to one another with respect to their plus and minus ends. Thus, while our data do not address if HSET localized between antiparallel microtubules, they show that a fraction of HSET is localized between parallel microtubules within the spindle during metaphase. These results, in combination with various in vitro data showing that members of this class of kinesin-related protein have two (or more) microtubule binding domains and are capable of bundling microtubules , suggest that HSET plays a role in cross-linking microtubules within the mammalian metaphase spindle. To determine if the HSET-specific antibodies were capable of disrupting mitotic spindle assembly in living cells, we microinjected αHSET-1 into both HeLa and CF-PAC1 cells. Injected cells were then monitored as they progressed through the cell cycle, fixed, and processed for immunofluorescence either in mitosis or after mitosis was completed. The fixed cells were stained for tubulin and for the injected rabbit antibody. Immunofluorescence analysis of 41 mitotic cells showed that 26 had normal bipolar mitotic spindles. The remaining 15 mitotic cells appeared to have abnormal spindles (data not shown), however, the abnormality was subtle in that the spindles were somewhat barrel-shaped with slightly broader poles than usual. This abnormality did not impede normal transit through mitosis, since 100% (32 out of 32) of αHSET-1–injected cells completed mitosis and formed typical pairs of G1 cells within a typical one hour time frame. This efficiency was similar to values obtained with the preimmune control antibody, where ∼90% of injected cells completed mitosis normally ( n = 18). These results suggest that either our antibody is not effective at perturbing HSET function in vivo or that inhibition of HSET has no severely deleterious effect on spindle morphology or function in vivo. Previous work on this class of kinesin-related motor had shown it to be involved in meiotic spindle assembly and function . To test if perturbation of HSET function blocked meiotic spindle assembly and function in mammalian cells, we injected αHSET-1 antibodies into mouse oocytes . Immunoblot analysis of total protein from mouse oocytes showed that HSET-H is the predominant isoform in these cells, and that our antibodies were specific for HSET-H in this cell type (data not shown). The oocytes were injected at the germinal vesicle stage and allowed to mature for 16 h, after which metaphase II arrest would normally occur. Injected oocytes were then processed for indirect immunofluorescence where we stained for chromatin, tubulin, and the injected antibody. In mock-injected oocytes, which completed meiosis and arrested at metaphase II, the meiotic spindles were typically barrel-shaped with broad poles and few astral microtubules. Eg5 localized strongly to the spindle poles of these cells and HSET localized along the length of the body of the spindle (data not shown). There were also numerous cytoplasmic asters (cytasters) scattered throughout the cytoplasm , but these did not immunostain for HSET or Eg5. Mock injection of oocytes did not affect the progression of meiosis, as ∼70% of cells proceeded through meiosis and arrested at metaphase II within the expected time frame . Oocytes injected with αHSET-1 also progressed through meiosis in the expected time frame, with >70% of αHSET-1–injected oocytes eliciting a first polar body and arresting at metaphase II . Oocytes injected with αHSET-1 and fixed during the first meiotic metaphase showed bipolar spindles in the center of the cell with spindle poles that were broader than in control cells . HSET was observed throughout the length of the spindle, as well as in small aggregates near the microtubule minus ends . The morphology of metaphase II spindles in αHSET-1–injected oocytes, however, was dramatically disrupted compared with either mock-injected cells or metaphase I spindles . The spindle poles were splayed, microtubule minus ends appeared to have lost cohesion, and overall, the spindles had lost bipolarity . In these injected cells, HSET was predominately localized in small aggregates near the microtubule minus ends . To verify that antibody injection was capable of blocking meiosis before metaphase II arrest, we injected antibodies specific to the motor Eg5. Injection of Eg5 antibodies into germinal vesicle stage oocytes blocked the formation of the first bipolar meiotic spindle, with >90% of cells arresting at prometaphase I . In these cells, an astral array of microtubules was assembled around the condensed maternal chromosomes . Therefore, perturbation of Eg5 function blocked the maturation of oocytes at meiosis I. In contrast, perturbation of HSET, while causing obvious defects in the structure of the metaphase I spindle, did not block progression of meiosis until metaphase II. Collectively, these data show that HSET is important for the formation of spindle poles in mammalian oocytes, although the loss of HSET activity was more deleterious to cells in metaphase II than metaphase I of meiosis. The results of these experiments indicate that our antibodies were capable of perturbing meiotic spindle assembly in mouse oocytes, but that they did not alter the normal assembly and function of the mitotic spindle in cultured cells. Mouse oocytes assemble spindles in the absence of conventional centrosomes , and we suspected that centrosomes provide additional structural stability to microtubule minus ends at spindle poles that masked any deleterious effect when HSET motor activity was inhibited in cultured cells. To test this hypothesis, we microinjected HSET-specific antibodies into cultured cells and treated those cells with taxol to induce microtubule aster formation. We reasoned that, if centrosomes stabilize the spindle so that HSET function was nonessential, then microtubule asters induced with taxol (many of which lack centrosomes) should be disrupted by our antibodies. For this experiment, we microinjected cells with either the preimmune antibody (control) or αHSET-1, treated the cells with 10 μM taxol, fixed, and processed the cells for immunofluorescence after they entered mitosis . We stained these cells with antibodies specific for tubulin and NuMA to highlight aster morphology. In cells injected with the preimmune antibody, multiple microtubule asters (13.7 ± 2.6 asters/cell, n = 10) were observed scattered throughout the cell cytoplasm . Each of these asters had NuMA concentrated at the core, consistent with taxol-induced asters in uninjected cells, indicating that mitotic aster formation was unaffected by microinjection. In contrast, cells injected with αHSET-1 display disorganized microtubule bundles extending throughout the cell cytoplasm, and only a few mitotic asters (1.5 ± 1.3 asters/cell, n = 10) after taxol treatment . NuMA associated with both the asters and the microtubule bundles in these cells, and staining with a human centrosome-specific autoimmune serum (courtesy of J.B. Rattner, University of Calgary, Calgary, Alberta, Canada) verified that each aster observed in these cells contained a centrosome (data not shown). Thus, HSET is essential for meiotic spindle assembly under acentrosomal conditions, but HSET is not essential for spindle assembly in cultured cells because any functional role that it plays is covered by the presence of centrosomes. Previously, we have shown that microtubules induced to polymerize with taxol in extracts prepared from mitotic HeLa cells organize into aster-like arrays . The organization of microtubule asters in this system requires the motor activity of cytoplasmic dynein and Eg5, and we proposed that a third motor activity was involved, based on the fact that microtubule asters formed in the complete absence of both cytoplasmic dynein and Eg5 . To determine if HSET has a functional role in organizing microtubule asters in this system, we used our antibodies to specifically perturb HSET activity. Initially, we attempted this by immunodepletion using HSET-specific antibodies. Unfortunately, for reasons that we do not understand, our antibodies maximally depleted only 30% of HSET. This was true regardless of the quantity of antibody used (up to 1 mg). In lieu of immunodepletion, we perturbed the function of HSET by adding our antibodies to the extract. Addition of the preimmune antibody (0.1 mg/ml final concentration) had no effect on the organization of microtubules into asters or on the concentration of NuMA at the aster cores . In contrast, addition of αHSET-1 (0.1 mg/ml final concentration) to the mitotic extract before or after the induction of microtubule asters blocked the formation of organized aster-like structures. Under these conditions, microtubules were not well-organized and were loosely aggregated in large, disorganized arrays with NuMA diffusely distributed throughout the microtubule aggregates . In addition to these morphological analyses, we separated the mitotic extract into soluble and insoluble fractions and examined the behavior of known aster components by immunoblot analysis . These blots showed HSET to be a bona fide aster component because a small percentage of HSET-L consistently associated with the insoluble aster-containing fraction. These blots also show that there was no difference in the efficiency with which any of the known aster components associate with the soluble or aster-containing insoluble fractions in the presence of αHSET-1. Thus, consistent with the data of Walczak et al. 1997 showing that XCTK2 is required for spindle assembly in vitro using extracts prepared from frog eggs, HSET is a component of microtubule asters assembled in this cell free system, and is required for both the formation and maintenance of aster-like arrays. To determine how HSET, cytoplasmic dynein, and Eg5 coordinate microtubule aster formation in this system, we used specific antibodies to perturb the function of each motor individually, as well as to perturb the function of every possible combination of two motors, and all three motors together . In this experiment, antibodies specific for Eg5 and cytoplasmic dynein were used to deplete those proteins from the mitotic extract, either alone or simultaneously. These depleted extracts, as well as a control extract, were then supplemented with either a preimmune antibody or αHSET-1 . Microtubule assembly was then induced with taxol, and the resulting structures were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin and NuMA. The extracts were also separated into soluble, insoluble, and immune pellet fractions, and the behavior of HSET, Eg5, and cytoplasmic dynein within these fractions determined by immunoblot . These immunoblots show that both dynein and Eg5 were depleted to ∼100% in each case. These blots also show that none of these motors coimmunoprecipitated with any of the other motors, consistent with our previously published results . Finally, the immunoblots show that neither the removal of Eg5 and cytoplasmic dynein, nor the addition of αHSET-1, had a detectable effect on the efficiency with which the other motors or NuMA and dynactin (data not shown) associated with the insoluble microtubule pellet fraction. As shown previously, addition of preimmune antibody to the extract had no effect on aster assembly, but addition of αHSET-1 prevented the assembly of mitotic asters . Depletion of Eg5 resulted in microtubule asters that were less tightly focused than the controls . The central core of asters assembled in the Eg5 depleted extract (4.5 ± 0.3 μm, n = 12) were also expanded, relative to the central core of asters in the control extract (2.3 ± 0.4 μm, n = 12), as judged by staining for NuMA. Addition of αHSET-1 to an Eg5 depleted extract resulted in microtubule asters that greatly resemble asters formed under control conditions . Asters formed in the absence of Eg5 and the presence of αHSET-1 were tightly focused, with NuMA well-concentrated at the central core (2.5 ± 0.4 μm, n = 12). This result shows that, while addition of αHSET-1 alone prevented microtubule aster formation , the HSET antibody did not block aster formation if Eg5 was absent . This result is consistent with the view that microtubule aster formation in this system requires a balance of forces . When HSET alone is perturbed, the balance of forces is upset so that asters cannot form. Microtubule aster formation can be restored under conditions where HSET is perturbed if the balance of forces is equilibrated by also removing the motor activity of Eg5. This result indicates that the minus end-directed activity of HSET antagonizes the plus end-directed activity of Eg5 during microtubule aster assembly in this system. We next tested the effect on microtubule aster assembly if both minus end-directed motors were perturbed. In the absence of cytoplasmic dynein, microtubules fail to organize into aster-like arrays and were randomly dispersed with NuMA distributed along the length of many of the microtubule polymers . Addition of αHSET-1 to the cytoplasmic dynein depleted extract yielded no microtubule asters and only random microtubule distributions . These results show that microtubule asters fail to form in the absence of HSET alone, cytoplasmic dynein alone, or both HSET and cytoplasmic dynein. The data presented in Fig. 6B and Fig. D , indicate that the activities of HSET and Eg5 act antagonistically in driving microtubule aster formation in this system. This antagonism is similar to the relationship that we showed previously for cytoplasmic dynein and Eg5 , which is reproduced here in Fig. 6E and Fig. G . These results show that both of these two minus end-directed motors antagonize Eg5 during microtubule aster formation. However, these results do not discriminate between the possibilities that these two motors act together to antagonize Eg5, or that these two motors act independently, with each antagonizing Eg5. To distinguish between these possibilities, we perturbed the function of all three of these motors, reasoning that if these two minus end-directed motors act together, then the perturbation of both minus end motors in an Eg5 depleted extract should yield results similar to the perturbation of either minus end-directed motor alone in an Eg5 depleted extract (i.e., mitotic asters should form). The results of perturbing HSET in a cytoplasmic dynein and Eg5 depleted extract show that aster-like arrays did not form, and that the microtubules were randomly dispersed . The lack of microtubule aster formation in the absence of all three motors is in stark contrast to the microtubule asters that form in the absence of either Eg5 and HSET or Eg5 and cytoplasmic dynein . This demonstrates that these two motors act independently of each other in antagonizing Eg5 activity in this system. We estimated the microtubule aster forming capacity of the mitotic extracts during these various depletion experiments by counting the total number of microtubule asters in 20 randomly selected microscope fields . These counts demonstrate that the microtubule aster forming capacity of the extracts depleted for Eg5, Eg5 and cytoplasmic dynein, or Eg5 with the addition of the HSET antibody were comparable to that of a control extract. On the other hand, if the extract was depleted of cytoplasmic dynein, or if the HSET antibody was added to the extract alone, extract depleted of cytoplasmic dynein, or extract depleted of both cytoplasmic dynein and Eg5, then virtually no microtubule asters were observed. Thus, the images shown in Fig. 6 are representative of the populations of microtubule structures observed under each condition tested. Collectively, the results from the experiments presented in Fig. 6 and Fig. 7 lead to three conclusions. First, the minus end-directed activity of HSET opposes the plus end-directed activity of Eg5 in a way that is similar to the opposition between cytoplasmic dynein and Eg5. Second, the minus end-directed activities of HSET and cytoplasmic dynein oppose the plus end-directed activity of Eg5 independently of each other. Third, although we cannot rule out a minor role played by other motors, the lack of microtubule organization in the absence of all three of these motors indicates that HSET, Eg5, and cytoplasmic dynein are most likely the primary motors responsible for building microtubule asters in this system. Finally, we tested if HSET functionally opposes Eg5 activity in vivo. For this experiment, we microinjected human CF-PAC1 cells with either antibodies specific for Eg5 or a combination of HSET antibodies and Eg5 antibodies. We monitored the injected cells and fixed and processed them for indirect immunofluorescence using antibodies specific for γ-tubulin to detect centrosomes and for the injected rabbit antibody. Cells injected with the Eg5 antibody alone formed monopolar spindles and arrested in mitosis . More than 75% of Eg5-injected cells had centrosomes that had not separated to any measurable degree ( Table ). In contrast, >68% of cells injected with both HSET and Eg5 antibodies displayed separated centrosomes ( Table ). Many of the double injected cells did not have a symmetric spindle at the time of fixation, as judged by the lack of a well-organized metaphase plate or the location of both centrosomes on the same side of the chromosomes. The centrosomes in these cells were clearly separated, but were frequently in different focal planes within the cell, which accounts for the variable intensity of each centrosome shown in Fig. 8 . In some instances, symmetric, bipolar spindles formed under these conditions , and we observed a small fraction of cells (4%) complete mitosis normally, forming pairs of G1 cells with recognizable midbodies (data not shown). These results show a statistically significant increase in centrosome separation in cells injected with antibodies to both HSET and Eg5, compared with cells injected with Eg5 antibodies only. Thus, with respect to centrosome separation, these data indicate that HSET and Eg5 oppose each other in vivo. Furthermore, these results indicate that centrosome separation can proceed under conditions where Eg5 function is blocked. Previous examination of mitotic spindles in cultured cells by EM has revealed a significant amount of bundling among spindle microtubules . Also, numerous articles in the literature have reported the visualization of structures cross-linking microtubules in spindles . Consistent with these early descriptive reports, we show here that the kinesin-related protein, HSET, is distributed throughout the main body of the spindle and localizes between microtubules in the metaphase spindle of cultured human cells. This localization, coupled with reports that this class of kinesin protein possesses two (or more) microtubule binding sites , is capable of generating extensive parallel microtubule bundles when expressed in Sf9 cells , and induces microtubule bundles in Xenopus egg extracts , indicates that HSET most likely participates in spindle assembly and function by promoting microtubule bundling through a cross-linking function. What role does microtubule cross-linking by HSET play during spindle assembly? HSET is a member of the kinesin-related proteins that possess minus end-directed motor activity. Coupling this motor activity to microtubule cross-linking activity generates a molecule with the potential to slide one microtubule relative to another. As proposed previously , molecules that combine such unidirectional microtubule motor and cross-linking activities have the potential to promote specific microtubule end convergence, and given that HSET is a minus end-directed motor, it would foster microtubule minus end convergence. During spindle assembly, this activity would participate in focusing microtubule minus ends at the poles, a view supported by the fact that spindle poles are poorly organized when HSET (or its homologues) are perturbed . This function for HSET would overlap that of cytoplasmic dynein, which also promotes microtubule minus end focusing at spindle poles . The work presented here, along with results from frog egg extracts , show that both of these minus end-directed motors participate in focusing microtubule minus ends. However, this function for HSET is only essential to spindles (or microtubule asters) assembled under acentrosomal conditions. Evidence presented here indicates that centrosomes compensate for HSET, rendering it nonessential for mitotic spindle assembly in cultured cells. This distinction in whether HSET activity is essential depending on the presence or absence of centrosomes is similar to the observation that flies carrying mutant ncd alleles show more severe spindle defects during female meiosis, compared with mitosis . We report here that the minus end-directed activity of HSET opposes the plus end-directed activity of Eg5 during microtubule aster assembly in vitro, and centrosome separation and spindle assembly in vivo. This antagonistic relationship is analogous to that seen for members of the KAR3 and bimC families of kinesin-related proteins in budding yeast , fission yeast , filamentous fungi , and Drosophila . Our work represents the first demonstration of such an antagonistic relationship between these classes of kinesin-related protein in a mammalian system. In cultured cells, frog egg extracts, Drosophila embryos, and fungi, centrosomes (spindle pole bodies) do not separate in the absence of bimC motor activity, and monopolar spindles result . In each of these experimental systems tested so far, the failure in centrosome separation in the absence of the bimC motor can be relieved by simultaneously perturbing the function of the KAR3 motor . As originally proposed by Saunders and Hoyt 1992 , a likely explanation for how these oppositely oriented motor activities establish and/or maintain centrosome (spindle pole body) separation involves the cross-linking of antiparallel microtubules projecting from each centrosome (spindle pole body). Sliding of these cross-linked antiparallel microtubules relative to each other by the plus end-directed motor (bimC) would push centrosomes apart, while the minus end-directed motor (KAR3) would draw the two centrosomes toward each other. When these two forces become unbalanced in the absence of plus end-directed motor activity, centrosome separation fails, due to the uncontested inward force generated by the minus end-directed motor. Centrosome separation can be restored in the absence of plus end-directed motor activity by reestablishing a balance to the forces acting on centrosomes (spindle pole bodies) by eliminating the activity of the minus end-directed motor. While this model fits much of the experimental data, there are features of centrosome separation in animal cells that are not fully consistent with such a hypothesis . First, Sharp et al. 1999a have found that centrosomes separate during prophase in Drosophila embryos before the release of KLP61F (Eg5) from the nuclear compartment. Furthermore, they reported that when KLP61F activity is perturbed by antibody microinjection, centrosomes separate efficiently during prophase, but subsequently collapse upon each other during later stages of mitosis, leading to the characteristic monopolar spindles . This suggests that the process of centrosome separation in Drosophila embryos, and perhaps animal cells, might have two distinct phases, an initial separation phase and a subsequent maintenance phase. These data also suggest that KLP61F is critical for the maintenance phase of centrosome separation, but not essential for the initial separation phase . The initial phase of centrosome separation may be driven by means involving other motor molecules that have been implicated in this process . Another striking difference between centrosome separation in fungal and animal systems was identified by Waters et al. 1993 . They showed that the forces acting to separate the mitotic asters in cultured cells are intrinsic to each aster, and that each aster moved independently from the other. This data contradicts the idea that the cross-linking and subsequent sliding of antiparallel microtubules projecting from the two centrosomes is involved in separating centrosomes in these cells. Here, we suggest an alternative viewpoint for the maintenance phase of centrosome separation which involves forces that motors, principally Eg5 and HSET, could exert along microtubules that are oriented parallel to one another within the spindle, and would therefore be contained within each centrosomal aster (half spindle). For example, Eg5 could associate with kinetochore fibers where microtubules have parallel orientations. Most microtubules in kinetochore fibers have their plus and minus ends anchored at the kinetochores and spindle poles, respectively. Other microtubules within these fibers, however, are not anchored to kinetochores, spindle poles, or both . The cross-linking and plus end-directed activities of Eg5 could generate a net poleward movement on the subset of unanchored microtubules within kinetochore fibers. This microtubule sliding would exert a force on the centrosome away from the chromosome, and consequently, away from the other centrosome. HSET would antagonize the activity of Eg5 in this context through an analogous mechanism using its minus end-directed activity. Whether HSET exerts a poleward (as for spindle pole organization discussed previously) or away from the pole force on unanchored microtubules within the spindle would depend on the orientation with which this asymmetric motor molecule cross-links microtubules . The idea that these two motors act on parallel microtubules, while counter to prevalent models, is supported by the localization of subsets of both HSET (this report) and Eg5 in the main body of each half spindle among microtubules with parallel orientations. In the end, the cross-linking and oppositely oriented motor activities of these two kinesin-related proteins may act to maintain centrosome separation in animal cells using mechanisms that involve forces generated on microtubules with both parallel and antiparallel orientations. | Study | biomedical | en | 0.999997 |
10525541 | β-Catenin protein containing an NH 2 -terminal 6-His tag was expressed in Sf9 cells using a baculoviral expression system, as described previously . Expressed protein was purified on NTA affinity resin (Qiagen). Eggs were obtained from HCG-treated Xenopus females (Nasco), and were fertilized by standard procedures. Cleavage blockade was achieved by a low-speed centrifugation procedure described by Newport and Kirschner 1982 , and is described briefly below. 45 min after fertilization, embryos (in 0.1× MMR) were layered onto a 50% Ficoll cushion (in 0.1× MMR) and centrifuged at 500 g for 10 min at 15°C. Noncentrifuged sibling embryos were maintained as a reference for developmental stage. After centrifugation, cleavage blocked (coenocytic), multinucleate embryos and nonblocked sibling embryos were incubated at 18°C. Several hours after centrifugation, coenocytic embryos that showed clear evidence of attempted cleavage (evidenced by furrows in the lipid cap) were selected for use in the β-catenin signaling assay. A schematic diagram of the cell-free assay used in these studies is shown in Fig. 1 . When sibling control embryos reached stage 8 (unless otherwise noted), 20 coenocytic embryos were placed in 1.8-ml centrifuge tubes (one tube per experimental condition). To minimize dilution of cytoplasm upon disruption, 0.5 ml Versilube (Andpak-EMA) was layered over the embryos, followed by a brief spin at 300 g , which effectively separates embryos from aqueous buffer. The overlying separated buffer droplet and Versilube oil was aspirated gently and, as completely as possible, from the embryos. Before crushing embryos, a number of components were added over the intact coenocytic embryos. Proteinase inhibitors (PMSF + protease inhibitor cocktail II) were added to give the following cytoplasmic concentrations: 1 mM PMSF, 2 μg/ml leupeptin, 4 μg/ml aprotinin, 10 μg/ml antipain, 50 μg/ml benzamidine, 10 μg/ml STI, 100 μg/ml iodoacetamide. To provide for regeneration of cytoplasmic ATP stores, an energy mix was added to give cytoplasmic concentrations of 7.5 mM creatine phosphate, 1.0 mM ATP, and 50 μM magnesium chloride. To block protein synthesis, cycloheximide was added to give a cytoplasmic concentration of 5 μg/ml. Indicated amounts of β-catenin protein or lithium were then added to the embryos, followed by a 7-min spin crush centrifugation at 16,000 g (maximum centrifugal force in Eppendorf microcentrifuge) at 4–8°C. After spin crush, tubes were incubated at 22°C for 1.5 h, which is sufficient for maximal induction and expression of target genes, as indicated by semiquantitative reverse transcriptase (RT)-PCR. Increasing incubation time beyond this point was found to have detrimental effects on reproducibility. Proteasome inhibitors ALLN and MG132 (both purchased from Sigma Chemical Co.; stocks were dissolved in ethanol at 25 mM) were included under certain experimental conditions in the cell-free assay. In these cases, the proteasome inhibitors were diluted 1:1,000 to give a cytoplasmic concentration of ∼25 μM. Total RNA was isolated from crushed embryos using Ultraspec RNA isolation reagent (Biotecx). RNA from 20 embryos was resuspended in 400 μl of DEPC ddH 2 O (to give RNA concentrations of ∼300 μg/ml). Detection of gene expression was carried out using standard RT-PCR procedures using 32 P-labeled dCTP and autoradiographic detection. Sequences of primer pairs used to detect the expression of various early Xenopus genes were as follows: EF-1 α upstream, 5′ CAGATTGGTGCTGGATATGC 3′; EF-1 α downstream, 5′ ACTGCCTTGATGACTCCTAG 3′; Siamois upstream, 5′ CGCGGATCCATGGCCTATGAGGCTGAAATGGAG 3′; Siamois downstream, 5′ GCTCTAGAGAAGTCAGTTTGGGTAGGGCT 3′; XTwin upstream, 5′ CATGACTTGTTGACTCTGA 3′; XTwin downstream, 5′ TGGCGTAGATCCCAGTAGA 3′; Xnr3 upstream, 5′ ATGGCATTTCTGAACCTG 3′; Xnr3 downstream, 5′ TCTACTGTCACACTGTGA 3′; Cerberus upstream, 5′ ATGTTACTAAATGTACTCAG 3′; Cerberus downstream, 5′ CTTGGCACCAGGCTTTTC 3′; Noggin upstream, 5′ ATGGATCATTCCCAGTGC 3′; Noggin downstream, 5′ TCTGTGCTTTTTGCTCTG 3′; Forkhead upstream, 5′ATGCTAAATAGAGTC-AAG3′; Forkhead downstream, 5′ GTAAGAGTATGGGGGCTT 3′; Xlim upstream, 5′ ATGGTTCACTGTGCTGGA 3′; Xlim downstream, 5′ GGGGTCACTGCCTGTTAC 3′; Follistatin upstream, 5′ ATGTTAAATGAAAGGATCCAG 3′; Follistatin downstream, 5′ TCTTCCCAGGGCCACAGTC 3′; Xotx2 upstream, 5′ ATGATGTCTTATCTCAAGC 3′; Xotx2 downstream, 5′ TGGCCTCCATTCTGCTGC 3′. Following standard electrophoretic and transfer procedures, Xenopus β-catenin was detected in Western blots using a polyclonal rabbit antibody directed against the NH 2 -terminal region of the protein. Proteins from crushed embryos were extracted with 1% NP-40 extraction buffer. For ConA precipitations of cadherin-bound β-catenin, 1/10 vol of a 1:1 slurry of ConA-Sepharose beads were added to protein extracts, and allowed to incubate 4 h overnight at 4°C. The efficacy of the ConA precipitations was indicated by the loss of detectable C-cadherin in Western blots. To test the effectiveness of cycloheximide inhibition of protein synthesis, 7 μCi of 35 S methionine (ProMix cell labeling mix; Nycomed Amersham, Inc., Pharmacia Biotech) was added to stage 8 coenocytic embryos before the spin crush step in the cell-free assay procedure. After 1.5 h, these samples were extracted in 1% NP-40, soluble material was separated electrophoretically, and visualized using fluorography. Immunoprecipitation of β-catenin was carried out using a rabbit polyclonal antibody directed against the NH 2 terminus of the protein, followed by incubation with protein A–Sepharose beads. In these studies, our goal was the development of a cell-free assay for analysis of the β-catenin signaling pathway. Such a system would require both transcriptionally active nuclei and cytoplasm that is competent to transmit a β-catenin dependent signal. Such a system would also require that, in the absence of added β-catenin (or alternative activators of the pathway), potential target genes of β-catenin signaling be either off or expressed at a low, basal level. Cleavage blockade of embryos would be expected to preclude any intercellular communications that are required for specific activation of dorsal genes, and would result in a single, freely diffusing cytoplasm. The induction of a number of genes known to be downstream targets in the β-catenin signaling pathway during early development is significantly reduced in coenocytic embryos, compared with normal embryos , although zygotic transcription occurs normally in these embryos . Therefore, the β-catenin mediated signaling events that induce target gene expression in normal embryos appear to be significantly reduced or disrupted in cleavage blocked embryos. We developed a novel cell-free assay that used endogenous nuclei and cytoplasm from cleavage-blocked embryos. Induction of the expression of Siamois , a known target gene of β-catenin signaling , was responsive to added β-catenin protein in a dose-dependent manner . The levels of exogenous β-catenin required for these inductions was relatively low, compared with total endogenous β-catenin, but roughly similar to the levels of the nonglycoprotein-associated (presumably cytosolic) pools of β-catenin . The addition of β-catenin into the crushed embryo system induced the expression of a number of genes previously described as direct targets of β-catenin signaling. β-catenin induced expression of Siamois , Xtwn , and Xnr3 . These inductions demonstrate that all required steps for β-catenin signaling, including nuclear localization, interaction with TCF, and activation of transcription at target sites can all occur in this disrupted assay system. To further characterize the β-catenin induced signaling events that occur in the cell-free assay system, we carried out a comprehensive analysis of various early marker genes, focusing on those expressed in the Nieuwkoop center or Spemann organizer. In addition to inducing Siamois , Xnr3 , and XTwin , β-catenin resulted in a weak, but reproducible, induction in the expression of Cerberus , a novel secreted factor expressed in the anterior endoderm that gives rise to head structures . All of the genes induced by β-catenin were induced in a protein synthesis independent manner, suggesting these inductive events are direct. The ability of β-catenin to directly target Cerberus is a novel observation, as previous studies have suggested a requirement for intervening synthesis of Xtwn or Siamois protein. Goosecoid , which has been shown to respond to β-catenin signaling through intervening expression of XTwin and Siamois , is not induced by β-catenin protein under conditions where Siamois , Xnr3 , and XTwin are induced. A number of other dorsal marker genes were evaluated , none of which were induced by β-catenin, either in the presence or absence of protein synthesis. It is known that lithium has dorsalizing effects on Xenopus embryos . While lithium is thought to inhibit the phosphoinositol cycle in Xenopus , it is likely that the dorsalizing activity of lithium occurs through activation of the Wnt pathway by direct inhibition of GSK-3 . In the prevailing turnover model for Wnt signaling, inactivation of GSK-3 by lithium results in blockage of the ongoing ubiquitin-dependent degradation of cytosolic β-catenin as a result of its dephosphorylation. This stabilization of β-catenin is thought to lead to its accumulation in the cytoplasm, which in turn allows for activation of downstream dorsal target genes. In the turnover model, continuing synthesis of β-catenin would provide the source of protein that would allow for enhanced cytosolic levels. In the cell-free assay, Siamois was induced by the addition of lithium . The inductive effect of lithium was dependent on β-catenin, as sequestration of β-catenin by addition of purified C-cadherin intracellular domain protein was inhibitory . In contrast, an equivalent amount of the juxtamembrane portion of the intracellular domain of C-cadherin, which does not bind β-catenin, was not inhibitory . Therefore, as in normal embryos, lithium can activate an endogenous signaling pool of β-catenin in the cell-free assay system. Induction of Siamois by lithium occurred in the absence of protein synthesis , suggesting that the β-catenin responsible for this signaling event is derived from preexisting pools, not from newly translated protein. This lack of a requirement for ongoing protein synthesis was surprising in the context of the current turnover model for β-catenin–dependent signaling, in which increased levels of cytosolic β-catenin are responsible for activation of the signaling pathway. It is thought that proteasome-dependent degradation of β-catenin in the absence of upstream signal serves to block accumulation of cytosolic pools. In other systems, the signaling activity of lithium and proteasomal inhibitors have been correlated with increased levels of β-catenin protein. In our in vitro assay system, there was no detectable increase in β-catenin levels (total or nonglycoprotein-associated) in response to activating levels of added lithium . However, it should be noted that the lithium induction of Siamois (approximately twofold) is relatively weak, compared with maximal induction brought about by β-catenin protein (greater than tenfold). Based on our titration of β-catenin protein , a response similar to that seen with lithium would require an approximate twofold increase over levels of cytoplasmic β-catenin. Thus, while we detect no such changes in levels in our experiments, it may be difficult to detect minor changes in β-catenin protein levels. The addition of proteasome inhibitors ALLN or MG132, like lithium, did not detectably alter β-catenin levels in our assay system (data not shown). In addition, inclusion of proteasome inhibitors did not lead to an overall increase in polyubiquitinated proteins, as assessed by Western blots using antiubiquitin antibodies (data not shown), suggesting that proteasomal degradation machinery may not be strongly active in our assay system. Furthermore, proteasome inhibitors did not activate Siamois , nor did they have any effect on lithium-induced activation of Siamois . Taken together, these results suggest that activation of β-catenin signaling by lithium likely involves mechanisms other than modulation of levels through regulation of proteasomal degradation. We have developed a novel cell-free assay system that recapitulates β-catenin–mediated signaling events occurring in the Xenopus embryo. From a developmental standpoint, this assay system has general utility in identification of transcriptional targets of early developmental signaling. In addition, this assay provides a potentially useful new tool for molecular analysis of the complex regulatory mechanisms that control β-catenin signaling. This cell-free assay system offers several clear advantages over cleaving embryos as a system for identification of direct transcriptional target genes. First, in the cell-free assay system, signaling occurs in a single, uniform cytoplasm in the absence of any intercellular communication, which could lead to secondary inductions. Secondly, as signaling components in the assay are introduced at a time when the embryo is able to respond transcriptionally, the assay allows for a precise timing of inductive events. This is in contrast with RNA injection into cleaving embryos, which requires injection before the onset of zygotic transcription. Because of these advantages, the cell-free assay holds promise not just for studies of β-catenin signaling, but may also be adapted for other signaling pathways in the early embryo. To confirm the efficacy of our assay system, we first evaluated the ability of β-catenin to induce expected target genes. Siamois , XTwin , and Xnr3 have all been reported to be direct targets of β-catenin signaling, and were all found to be specifically induced in the cell-free assay system. Thus, all required events for induction of target genes, including nuclear localization and interaction with TCF family members, can occur in the cell-free system. In addition to known target genes, the assay allowed us to identify Cerberus as an additional direct target of β-catenin signaling. Previous reports have shown that activation of the β-catenin signaling pathway results in induction of Cerberus . These whole embryo injection studies suggested that Cerberus may be induced through the secondary inductive effects of the Siamois gene product, rather than direct activation by β-catenin . It is clear in our assay, however, that induction of Siamois or Twin proteins is not required for the Cerberus response to β-catenin, as the Cerberus induction is protein synthesis independent. There are two possible explanations for these differing results. First, as there is some basal level of expression of Siamois and Twin in our assay, it is possible that β-catenin/TCF and Siamois and/or Twin protein cooperate at the Cerberus promoter. Alternatively, in the embryo, it is possible that Siamois may activate β-catenin signaling, which then directly activates Cerberus . Indeed, there is evidence that Siamois induces itself by a feedback mechanism and, given the strong direct inductive effects of β-catenin on Siamois , it is not unreasonable to consider that this may occur indirectly via β-catenin. While we observed a number of direct inductions of dorsal genes, we found no evidence for secondary inductions (that is, inductions that require intervening cell-cell communications or protein synthesis) under the conditions used for the assay. Particularly notable, in this regard, is the absence of Goosecoid induction. Goosecoid has been reported to involve wnt/β-catenin responsive elements, as well as activin/TGFβ responsive elements . Wnt/β-catenin responsiveness has been attributed to direct binding of Xtwn (and likely Siamois ) at the Goosecoid promoter . Since XTwin mRNA expression is strongly induced in the cell-free assay system, the absence of Goosecoid induction is of interest. While we cannot rule out technical reasons for the absence of Goosecoid induction (i.e., insufficient time for secondary inductions or inappropriate cytoplasmic environment), it is possible that induction at the Goosecoid promoter normally requires additional input that in normal embryos would derive from a Nieuwkoop center signaling activity. In addition, none of the other early dorsal marker genes we evaluated were induced by β-catenin, either in the presence or absence of protein synthesis. This would suggest that intervening cell–cell communication or synthesis steps that are required for activation of these genes do not occur in the cell-free assay system. In addition to identification of target genes, we also used the cell-free assay to examine the source of endogenous signaling pools of β-catenin after addition of lithium. While lithium is known to exert effects on phosphoinositol metabolism in a number of systems, more recent evidence suggests that the dorsalizing effect of lithium on early Xenopus embryos occurs primarily through lithium's inhibitory effects on GSK-3 . According to the current turnover model for regulation of β-catenin signaling , the downregulation of GSK-3 activity results in the stabilization of free cytoplasmic pools of β-catenin in dorsal cytoplasm, and subsequent activation of dorsal genes. In Drosophila , activation of the Wnt pathway can lead to a net increase in β-catenin in cells, presumably through accumulation of newly synthesized protein . The addition of lithium in our assay system results in an induction of Siamois expression. This induction requires the participation of free pools of β-catenin, as is indicated by the sensitivity of this response to added C-cadherin intracellular domain protein. These observations indicate that, in the cell-free assay system, lithium is capable of generating an endogenous signaling pool of β-catenin. In addition, the insensitivity of the inductive response to protein synthesis inhibition suggests that accumulation of newly synthesized β-catenin is not required for β-catenin signaling activity. Rather, it appears that actively signaling β-catenin protein can be wholly derived from a preexisting, nonsignaling pool (for instance, pools associated with cadherins or cytoplasmic molecules, such as axin and APC), with no requirement for newly synthesized protein. In these studies, lithium activates endogenous β-catenin signaling in the absence of an input of protein from new synthesis and without detectable increases in cytoplasmic levels of β-catenin. Furthermore, while in some systems proteasomal inhibitors have been shown to activate β-catenin signaling , proteasomal inhibitors in our assay had no effect on levels of β-catenin and did not influence the activation of the signaling pathway with lithium. Thus, signaling in this assay does not appear to rely on the accumulation of cytoplasmic β-catenin brought about by regulation of the proteasomal degradation pathway. Rather, these results could be explained by an alteration in the intrinsic signaling activity of cytoplasmic β-catenin. Indeed, recent studies in our laboratory have indicated that putative GSK-3 phosphorylation sites in the NH 2 terminus of β-catenin may regulate its signaling activity independent of levels (Gumbiner, B.M., manuscript submitted for publication), even in the whole embryo. These findings suggest the possibility of the existence of an alternative mechanism of activation that may act independently or in parallel with regulation of cytoplasmic levels of β-catenin. By allowing direct access to the cytoplasm, the described cell-free assay makes possible a wide range of potential biochemical manipulations in addition to the applications presented here. For example, conditions of this assay could be adapted to allow for testing the effects of immunodepletion of associated components of the β-catenin signaling pathway. Also, the assay is well-suited to allow for assessing the signaling activity of exogenously derived cytoplasm, or for testing cytoplasmic fractions or various purified components for regulatory activities. | Study | biomedical | en | 0.999997 |
10525542 | The generation of the polyclonal antibody anti-CRYPα and its purification have been previously described . The polyclonal anti-NCAM antibody and its function-blocking activity have been characterized . The monoclonal antibody against β1-integrin was JG22 . The CRYPα1-AP and Ig3-AP fusion proteins were generated by subcloning CRYPα1 fragments spanning amino acids 1–721 and 1–316 into the APtag2 vector . AP was generated by transfecting the APtag4 vector . All three vectors were transfected into cos7 or 293T cells using Superfect TM (Qiagen) and the proteins were collected after 6 d in the conditioned medium. Medium was passed through an anti-AP agarose column (Sigma), and the fusion protein eluted using 0.1 M glycine-HCl, pH 2.5, with immediate buffering to pH 8 with Tris-HCl. The protein was dialyzed against TBS buffer and stored at 4°C. Laminin-1 and matrigel were obtained from Becton Dickinson. N-cadherin was purified as described . E6 retinal cryosections were prepared as previously described and incubated with α1-AP. The AP staining method was also previously described . The basal membrane assay was performed as described in Halfter et al. 1987 . The basal lamina was prepared from E7 retina. In the first step, the retina is flatmounted on a nitrocellulose filter (Sartorius AG). The filter with the attached retina is then put upside down on a poly- l -lysine (PLL)-coated glass coverslip . Two small metal bars are put on the filter before it is incubated for 10 min at 37°C. Afterwards the filter is lifted up while the basal lamina sticks to the glass surface . To remove the glial endfeet from the basal lamina, glass coverslips were washed with 2% Triton X-100 for 5 min. The Triton was removed after three 10-min wash steps with Hank's solution. For experiments with laminin as a growth substrate glass coverslips were coated with laminin at 37°C for 2 h and afterwards washed with Hank's. Coating of glass coverslips with matrigel was carried out according to the manufacturer's protocol. For experiments with N-cadherin as a growth substrate, PLL-precoated glass coverslips were coated with N-cadherin at 37°C for 3 h, resulting in a PLL/N-cadherin substrate mix. Retinal explants as outgrowth source were prepared from E6 retinae as previously described . Cultures were incubated for 24 h at 37°C in 1 ml F12 medium containing methylcellulose and various amounts of preadded different antibody in a humidified chamber (5% CO 2 ). After incubation cultures were immediately fixed in 4% paraformaldehyde (dissolved in 0.1 M phosphate buffer) for 10 min at room temperature. They were permeabilized with 0.1% Triton X-100 in PBS and blocked by 1% BSA in PBS. Stainings with Alexa-labeled phalloidin were performed according to the manufacturer's protocol (Molecular Probes Inc.). The fixed cultures were finally covered with moviol. Analysis was done using an Axiophot (Zeiss) fluorescence microscope using a Sony CCD-camera together with the Analysis program (SIS). To quantify outgrowth real-time pictures were taken and directly measured on the screen. The average axon length of a culture was determined as the distance from the explant to the region reached by at least 60% of the axons (longer and shorter neurites were not considered) . Growth cone morphology was analyzed by measuring two diameters of a growth cone using a 100× objective . The d1 parameter represents the length of the growth cone in μm, measured from the growth cone-axon neck to the border of the leading lamellae, and the d2 parameter represents its width, characterized mainly by the extension of its lamellipodia. The data were analyzed by regression analysis, ANOVA, and Student's t test using the EXCEL 98 program (Microsoft) and StatView 4.5 (Abacus Concepts Inc.). Immunostaining with CRYPα-specific antibodies strongly labels retinal axons inside the retina, optic nerve, and tract, and in the optic tectum of the chick embryo . CRYPα is already present at E4 on retinal axons and growth cones. Two isoforms of CRYPα, CRYPα1 and α2 , were described, both containing 3 Ig-like domains but differing in the number of fibronectin type III-like domains (FNIII). α1 has four and α2 has eight FNIII repeats, respectively . Until E7, only CRYPα1 is expressed in retinal ganglion cells, whereas CRYPα2 is coexpressed by E8 . We suggest, therefore, that for the early stages of retinal optic fiber growth, CRYPα1 is the relevant isoform. To examine the expression of potential ligands of this PTP, the extracellular domain of the CRYPα1 isoform was fused to the enzyme alkaline phosphatase and the fusion protein α1-AP was used to analyze the localization of the ligands in the developing chick retinotectal system . Using α1-AP , a ligand was detected at E6 in the basal membrane of the retina , the optic stalk and the optic chiasm and in many basal membranes throughout the embryonic brain (Stoker, A.W., unpublished observations). The expression of the putative ligand in the retinal basal membrane prompted us to use this membrane as a growth substrate for retinal axons and to analyze what role the interaction of CRYPα with its ligand has in retinal axon growth. To this end, retinal basal membranes were isolated according to a published method and retinal strips were explanted. Retinal axons were grown on retinal basal membranes or on laminin for 24 h either in the presence of polyclonal antibodies directed against the two outermost Ig-like domains of CRYPα , or in the presence of α1-AP, thereby disturbing ligand-receptor interactions. Importantly, the IG2 antibody is known to block the binding of α1-AP to the basal membrane ligand and therefore is predicted to block CRYPα ligand interactions in general. A fusion protein containing the 3 Ig domains of CRYPα was used as a negative control, as previous experiments suggested that this protein does not to bind to the CRYPα ligand . To analyze the effect of disturbing the CRYPα-ligand interaction, the average length of retinal axons leaving the explant was measured from the edge of the explant to the front of the majority of axons . The IG2 antibody and α1-AP strongly reduced the length of retinal axons, in a dose-dependent manner on BM . We obtained the same result in preliminary studies with IG2 antibodies on the tectal membrane stripe assay with tectal membrane preparations (Ledig, M.M., and B.K. Mueller, unpublished data). Outgrowth of both nasal and temporal axons was reduced, but there was no effect on the decision behavior (data not shown). 125 pmol of IG2 reduced retinal axon length by 63% on basal membrane and by 25% on LN . 26 pmol α1-AP reduced retinal axon length on BM by ∼50% . No comparable effect was observed with Ig3-AP on BM (data not shown) or with α1-AP or Ig3-AP when retinal axons were grown on LN . A slight dose-dependent effect, caused by the alkaline phosphatase (AP), was observed on both LN and retinal BM, but only when used at high levels . 125 pmol of NCAM antibody was used as a control for antibody binding and did not influence retinal axon length on LN or BM . Moreover, there were also no obvious effects on fasciculation by IG2 or by the polyclonal control antibody NCAM (data not shown). A second antibody, directed against the β1-integrin chain (JG22), was used to compare the effect of CRYPα1 to one of the major receptors for ECM molecules. Using this antibody significantly reduced retinal axon length in a dose-dependent manner on both substrates. 125 pmol of JG22 blocked retinal axon growth on LN almost completely . On BM retinal axon length was reduced by 71% . This suggests the presence of different outgrowth promoting activities in basal membranes, mediating their effects by at least two different receptor systems, the integrins, and the RPTP CRYPα. The d1 and d2 parameters were used to measure growth cone extensions . Retinal axons growing on the retinal basal membrane or on laminin exhibit a morphology with few filopodia but elaborate lamellipodia . In the presence of increasing amounts of IG2 or α1-AP, lamellipodia are gradually retracted and the number of long filopodia is increased . This striking transition from a lamellipodial to a filopodial growth cone morphology was most evident after adding 125 pmol IG2 or 26 pmol of α1-AP ( Table ). Significantly, this was only observed on retinal BM but not on LN . The transition in growth cone morphology was reflected in a >40% reduction of the growth cone width (d2) without any significant change of its length (d1) ( Table ). Neither control antibody anti-NCAM (125 pmol) nor AP (30 pmol) influenced retinal growth cone morphology on either substrate . A similar change in growth cone morphology was observed after adding 62.5 or 125 pmol of the β1 integrin antibody JG22 . In contrast to inhibition of the CRYPα ligand interaction, the morphological change seen with JG22 was not restricted to growth cones on BM but was also found for growth cones growing on LN . Therefore, blocking the interaction of the RPTP CRYPα with its ligand from either the receptor or the ligand side, not only reduced retinal axon length but induced a significant shift in the morphology of retinal growth cones to a more filopodial appearance. This suggests that CRYPα action influences the maintenance of lamellipodia when growth cones are migrating on the intact retinal BM substrate. The retinal basal membrane contains the endfeet of the Mueller glia cells. These endfeet are visible under microscopic bright field illumination and are stained by Alexa-phalloidin . Using such a staining procedure, endfeet appear as dot like structures with a diameter of ∼2 μm and with a ring-like F-actin organization. Incubating the α1-AP protein on E7 basal membranes resulted in staining of only these glial endfeet . This suggests that the putative ligand for CRYPα1 is located on the glial endfeet. We tested this possibility by removing the endfeet from the retinal BM. Removal of the endfeet was performed by washing the retinal basal membrane with 2% Triton X-100 in PBS according to a published protocol and resulted in a completely transparent basal membrane (BM-Ef). In accordance with our hypothesis, there was no detectable staining of BM-Ef using the α1-AP protein . Washing away the endfeet demonstrated the enormous outgrowth-promoting potential of the ECM. Whereas the average axon length in controls was almost the same on LN and BM, removal of the endfeet caused an increase in the average axon length of >40% . However, growth on BM-Ef was much less susceptible to inhibition with IG2. IG2 inhibited axon growth on complete BM by 63%, but inhibition of growth on BM-Ef was slight and similar to that seen on LN . As an additional control for specificity of inhibition by IG2 antibodies, we tested matrigel, an acellular basement membrane preparation from EHS sarcoma cells that lacks detectable CRYPα ligand (McKinnell, I., and A. Stoker, unpublished work). This was considered appropriate because most brain BMs at stage E6 are ligand-positive, and other BMs are very difficult to isolate. Axon growth on matrigel was as prolific as on BM-Ef, and was completely unaffected by IG2 . Finally, anti N-CAM antibody had no influence on retinal axon growth on any substrate . These results suggest that the ligand of the PTP CRYPα is located predominantly on retinal glial endfeet structures. On intact BMs, the anti-β1-integrin antibody (JG22) reduced outgrowth by ∼70%, suggesting that, in addition to CRYP-α interactions, ECM/integrin interactions are important regulators of axon growth on this tissue. As expected, inhibition of axon growth by JG22 was even greater when BM-Ef was tested, and outgrowth on LN was completely blocked. In contrast, axon growth on matrigel was inhibited only 48% by JG22. This result suggests that matrigel contains growth-promoting substances in addition to the well-characterized ECM components that are completely susceptible to inhibition by integrin antibodies . PLL/N-cadherin mediated outgrowth was not affected by either IG2 or JG22 . Comparison of the morphology of retinal growth cones on the three different substrates (LN, BM, and BM-Ef) revealed that growth cones on LN and BM-Ef are more elaborate, possessing larger d1 and d2 parameters, than growth cones on BM . Blocking the interaction of the RPTP CRYPα with its ligand-affected growth cone morphology on BM but not on BM-Ef , LN , or matrigel (data not shown). Growth cone morphology on BM-Ef was affected by JG22 in the same way as on LN . For statistical data see Table . In recent years, evidence has accumulated that RPTPs play important roles in axon growth and guidance . Nearly all of this evidence comes from studies in invertebrates, especially from Drosophila and leech. Far less is known about the role of RPTPs in the nervous system of vertebrates. Here we present data from in vitro experiments in which we examined the role of the RPTP CRYPα in the growth of retinal axons towards the optic fissure. Previous experiments have shown that CRYPα is expressed at very early stages on retinal axons and growth cones . The finding that a currently unknown ligand is present on basal membranes of the eye, optic nerve, optic tract, and tectum, pointed to an important role of this receptor and its ligand in intraretinal axon growth and during growth of retinal axons towards their target, the tectum opticum . Blocking the CRYPα-ligand interaction from both the receptor (IG2 antibodies) and the ligand side (α1-AP) induced dramatic changes in growth cone morphology and retinal axon length on the in vivo-like BM substrate. No effects on growth cone morphology and only weak effects on outgrowth were observed on a laminin substratum, on the physiological ECM substratum matrigel and on detergent-washed, endfeet-free basal membranes. We also observed no changes in outgrowth or growth cone morphology when we used Ig3-AP, a truncated form of CRYPα with no ligand binding capacity . Taken together with the α1-AP staining pattern on BM we suggest that the elusive CRYPα ligand is found predominantly on the surface of endfeet of Müller glia cells. The ligand itself exerts growth promoting and growth cone lamellipodia-stimulating activities. We have demonstrated that the glial endfeet of the retinal BM contain a balance of positive and negative cues. For example, removal of the glial endfeet leads to an enormous increase in axon outgrowth on BMs, whereas only on intact BMs do antibodies to CRYPα and the CRYPα1-AP fusion protein induce dramatic reductions in outgrowth. Previous data have demonstrated that the net outcome of these factors is indeed permissive for retinal axon growth . In the presence of these endfeet, the disturbance of CRYPα-ligand interactions causes a strong decrease of axon outgrowth. This suggests that during normal, intraretinal axon outgrowth, the growth-facilitating or -promoting role of CRYPα is in constant balance with negative influences on the growth cone. Therefore, this work places the RPTPs in a critical position in the regulation of intra-growth cone phosphotyrosine levels and maintenance of axon outgrowth . Glial endfeet are present on retinal basal membranes , but their molecular components are not completely characterized. The laminin/nidogen complex is present in the BM . It was recently shown that this complex binds in vitro to the fifth fibronectin type III domain of the long isoform of LAR , an RPTP structurally related to CRYPα2. However, this isoform of LAR is not expressed in the nervous system. We would not expect a similar interaction of CRYPα1 with laminin, as in vitro binding assays failed to detect an interaction of CRYPα1 or CRYPα2 with laminin 1 or 2 . In the same in vitro assays, matrigel did not bind to CRYPα. Recent work also suggests that CRYPα does not interact homophilically . These data underline our idea that the CRYPα ligand is a currently unknown transmembrane, membrane-anchored, or membrane-associated molecule at the surface of the endfeet of Mueller glia cells . On BM-Ef, neurite growth signals are mainly integrated via β 1 integrin receptors . Blocking the β 1 -subunits of the integrin receptors results in an enormous reduction of outgrowth. Due to the difficulties in isolating a native, ligand-negative basal membrane, we chose matrigel as a physiological control substrate that lacks detectable α1-AP binding activity. The results obtained on matrigel are largely the same than on BM-Ef, except that outgrowth on matrigel is less affected by blocking the integrin receptor. This suggests, unsurprisingly, that matrigel matrix contains additional growth-promoting components aside from laminin and the putative CRYPα ligand. Blocking the interaction of CRYPα with its ligand as well as the integrin receptor resulted in a dose-dependent loss of growth cone lamellipodia and in a transition towards a more filopodial morphology. Although this could relate to the reduced growth rate in both treatments, this does not imply that a filopodial morphology is always disadvantageous for growth cone migration . The data also indicate that both integrins and CRYPα can both influence lamellipodial dynamics, either independently or synergistically. What could be the link between CRYPα and lamellipodia? Well-known molecular regulators of lamellipodia and filopodia in fibroblasts are the small GTP-binding proteins of the Rho family, Rac and Cdc42, respectively . Available evidence suggests that they exert similar functions in neuronal growth cones . Control of the growth cone actin cytoskeleton by RPTPs could be achieved by interacting adapter proteins, like Trio. Trio binds to the intracellular domain of the CRYPα family member, LAR, and has two guanine exchange factor domains specific for Rac and Rho . Furthermore, the Caenorhabditis elegans Trio homologue, UNC-73, was shown to be required for axon growth and guidance . The phenotypic similarities of null mutants for DLAR and the Drosophila Rac suggest their interaction in guidance of intersegmental nerve b . Further evidence for an important function of RPTPs in controlling the actin cytoskeleton comes from two recent papers showing that profilin is regulated by DLAR involving the cytoplasmic tyrosine kinase abl and its substrate enabled . Trio could also provide a connection between DLAR and profilin via Rho and phosphatidylinositol 4-phosphate 5-kinase . From our data that blocking of CRYPα-ligand interactions on BM leads to changes in the actin cytoskeleton and to lamellipodia formation, it seems most likely that the CRYPα downstream signaling pathway also involves Rac. This may be related to a similar process as shown for integrins . There is likely to be considerable cross-talk between RPTPs and other extracellular receptors. This is especially important for recognition of multiligand complexes consisting of cell adhesion molecules and other growth cone guidance activities . The downstream signaling pathways of both CRYPa and integrin receptors seem to converge as well. There are several examples for an interplay of RPTPs and integrins , and it is quite likely that the same is true for CRYPα. The LAR-interacting protein (LIP) localizes type II RPTPs to focal adhesions , and, therefore, brings them into the close neighborhood of integrins. An interplay between RPTPs and integrin could then happen on the level of the small GTPases and Src-family of cytoplasmic kinases , both of which are shared in downstream pathways. This could result in the joint control of adhesive and deadhesive properties of cells or growth cones . Such a relationship has been shown, for example, with the T and B cell RPTP CD45 and integrins . Based on the data we have presented, we can formulate a basic model for some of the signaling processes downstream of CRYPα and integrins in retinal growth cones . It is clear that laminin-integrin interactions alone can stimulate growth cone migration and lamellipodia formation in the absence of other incoming signals . On a complete BM, however, we suggest there is a positive, CRYPα-dependent signal and a balancing negative signal(s) coming from an independent ligand-receptor complex. The latter signal could partly suppress integrin-promoted outgrowth and lamellipodia formation, by downregulating, directly or indirectly, the Rac pathway. This is counteracted by the positive CRYPα signal. Thus, loss of CRYPα signal in our experiments induces a significant block in neurite outgrowth. The modest influence of the IG2 antibody on a pure laminin substrate could be partly explained by a low level of activity in both the CRYPα and negative signals, in the absence of their cognate ligands. CRYPα antibodies would then cause a only slight suppression of integrin-mediated outgrowth, without grossly altering lamellipodia. This model needs to be tested further, but possible candidates for the negative signals would include nongraded Eph receptors, and their ephrin ligand, which are known to be present on glial endfeet . Another interesting possibility could be that there are some cis-interactions between integrins and CRYPα on the growth cone. PTPμ, another membrane tyrosine phosphatase, was recently shown to mediate homophilic trans-interactions , and to be involved in retinal axon growth . PTPμ interacts with three different, calcium-dependent cell adhesion molecules, N-, R-, and E-cadherin . It was shown that N- and R-cadherin promote outgrowth of neurites . Used as a substrate, PTPμ stimulated neurite outgrowth of E8 chick RGC neurites and on an N-cadherin substrate; downregulation of this phosphatase resulted in a significant decrease of the retinal neurite length, suggesting that the phosphatase activity of PTPμ is important for growth of RGC neurites on N-cadherin . RPTPβ/PTPζ is another outgrowth-inducing RPTP . It binds to other members of cell surface recognition complexes, among them contactin/F11 and Nr-CAM, enabling signal transduction into the growth cone . But the relevance of this RPTP for the development of the retinotectal system remains to be elucidated. During formation of the retinotectal map, there now appear to be, besides the integrins and N-cadherin, at least two different RPTPs involved in outgrowth of retinal ganglion cell axons: CRYPα and its yet undefined ligand, and PTPμ. Our results suggest that besides the RTKs , RPTPs such as CRYPα play an extremely important, complementary role in promoting formation of the visual system. It remains to be seen if gene deletion of RPTPs such as the mammalian orthologue of CRYPα, RPTPσ , results in disturbance of the retinotectal projection. | Study | biomedical | en | 0.999997 |
10525543 | Peptides were synthesized, purified, and confirmed by mass spectrometry at the Protein and Nucleic Acid Chemistry Laboratory of Washington University School of Medicine as described previously . The amino acid sequences of the TSP1 peptides and preparation of human platelet TSP1 were as described . Rat tail collagen-I was obtained from Collaborative Biochemical Products. The signal transduction inhibitors and activators were purchased from CalBiochem-Novabiochem. Both anti-α2β1 antibodies (function stimulating, JBS2, and blocking, BHA2.1) were from Chemicon International. Anti-ERK antibody was from Upstate Biotechnology Inc. Anti-active (-phosphorylated) ERK antibody was from Promega. Anti-ERK polyclonal antibody for Western blotting was from Santa Cruz Biotechnology, Inc. cAMP enzyme immunoassay (EIA) system (dual range) kits and γ-[ 32 P]ATP were from Amersham. Myelin basic protein and anti–mouse IgG agarose were from Sigma Chemical Co. The plasmids 3CH134 encoding MAP kinase phosphatase 1 (MKP-1) and the catalytically inactive mutant of MKP-1 were generously provided by Dr. N. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Human and mouse aortic SMC were isolated by the explant method and cultured as described . Cells were maintained in a humidified 37°C and 5% CO 2 environment in MEM with 20% fetal calf serum, and identified by immunostaining of α-actin . Passages 2–10 were used for experiments. Cell transfection using Lipofectamine was as described . In brief, cells at 50–60% confluence were transiently transfected with 20 μg cDNA of each plasmid for 5 h, and recovered in growth medium for 2 d before they were harvested, tested for viability, and counted for use in assays. Depletion of ERK from cells was performed according to the procedure of Klemke et al. 1997 . In brief, SMC grown to 60% confluence in 100-mm tissue culture dishes were incubated in Opti-MEM medium (GIBCO BRL) containing Lipofectamine (10 μg/ml) and 1.5 μM ERK antisense (5′-GCC GCC GCC GCC GCC AT-3′) or control (5′-CGC GCG CTC GCG CAC CC-3′) phosphorothioate oligonucleotides for 5 h at 37°C. Cells were then rinsed, incubated for 2 d in fresh culture medium containing oligonucleotide, and then tested in migration assays as described above and processed for Western blotting with anti-ERK antibody. Chemotaxis assays were conducted in 48-well micro-Boyden chambers (Neuroprobe) using 8-μm PVP-free, polycarbonate filters (Nucleopore). Filters were precoated by soaking in 100 μg/ml gelatin at 37°C overnight, followed by washing twice in PBS. SMC were harvested with trypsin/EDTA, washed, and diluted in MEM with 0.1% BSA to a final concentration of 3–5 × 10 5 cells/ml. Chemoattractants were diluted in the same solution. Signal transduction inhibitors or activators were added to the cell suspension directly at the concentration indicated. The assembled chamber was incubated for 6 h at 37°C. Filters were fixed, stained, and mounted. Cells were counted in five high-power fields (HPF) in each of the triplicate wells for each condition. Human SMC were harvested by EDTA/trypsin. After challenging with different treatments, either on matrix protein-coated 35-mm dishes or in suspension for indicated times, SMC were lysed directly at 4°C for 30 min in RIPA buffer including phosphatase inhibitors (50 mM Tris, pH 7.4, 0.15 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM Na 3 VO 4 , and 20 mM β-glycerophosphate) and protease inhibitors (10 μg/ml each of antipain, pepstatin A, chymostatin, leupeptin, soybean trypsin inhibitor, and aprotinin). The clarified cell lysates were incubated with 3 μg/tube of anti-MAP kinase antibody overnight at 4°C and then immunoprecipitated with anti–mouse IgG agarose for 1 h at 4°C. The precipitates were extensively washed with 1:3 diluted RIPA buffer three times and then two times with PBS. The immune complexes were resuspended in 60 μl kinase assay buffer (20 mM Hepes, pH 7.4, 1 mM EGTA, 20 mM β-glycerophosphate, 0.1 mM Na 3 VO 4 , 1 mM dithiothreitol, and 10 mM MgCl 2 ) including 20 μg myelin basic protein, 50 μM ATP and 5 μCi γ-[ 32 P]ATP, and incubated at 30°C for 30 min. The reaction was stopped by adding 20 μl of 100 mM EDTA (pH 7.0). After centrifugation to pellet the beads, aliquots (20 μl) of the supernatants were spotted onto P81 filters . The filters were washed 10 times with 1% phosphoric acid and then rinsed with 90% ethanol. The amount of 32 P incorporated into myelin basic protein was determined by scintillation counting. Human SMC were treated as described above and lysed in RIPA buffer. Clarified cell lysates (50 μl) were added to 2× SDS-sample buffer and run on 12% Tris-glycine gels under reducing conditions. The proteins were transferred electrophoretically onto nitrocellulose membranes which were blocked with 3% BSA plus 3% dried milk in TBS buffer with 0.1% Tween-20 for at least 1 h, and subsequently incubated with antibody against ERKs overnight at 4°C. The membranes were washed extensively and incubated with peroxidase-conjugated secondary antibody for 1 h. Detection was by chemiluminescence with an ECL kit (Amersham). Human SMC were harvested with EDTA/trypsin, resuspended in serum-free MEM at 2.5 × 10 5 /ml, and incubated with peptides or inhibitors for the indicated times. Cells were pelleted and cAMP was rapidly extracted twice with the addition of 100 μl of ice-cold ethanol (70%) for 30 min. Supernatants were collected and evaporated in a SpeedVac. The samples were dissolved in 5 ml assay buffer. The levels of cAMP were determined using a cAMP enzyme immunoassay system kit from Amersham. We previously found that either soluble type I collagen or the TSP1 peptide 4N1K alone are relatively weak chemoattractants of human aortic SMC migrating on a matrix of gelatin. However, in combination they provoke a strong chemotactic response. This response is, in fact, chemotactic (not chemokinetic) and depends on the function of both α2β1 and IAP . To determine if the α2β1 integrin-dependent migration of SMC stimulated by 4N1K is strictly dependent on IAP, we examined the response of IAP-deficient SMC isolated from aortae of IAP −/− mice . As shown in Fig. 1 , both 4N1K and collagen can induce migration of wild-type SMC (IAP +/+ ), and 4N1K plus soluble collagen together provoke a strong response, ∼200% compared with control cells, similar to the response of human SMC . However, 4N1K does not induce migration of IAP-deficient SMC. When soluble collagen is added along with 4N1K, the level of response is equal to the effect of collagen alone which reflects the action of α2β1 in the unstimulated state . This response to collagen also indicates that there is no significant defect in the ability of the IAP −/− SMC to adhere to collagen. This was confirmed in quantitative cell adhesion assays (not shown). Thus, 4N1K stimulation of chemotaxis with or without soluble collagen is receptor mediated and is strictly dependent on the presence of IAP. This also rules out a number of trivial explanations (e.g., aggregation of collagen) for the enhanced chemotaxis. Our previous work suggested the hypothesis that IAP stimulates α2β1 integrin function in SMC by activating the integrin in some way . If this is in fact the case, 4N1K may not need to be present during the chemotaxis assay and pretreatment of SMC with 4N1K might sensitize the cells to respond better to a gradient of soluble collagen. Thus, we compared the effects of pretreatment of SMC with 4N1K and with an anti-α2β1 mAb known to activate the integrin on other cell types . As shown in Fig. 2 , cells pretreated with 4N1K for 30 min and washed before placing them into the chemotaxis apparatus displayed significantly increased migration to soluble collagen. Pretreatment with the control peptide 4NGG (KRFYGGMWKK) had no effect. This effect of 4N1K is not due to residual bound peptide since direct binding assays using 125 I-4N1K reveal a very fast off rate (Dimitry, J.B., and W.A. Frazier, unpublished data). Similar results to those with 4N1K were observed when the cells were preincubated with the α2β1 function stimulating antibody: the migration to collagen increased more than threefold in three experiments . An α2β1 functional blocking mAb was tested in the same experiment, and it effectively inhibited cell migration . These data support the notion that ligation of IAP activates α2β1 for a period of time and it is this activation of α2β1 that is responsible for the enhanced migration of SMC towards soluble collagen . Chemotaxis is complex process in which cell adhesion and deadhesion are regulated in a dynamic way . In an attempt to assess the effects of 4N1K stimulation of IAP on a more direct index of integrin function, we tested the effect of 4N1K on the adhesion of SMC to gelatin and collagen-coated surfaces. As expected, collagen is a much better ligand for α2β1 than gelatin . These SMC adhere to both collagen and gelatin via α2β1 as determined with a function-blocking mAb (not shown). For both immobilized ligands, 4N1K significantly decreases stable adhesion of the SMC. At submaximal densities of collagen and at all densities of gelatin, this inhibition is as much as 50%. Thus, the effect of 4N1K (and TSP1) on SMC chemotaxis, on gelatin, and collagen , may represent a case in which loosening of tightly adherent cell contacts promotes migration as pointed out by Dimilla et al. 1991 and Lauffenburger and Horwitz 1996 . This deadhesion effect of 4N1K and TSP1 has been seen previously in C32 cells adhering to vitronectin , even though stimulation of IAP in C32 cells causes enhanced cell spreading which uses much the same cellular apparatus as cell motility. Thus, it is not clear at this point just what the state of the integrin is when it is activated via the IAP pathway. By analogy with the platelet system, perhaps α2β1 is in a state in which it binds soluble collagen better . Why this should result in decreased adhesion of cells to immobilized ligand is not obvious, but may relate to changes in the mobility or clustering of the integrin on the cell surface . In platelets , C32 melanoma cells , and endothelial cells , IAP stimulates the activity of β3 integrins when ligated with TSP1 or 4N1K peptide. This results in platelet aggregation, C32 cell spreading, and endothelial cell chemotaxis. In all of these cases, the action of IAP is abrogated by pertussis toxin treatment of the cells . Therefore, we tested the effect of pertussis toxin treatment on the response of the SMC to 4N1K and collagen. Fig. 4 shows that treatment overnight with 50 ng/ml pertussis toxin inhibited random migration to some extent, but completely eliminated the directional migration in response to 4N1K, collagen, or 4N1K plus collagen. Thus, the effects of IAP stimulation on α2β1 integrin function, like β3 integrins, appear to be mediated via a heterotrimeric Gi protein. Since IAP signaling is mediated via activation of Gi, the alpha subunit of Gi might inhibit adenylate cyclase activity . Thus, we examined the effect of IAP stimulation on intracellular levels of cAMP in SMC. As seen in Fig. 5 A, 4N1K alone evoked a rapid decrease in cAMP levels to less than half their original value in 20 min while the control peptide 4NGG had no significant effect. Interestingly, soluble collagen alone caused a substantial but transient decrease in cAMP levels while 4N1K plus collagen, the most potent chemotactic stimulus, rapidly decreased intracellular cAMP to very low levels that did not rebound by 40 min . As seen in Fig. 5 B, pertussis toxin pretreatment of the SMC increased control (prestimulus) levels of cAMP as expected due to attenuation of inhibitory signals. The toxin also prevented the marked decrease in intracellular cAMP (at 40 min) caused by 4N1K with or without collagen. These results strengthen the conclusion that, in SMC as in other cell types, IAP acts via Gi. They also suggest that a decreased level of intracellular cAMP may be necessary for the response. To test this idea, SMC were treated with forskolin, a direct activator of adenylate cyclase. As shown in Fig. 6 , forskolin strongly inhibits the migration of SMC toward 4N1K or collagen alone and to 4N1K plus collagen. We also tested the cell-permeable cAMP derivative 8-Br-cAMP, and found that it also inhibited SMC chemotaxis to all three stimuli . Thus, 4N1K/IAP induces SMC migration via activation of Gi, a necessary effect of which is decreased cytoplasmic cyclic AMP levels. In many cell types, integrin-mediated attachment of cells to matrix proteins results in activation of MAP kinase p42/44 or ERK . Furthermore, it has been reported that ERK can regulate αvβ3-dependent haptotaxis of FG carcinoma cells on vitronectin. Thus, we asked whether IAP modulation of SMC chemotaxis might involve regulation of ERKs. When SMC adhere to gelatin (or collagen, not shown), they display a transient burst of ERK activity that peaks ∼10 min after plating and then returns to baseline levels . 4N1K completely inhibited the increase in MAP kinase activity at 10 min and continued to decrease the baseline level of activity over time . The control peptide 4NGG had no effect . Western blotting using an antibody that detects the phosphorylated state of ERK confirmed this time course of activation (not shown). The inhibition of ERK activity appears to be an active process since even the basal level of kinase activity significantly declines over time with 4N1K treatment even under conditions in which cells are actively engaging the matrix . To determine if engagement of the integrin is required for IAP-mediated inhibition of ERK activity, we harvested SMC and treated them with 4N1K in suspension. As seen in Fig. 7 B, the level of ERK activity in suspended SMC is, as expected, somewhat lower than in SMC attaching to collagen. Nonetheless, 4N1K caused a significant inhibition of ERK activity in suspended SMC while 4NGG had no effect. Significantly, collagen had no effect on ERK activity under these conditions, and collagen did not synergize with 4N1K to further reduce ERK activity . Since it is not clear to what extent soluble collagen can bind to α2β1 under these conditions, we also treated suspended SMC with the α2β1 integrin-activating mAb used above. Neither the mAb alone nor in combination with a cross-linking anti–mouse IgG had any effect on ERK activity. Thus, ligation of IAP alone is sufficient to inhibit ERK activity and ligation and/or cross-linking of α2β1 alone is not. To test whether ERK in SMC can be regulated by Gi, SMC were treated with pertussis toxin (50 ng/ml) overnight. These same conditions lead to a blockade of IAP-dependent chemotaxis and an increase in intracellular cAMP levels . The activation status of ERK was then determined by MAP kinase immune complex kinase assays. Fig. 8 shows that pertussis toxin treatment significantly activates ERK in SMC, and the activation is concentration dependent. These results were confirmed by Western blot using an antibody that detects the phosphorylated state of ERK (not shown). Thus, blockade of a Gi-dependent pathway stimulates ERK activity indicating that an inhibitory input has been attenuated, just as in the elevation of intracellular cAMP levels by pertussis toxin . However, elevated cAMP does not cause the increase in ERK activity. We find that treating SMC with forskolin does not elevate ERK activity . Since 4N1K had a strong inhibitory effect on ERK activity, the effect on chemotaxis of inhibiting ERK by other means was tested. This was done in several ways. First, SMC were treated with the MAP kinase kinase (MEK) inhibitor PD98059 resulting in a modest but significant stimulation of chemotaxis to 4N1K and 4N1K plus soluble collagen . In another approach, we downregulated the amount of ERK protein in SMC using an antisense oligonucleotide based on the ERK mRNA sequence . Human SMC were treated with antisense and control (scrambled) oligonucleotides in the presence of lipofectamine for 5 h followed by a 2-d recovery period . As shown in Fig. 10 A, treatment with the antisense ERK oligonucleotide resulted in increased migration to collagen, 4N1K, and 4N1K plus collagen compared with cells treated with the scrambled oligonucleotide. The same samples were subjected to Western blot analysis with an anti-ERK antibody. As shown in Fig. 10 B, the ERK protein levels were in fact substantially decreased in cells treated with the antisense ERK oligonucleotide (lane AS). ERK levels were the same in untreated cells and in those treated with lipofectamine and the control scrambled oligonucleotide (lane SC). As seen in Fig. 7 A, 4N1K causes a progressive decrease in ERK activity in SMC suggesting an active suppression of ERK activity. This is normally accomplished in cells by MAP kinase phosphatases which serve to balance and attenuate the activation of MAP kinases . Thus, in a separate approach, we transiently transfected SMC with plasmid 3CH134 encoding MAP kinase phosphatase-1 (MKP-1) which can dephosphorylate MAP kinases activated by serum or v-raf . To determine if the MKP-1 was active in the transfected SMC, ERK activity was tested in cells replated on gelatin-coated surfaces after the cells were recovered for 48 h. As shown in Fig. 11 A, MAP kinase (primarily p42) phosphorylation was increased when control cells were attached to a gelatin-coated surface for 10 min (control). By 30 min, phosphorylation had returned to near basal levels . Treatment with PMA sustained phosphorylation through 30 min. Transfection with the vector control gave the same result as the control. However, in cells expressing wild-type MKP-1 (WT), the attachment-induced increase in ERK phosphorylation at 10 min was essentially blocked. Interestingly, in SMC transfected with the mutant MKP-1 in which the essential catalytic cysteine is replaced , phosphorylation of ERK is sustained through 30 min , indicating that MKPs likely play a normal role in effecting the decrease in ERK phosphorylation in SMC. The same transfected cells were also subjected to chemotaxis assays in which they were stimulated with soluble collagen alone, 4N1K alone, or 4N1K plus collagen . In the MKP-1–transfected cells, all three stimuli were about twofold more effective chemoattractants than in the vector control or mutant MKP1 transfected SMC. The mutant MKP-1, which causes a prolonged activation of ERK , has the same chemotactic response as the control. Thus, it appears that the transient nature of ERK phosphorylation may not be essential for the normal level of chemotaxis. The significant result here is that when ERK activation is attenuated, the chemotactic response to all three stimuli is enhanced. Thus, three different methods of decreasing ERK activity all result in enhanced chemotaxis toward collagen and magnify the effect of 4N1K. IAP is a widely distributed membrane protein implicated in modulating integrin functions required for Ca 2+ fluxes , phagocytosis , transendothelial , and transepithelial migration of PMNs, endothelial cell migration , as well as integrin-mediated C32 cell spreading and platelet spreading and aggregation . Many of these functions involve modulation of β3 integrins. We have shown that TSP1, through its IAP-binding motif, the 4N1K peptide, is able to modulate the activities associated with the α2β1 integrin such that it can promote chemotaxis of human, rat , and mouse (this report) arterial SMC toward soluble collagen-I. This effect of TSP1 and 4N1K on α2β1-mediated chemotaxis requires IAP. Chemotaxis of IAP-deficient SMC toward soluble collagen cannot be stimulated by 4N1K even though these cells adhere to gelatin and collagen as well as IAP +/+ control cells. These experiments represent the most direct proof to date that TSPs, via the 4N1K peptide, act through IAP to augment β1 integrin function. How does this occur? The effect of 4N1K-IAP on α2β1-dependent migration on gelatin can be conceptualized in two different ways. First, IAP may alter the affinity state of α2β1, either by direct physical interaction or via inside out signaling. Our previous data from platelets support such an affinity modulation model. The ability of 4N1K (or TSP 1 or its recombinant COOH-terminal domain, McDonald, J.F., X.-Q. Wang, and W.A. Frazier, unpublished data) to enhance SMC chemotaxis to soluble collagen is reminiscent of the activation of platelet αIIbβ3 by 4N1K/TSP-1 . This effect in platelets is also dependent on IAP since platelets from IAP deficient mice do not aggregate in response to 4N1K or TSP 1 . The fact that 4N1K pretreatment can stimulate migration to soluble collagen is suggestive of a change in the activation status of α2β1 that lasts long enough to be manifested during the several hours required for cells to migrate through the filters. An activating mAb against α2β1 gives the same result, i.e., enhanced chemotaxis to soluble collagen, which is a poor ligand for the basal state of the integrin just as soluble fibrinogen is a terrible ligand for αIIbβ3 on quiescent platelets . Thus, the enhanced chemotaxis toward soluble collagen by 4N1K may be analogous to the stimulation of soluble fibrinogen binding to platelet αIIbβ3 by 4N1K which leads to platelet aggregation. In platelets, the binding of the ligand mimetic mAb PAC-1 can be used as an index of αIIbβ3 affinity. In the case of α2β1, there is no such convenient marker for a higher affinity/avidity state of the unliganded integrin. The effect of 4N1K to decrease cell adhesion as measured in a static adhesion assay appears to argue against a simple activation of ligand binding by the integrin, since this would normally be expected to enhance not inhibit cell adhesion. This result speaks to some change in the state of the integrin's valency or affinity, but the nature of this change is unclear at the present time. The second hypothesis, which need not be exclusive with affinity modulation, is that IAP signaling is integrated with α2β1 signaling at one or more levels inside the cell. Both collagen and 4N1K lead to a drop in intracellular cAMP levels. With collagen alone, this effect is transient, whereas 4N1K leads to the sustained suppression of cAMP which is enhanced by coadministration of collagen. This effect is Gi dependent, as shown by experiments with pertussis toxin, and independent of adhesion and integrin engagement, as shown by experiments with suspended cells. The decrease in cAMP levels and the inhibition of ERK activity can both have profound effects on the cell's motility apparatus , and perhaps on other events upstream that connect integrin engagement to cell motility. The precipitous drop of cAMP levels in the SMC upon treatment with 4N1K is likely to be due to a direct action of Giα [GTP] on adenylate cyclase. In platelets and in leukocytes, high levels of cAMP appear to block the transition to a more active state of the integrins . Thus, our observation that a prominent effect of IAP stimulation in SMC is a profound drop in intracellular cAMP levels may well be related to removing a brake on α2β1 function. This effect is pertussis toxin sensitive and thus clearly requires Gi function . We have found an identical, IAP-dependent, pertussis toxin-sensitive depletion of cAMP in platelets where other costimulators of integrin activation such as thrombin, epinephrine, and ADP act via G proteins to lower cAMP levels . Soluble collagen alone also causes a transient fall in cAMP in SMC. Together, 4N1K and collagen synergize, resulting in a more profound and prolonged decrease of cAMP levels, in parallel with the more pronounced chemotactic response elicited by the combined stimuli. This suggests that the integrin and IAP work together to activate Gi. In fact, we have recently found that 4N1K or TSP1 ligation of IAP on platelets synergizes with collagen ligation of α2β1 to induce platelet activation and aggregation . That a heterotrimeric complex of integrin and IAP is the functional signaling unit is further indicated by the existence of a detergent-stable integrin/IAP complex in SMC , in platelets , and in melanoma cells which is physically associated with Gi . In addition, Gi can be copurified with IAP and its integrin partner by affinity chromatography on immobilized 4N1K, and by immunoprecipitation with anti-IAP mAbs. Furthermore, 4N1K stimulates the binding of γ-[ 35 S]GTP to cell membranes and this stimulation requires IAP since it is not observed in membranes from cells of IAP knockout mice . This result with isolated membranes makes it unlikely that IAP signals the synthesis of an autocrine factor which then indirectly activates Gi through its own Gi coupled serpentine receptor. If ligation of IAP is required to activate Gi, how then does collagen alone cause a decrease in cAMP levels ? Our preliminary data suggests that cross-linking of the integrin-IAP complex by collagen may responsible. Another immediate result of IAP stimulation with 4N1K is a suppression of ERK activity and phosphorylation . That this suppression of ERK activity is causal for chemotaxis is indicated by the fact that three different methods of inhibiting ERKs all result in enhanced chemotaxis . A mechanism of integrin modulation that involves ERKs is suggested by experiments of Hughes et al. 1997 in which a chimeric integrin expressed in CHO cells was engineered to be in an activated state as determined by the binding of the ligand-mimetic mAb, PAC-1. Then cDNAs were expressed in the CHO cells and screened for inhibition of PAC-1 binding. Several inhibitory or integrin deactivating cDNAs were identified and all encoded activators of the ERK pathway such as activated H-ras and raf-1. These results suggest that MAP kinase, specifically ERKs, inhibit integrin activation of the sort required for binding of soluble ligands. Interestingly, it has been reported that activation of αIIbβ3 and binding of soluble fibrinogen to platelets coincides with inhibition of ERKs . Whether ERK inhibition might cause αIIbβ3 activation was not determined. We used three very different ways of decreasing ERK activity and all three led to an increased chemotactic response of SMC. The modulation of α2β1 activity in SMC by IAP suggested by our data may be the first physiological example of the ERK regulation of integrin affinity/avidity. Klemke et al. 1997 have reported that α2β1-dependent migration of FG carcinoma cells on a collagen substrate required activation of MAPK/ERK. While their assay used a Boyden chamber apparatus like our chemotaxis assays, the filters on which the cells migrated were coated with fibronectin only on the bottom side. Their assay was thus a haptotaxis assay which measures increased adhesion to the matrix protein coated on the underside of the filter. Thus, their result is that activation of ERKs results in increased integrin-mediated adhesion to matrix, not increased chemotaxis as observed here for SMC. In fact, we observe that activation of IAP with 4N1K results in decreased adhesion concomitant with increased chemotactic migration. Importantly, Klemke et al. 1997 found that inhibition of ERKs did not prevent spreading of the FG cells, a cellular function closely allied to cell migration. Thus, our results are, in fact, consistent with those of Hughes et al. 1997 and Klemke et al. 1997 , and reinforce the idea that activation of MAPK/ERKs inhibits binding of soluble ligands to integrins and also stimulates integrin-mediated adhesion which can inhibit cell migration. The finding that ERKs can phosphorylate MLCK may also support the second model for IAP action to regulate cell motility at downstream sites. The mechanism by which IAP inhibits ERK activity is not clear. We find that ERK activity is stimulated by disabling Gi with pertussis toxin suggesting that under normal growth conditions, some tonic input acts through Gi to exert a negative influence on ERK activity in SMC. This negative regulation may be due to βγ since expression in SMC of the β-ARK COOH-terminal domain that sequesters βγ also stimulates ERK activity in randomly cycling, proliferating cultures of SMC (Wang, X.-Q., and W.A. Frazier, unpublished data). This result is in contrast to recently published data on the role of βγ as a positive effector of ERK activation in serum-starved SMC stimulated with serum or growth factors . However, the effects of βγ sequestration are potentially quite complex, impacting not only downstream targets of βγ, but also receptor desensitization and down regulation . Thus, it is conceivable that βγ sequestration may differ in its net result when tested in proliferating vs synchronized cells stimulated to enter the cell cycle. Whatever the mechanism, the essential role of a Gi protein in the stimulation of chemotaxis by 4N1K is clearly demonstrated by the effect of pertussis toxin treatment of the SMC. The downstream events that lead to the effects on cell motility that we observe could be mediated by the Giα subunit or the βγ heterodimer released upon activation and dissociation of Gi . The inhibition of chemotaxis by forskolin and 8-Br-cAMP implies that decreased cAMP (and lower PKA activity) is necessary to allow directed migration . This observation is consistent with other studies of SMC chemotaxis and with the inhibitory effect of high cAMP levels on activation of leukocyte integrins and the long known inhibitory effect of cAMP on activation of the platelet integrin αΙΙbβ3 . Another well known effect of cAMP is the PKA-mediated inhibition of cell motility via phosphorylation of myosin light chain which would also put a brake on chemotaxis until cAMP levels are reduced. It has been reported that adenylate cyclase is downstream of MAP kinase in SMC, since activated ERKs can increase cAMP levels. This occurs via ERK-mediated phosphorylation and activation of PLA2 leading to prostaglandin production, and autocrine adenylate cyclase stimulation via Gs . In addition to 4N1K, PD98059 also decreases cAMP levels in SMC (our unpublished data). Thus, when ERK is inhibited, cAMP levels follow. However, manipulation of cAMP levels does not appear to affect ERK , consistent with the idea that ERK regulation is upstream of adenylate cyclase in SMC . Thus, activation of Gi can lower cAMP levels in two ways: direct inhibition of adenylate cyclase by Giα and inhibition of ERKs leading to decreased stimulation of cyclase via the prostaglandin pathway. It appears that both decreased cyclic AMP levels and lower ERK activity are necessary for IAP modulation of SMC motility. The data reported here indicate a novel role for TSP family members (all of which contain the IAP binding sequence) and IAP in the regulation of SMC migration. This may occur via modulation of a β1 integrin mediated by inhibition of MAP kinase, and a concomitant decrease in intracellular cyclic AMP levels. Both of these signaling events are tremendously pleiotropic, and both PKA and ERKs have been shown to impact regulation of cell motility via direct phosphorylation of myosin light chain kinase and/or myosin light chain . Thus, IAP may act at multiple levels to modulate integrin function as well as facilitating signaling downstream of integrin ligation. There is ample literature implicating TSP1 in the proliferation and chemotaxis of cultured SMC and in atherogenesis and restenosis . Our data suggest the possibility that manipulation of TSP1 signaling through IAP may be of therapeutic benefit. | Study | biomedical | en | 0.999997 |
10525544 | Herbimycin A, bisindoylmaleimide, KT5720, KT5723, SB 203580, PD 98059, and SKF 86002 were obtained from Calbiochem-Novabiochem Corp. Cycloheximide was obtained from Sigma Chemical Co. (R)-(+)-perillyl alcohol (POH) was obtained from Aldrich and anisomycin was obtained from Boehringer Mannheim. α2 integrin cDNA corresponding to nucleotides 1–4559 in the published sequence and the X2C5PFNeo plasmid were provided by Dr. M. Hemler (Dana Farber, Boston, MA). The pAWneo2 expression vector was provided by Dr. A. Weiss (University of California San Francisco, San Francisco, CA) . The oligonucleotides used were purchased from Kebo Lab. The glutathione-S-transferase (GST)–tagged ATF2 (residues 1–109) , the pRSV-MKK3(ala) and pcDNA-MKK4(ala) expression plasmids were provided by Dr. R. Davis (University of Massachusetts, Worcester, MA), and flag-tagged p38 isoform expression vectors were provided by Dr. J. Han (Scripps Research Institute, La Jolla, CA). The pCEV29RhoA Asn19 , pCEV29Rac1 Asn17 , and pCEVCdc42 Asn17 were provided by Dr. J.C. Lacal (CSIC, Madrid, Spain). Bacterial expression of GST-ATF2 was done as described . The c-Jun protein was provided by Dr. E. Coffey (Åbo Akademi University, Turku, Finland). Bacterial expression of GST-ATF2 was done as described . Antibody against α2 integrin, 12F1 was provided by Dr. V. Woods (University of California, Medical Center, San Diego, CA). Polyclonal antisera against α1 integrin cytoplasmic tail was purchased from Chemicon International, Inc., and antibody against α1 integrin used in flow cytometry, SR-84, was a gift from Dr. W. Rettig (Boehringer Ingelheim, Germany). Human osteosarcoma cell line Saos-2 was obtained from the American Type Culture Collection. The cell cultures were maintained in DME supplemented with heat inactivated 10% FCS (GIBCO-BRL), 2 mM glutamine, 100 IU/ml penicillin-G, and 100 μg/ml streptomycin. The α2 integrin expression construct was prepared as described previously . The mutant α2 subunit, in which the cytoplasmic tail has been replaced with the corresponding α1 integrin sequence, was prepared in the following way. A silent point mutation that introduces a new NheI recognition site was made with the Altered sites II in vitro mutagenesis system (Promega) according to the manufacturer's instructions. The mutant oligo used was ACCAGAGCTAGCAGCAAAAGG. The α2 cDNA was digested with NheI and SacI cleaving the region corresponding to the α2 cytoplasmic tail. The α1 DNA was obtained by annealing sense and antisense synthetic nucleotides corresponding to nucleotides 3506–3543 in the published α1 sequence flanked with corresponding α2 sequences; antisense oligo (TTTTGCTGCTAGCTCTGGTTGCAATTTTATGGAAGCTCGGATTCTTCAAAAGACCA-CTGAAAAAGAAAATGGAGAAATGAGAGCTCAGTAGCTG), sense oligo (TCAGCTACTGAGCTCTCATTTCTCCATTTTCTTTTTCAGTGGTCTTTTGAAGAATCCGAGCTTCCATAAAATTGCAACCAGAGCTAGCAGCAAAA). This synthetic DNA fragment was digested with NheI and SacI restriction enzymes and ligated with the cleaved α2 cDNA. The correctness of the construct was checked by sequencing. Stable transfections were performed with the calcium polybren/DNA method on confluent 60-mm dishes. Incubation with 5 μg DNA and 5 μg polybren in 1 ml 10% FCS/DME per dish was carried out for 6 h, agitating the dishes once an hour. DMSO (30% in FCS) shock was done for 3 min, cells were washed twice with PBS, and culture medium was added. Neomycin analogue G418 (Life Technologies, Inc.) was added to the culture medium in a concentration of 400 mg/ml. G418-resistant cell clones were selected for 2–3 wk, isolated, and analyzed for their expression of α2 integrin. Control cells were transfected with the pAWneo2 plasmid only. Transfected cells were cultured in 10% FCS/DME containing 2 mM glutamine, 100 IU/ml penicillin-G, 100 μg/ml of streptomycin and 200 mg/ml G418. Collagen gels were prepared from bovine dermal collagen, which contains 95% type I collagen and 5% type III collagen (Cellon). 8 vol of Cellon were mixed with 1 vol of 10× concentrated DME and 1 vol of 10× concentrated NaOH (0.05 M) in Hepes buffer (0.2 M) and kept on ice. Cells were trypsinized, resuspended in 1/10 gel volume culture media DME supplemented with 10% FCS, mixed into neutralized Cellon solution, and transferred into 6-well plates. The collagen was allowed to polymerize for 2 h at 37°C, after which the culture media containing 10% FCS was added, the gels were detached from the sides of the wells, and incubation was continued for the times indicated. Cells were also cultured on plastic as a monolayer in culture media containing 10% FCS. In experiments involving inhibitors, the cells were pretreated with the inhibitor for 30 min at room temperature before mixing the cells with the neutralized Cellon solution, also supplemented with the inhibitor at the concentrations indicated. After polymerization, culture media containing 10% FCS and the inhibitor was added and the gels were detached from the sides of the dishes. Incubation was continued for 48 h. When studying collagen gel contraction, cell culture wells were photographed after 48 h and the surface areas of the gels were measured from the prints. The cells grown on immunofluorescence glass slides (CML) covered or uncovered with collagen film (Cellon) were rinsed with PBS and fixed with methanol at −20°C for 5 min. The cells grown inside collagen gels were excised from culture wells, embedded in OCT compound (Tissue-Tek; Miles Scientific), and frozen in isopentane chilled with liquid nitrogen. Sections of 10-μM thickness were cut and picked up onto microscope slides and treated as described above. The slides containing the fixed samples were incubated in 2% BSA in PBS and monoclonal anti–CD49b antibody (Chemicon International Inc.) was added to the same solution and incubated 30 min at room temperature. After rinsing, the cells were incubated with anti–mouse–FITC conjugate (Dako A/S) for 30 min and mounted before observation and photography. For staining of the actin filaments, plastic coverslips (Nunc) were coated with PBS containing 16 μg/ml type I collagen overnight at 4°C and blocked with 1% BSA in PBS for 1 h at 37°C. Cells were allowed to adhere and spread on collagen coated coverslips for 24 h in DME. Coverslips were washed once with PBS and fixed with 2% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and incubated with rhodamine-conjugated phalloidin (Sigma Chemical Co.) 1:1,000 in PBS for 30 min. Cells were washed with PBS and mounted before observation and photography. Total cellular RNA was isolated using the Qiagen RNeasy kit. Total RNA was separated in formaldehyde-containing agarose gels, transferred to nylon membranes (Zeta-probe; Bio-Rad Laboratories), and hybridized with 32 P-labeled (Amersham) cDNA probes. The following cDNAs were used: human α2 integrin , human collagen α1(I) , human collagen α2(I) and 28S (a gift from R. Penttinen, University of Turku), and rat glyceraldehyde-3-phosphate dehydrogenase . Cells were grown to early confluence, detached with trypsin-EDTA, and trypsin activity was inhibited by medium supplemented with serum. Cells were washed with PBS, pH 7.4, and then incubated with PBS containing 10 mg/ml BSA, 1 mg/ml glycine, and 0.02% NaN 3 for 20 min at 4°C. Cells were collected by centrifugation, exposed to saturating concentration of mAb against α2 integrin (12F1) or α1 integrin (SR-84) in BSA/PBS (BSA concentration 1 mg/ml) containing NaN 3 for 30 min at +4°C, and stained with rabbit anti–mouse IgG coupled to fluorescein (1:20 dilution; Dako A/S) for 30 min at 4°C. Cells were washed twice with PBS containing NaN 3 and suspended in the same buffer. To measure the amount of α2 integrin on the cell surfaces, the fluorescent excitation spectra were analyzed by using a FACScan apparatus (Becton Dickinson). Control samples were prepared by treating the cells without primary antibodies. Cells were metabolically labeled with 100 μCi/ml of [ 35 S]methionine (Tran[ 35 S]-label, ICN Biomedicals Inc.) for 16 h in methionine-free minimum essential medium. Cell monolayers were rinsed on ice with a solution containing 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 25 mM Tris-HCl, pH 7.4, and then detached by scraping. Cell pellets were obtained by centrifugation at 500 g for 5 min. Cells were solubilized in 200 μl of the same buffer containing 100 mM N -octyl-β- d -glucopyranoside (Sigma Chemical Co.) on ice with occasional vortexing. Insoluble material was removed by centrifugation at 10,000 g for 5 min at 4°C. Radioactivity in cell lysates was counted and an equal amount of radioactivity was used in immunoprecipitation assays. Triton X-100 (0.5% vol/vol) and BSA (0.5 mg/ml) were added to the supernatants, which were precleared by incubation with 50 μl of packed protein A–Sepharose (Pharmacia LKB Biotechnology Inc.). Supernatants were immunoprecipitated with antiintegrin antibody for 12 h at 4°C followed by incubation with secondary antibody (rabbit anti–mouse, DAKO), when 12F1 was used. Immune complexes were recovered by binding to protein A–Sepharose and washing the beads four times with 25 mM Tris-buffered isotonic saline, pH 7.4, containing 0.5% Triton X-100 and 1 mg/ml BSA and twice with 0.5 M NaCl and 25 mM Tris-HCl, pH 7.4. The immunoprecipitates were analyzed by electrophoresis on SDS-containing 6% polyacrylamide gels under nonreducing conditions followed by fluorography. The cells were lysed with NP-40 (ICN) and the nuclei were isolated by centrifugation (12,000 g ) for 3 s at 4°C. The nuclei were incubated in the presence of 100 μCi of [α- 32 P]UTP (3,000 Ci/mmol, NEN) for 30 min at room temperature as described previously . Radiolabeled RNA was hybridized with 2 μg of nitrocellulose-fixed plasmids: cDNAs for human α1(I) collagen, human α2(I) collagen, GAPDH, and pBluescript (Stratagene). The hybridization and washing conditions used were as described previously . Quantitation was performed with GS-250 Molecular Image System (Bio-Rad Laboratories) and the results were corrected for the levels of GAPDH transcripts in the same samples. The coating of a 96-well immunoplate (Maxi Sorp; Nunc) was done by exposure to 0.2 ml of PBS, pH 7.4, containing 0.1 μg/cm 2 (1.64 μg/ml) type I collagen (from lathyric rat skin, Boehringer Mannheim) for 12 h at 37°C. Residual protein absorption sites in all wells were blocked with 1% BSA in PBS for 1 h at 37°C. BSA was also used to measure the nonspecific binding. Cells were detached by using 0.01% trypsin and 0.02% EDTA. Trypsin activity was inhibited by washing the cells with 1 mg/ml of soybean trypsin inhibitor (Sigma Chemical Co.). In cell spreading assays, cells were suspended in DME with 50 mM cycloheximide (Sigma Chemical Co.), transferred into each well, and incubated for 35 min at 37°C. The wells were washed with PBS and fixed with 8% formaldehyde and 10% sucrose in PBS for 30 min. The total number of cells attached per one microscopic field and the percentage of spread cells were counted. A spread cell was characterized as one having a clearly visible ring of cytoplasm around the nucleus. The activation of ERK1 and 2, JNK/SAPK, and p38 MAPK was determined by Western blotting using antibodies specific for the phosphorylated, activated forms of the corresponding MAPKs (New England Biolabs). The control blots for the total (phosphorylated and nonphosphorylated forms) protein levels were done by using antibodies recognizing the corresponding MAPKs (p38, ERK2, New England Biolabs; JNK1, Santa Cruz Biotechnology). Saos cell clones were either grown in monolayer for 24 h or seeded in collagen gels as described earlier. Once polymerized, the gels were detached from the dish and incubated for the time indicated. The cells were released from the gels as described above and lysed in 100 μl of Laemmli sample buffer. Cells grown in monolayer were washed once with warm PBS and lysed in 100 μl of Laemmli sample buffer. The positive control treatment for the JNK Western blot was done by treating cells in suspension with 10 μg/ml anisomycin (Boehringer Mannheim). The samples were sonicated, fractionated by 10% SDS-PAGE, and transferred to a Hybond ECL membrane (Amersham Corp.). Western blotting was performed as described previously , with phosphospecific antibodies in dilution 1:1,000. Specific binding of antibodies was detected with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL) detection system (Amersham). Subconfluent Saos cell clones plated on 60-mm dishes were transfected using 4 μl of Fugene 6 transfection reagent (Boehringer Mannheim) and 2 μg of either eukaryotic expression vector alone (pcDNA3; Invitrogen Corp.) or the same vector containing the flag-tagged p38 isoform . Cotransfection of dominant negative signaling proteins was done by using 12–16 μl of Fugene 6, 3–8 μg of empty expression vector or the same vector containing the effector mutant and 2 μg of flag-tagged p38α. 36 h after transfections, the cells were treated with collagen gel for 3 h as described earlier or left untreated. Cells were solubilized with RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 0.5 mM DTT, 1 mM PMSF in PBS, pH 7.4) supplemented with leupeptin, antipain, and pepstatin, 2 μg/ml each. The extracts were centrifuged 3,000 rpm for 15 min at 4°C. 1 μg of M2 antibody (Eastman Kodak Co.) conjugated to protein G–Sepharose (Pharmacia-LKB Biotechnology) was used for immunoprecipitation. The immunoprecipitates were washed twice with RIPA buffer, once with LiCl wash buffer (500 mM LiCl, 100 mM Tris, pH 7.6, 0.1% Triton X-100, 1 mM DTT) and once with kinase buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl 2 , 0.1% Triton X-100, 1 mM DTT). The kinase assay was initiated with 2 μg GST-ATF2 as substrate and 50 mM MgCl 2 , 25 μM ATP, 3 μCi γ-[ 32 P]ATP in a final volume of 50 μl. The reactions were terminated after 30 min at 37°C by the addition of Laemmli sample buffer. The phosphorylation of substrate protein was examined after SDS-PAGE by autoradiography. For Western blotting, samples of lysed cells were fractionated by 10% SDS-PAGE and transferred to Hybond ECL membrane (Amersham Corp.). Western blotting was performed as described previously , with M2 antibody (Eastman Kodak Company) in dilution 1:750. Specific binding of antibodies was detected with peroxidase conjugated secondary antimouse antibody and visualized by the ECL detection system (Amersham). The JNK kinase assay was done as described in Westermarck et al. 1998 with some modifications, namely the antibody used was anti-JNK1 (Santa Cruz Biotechnology) and the immunoprecipitation was done with protein G–Sepharose. The cells were brought into suspension, counted, and diluted so that equal number of cells from both clones were used. The cells were then either lysed immediately, treated with anisomycin for 10 min or seeded inside collagen for 3 h. The cells were brought into suspension, counted, and diluted so that equal number of cells from both clones were used. The cells were either lysed immediately or allowed to adhere to fibronectin (1 μg/cm 2 coated on a 60-mm dish overnight at +4°C and blocked with 0.1% heat-inactivated BSA for 1 h at +37°C) or seeded inside collagen for 1 h. The assay was done as described previously . The antibody used for immunoprecipitation was polyclonal anti-FAK (Upstate Biotechnology Inc.). The filter was first blotted with antiphosphotyrosine (4G10; Upstate Biotechnology Inc.), stripped according to manufacturer's instructions, and reprobed with anti–FAK antibody. Alternatively, total cell lysates were fractionated by SDS-PAGE and the filter was first blotted with an antibody mixture detecting all the phosphorylated forms of FAK (Biosource, Hopkinton, MA), following stripping and re-probing with anti-FAK antibody. Initially, we made stable transfectants expressing wild-type α2 or chimeric α2/α1 subunit using Saos-2 cells, which have endogenous α1β1 but no α2β1 . Flow cytometric analysis using anti–α2 mAb confirmed that both the wild-type and the chimeric receptor were expressed on the surface of the transfected cells. Based on the flow cytometric measurements, we selected cell clones with similar expression levels of wild-type α2 or chimeric α2/α1 integrin to be used in the experiments . Also, the cell clones used in the experiments and not shown in the Fig. 1 B were tested. In addition, we measured the expression levels of α1β1 from both mock-transfected and α2-transfected cells to ensure that the α2 cDNA transfections did not alter the expression of the endogenous α1β1. The average levels of α1 integrin on four independent single cell clones tested were 60.8 ± 3.8 (arbitrary units) in mock-transfected cells and 68.3 ± 8.6 in α2-transfected cells. The corresponding values for α2 integrin were 13.5 ± 0.7 (represents negative background level fluorescence) and 362 ± 54. Immunoprecipitation experiments were performed to confirm that the chimeric α2/α1 subunit was associated with the endogenous β1 subunit to form a typical heterodimeric αβ complex. The chimeric structure of the heterodimer was verified using an antibody recognizing the α1 cytoplasmic tail (not shown). Deletion of the α2 cytoplasmic domain has been shown to result in ligand-independent recruitment of the integrin to preformed focal contacts . To study if swapping of the α2 cytoplasmic domain to the significantly shorter α1 tail results in indiscriminate integrin recruitment into focal adhesion sites formed by other integrins, we studied both wild-type α2 and chimeric α2/α1 in Saos-2 cells spread on serum proteins. Both clones showed a diffuse staining pattern indicating that the receptors do not localize to focal contacts ligand independently . When these clones were allowed to adhere to and spread on collagen in serum-free conditions both wild-type and chimeric receptors were able to form focal contacts indicating that the α2 chain cytoplasmic tail can be replaced with the corresponding α1 sequence without affecting cellular localization of the receptor. Also, the formation of actin stress fibers on collagen was similar in both clones . In cells cultured inside three-dimensional collagen, no aberrant clusters of integrins were seen and both receptors showed a diffuse staining pattern . Cell adhesion to type I collagen was studied to determine the effect of swapping the tails on the adhesive properties of the integrin. Adhesion mediated by the chimeric α2/α1β1 receptor was found to be equivalent to that of the wild-type receptor . Both the wild-type α2-transfected cells and the α2/α1 chimera–transfected cells adhered to collagen at the early time point studied (1 h) more efficiently than the mock-transfected cells. When studying focal contact formation by α2 wild-type and chimeric α2/α1–transfected clones, we noticed that even though cell adhesion to collagen was not influenced by the replacement of the α2 cytoplasmic tail with the α1 tail, the efficiency of the clones to spread on collagen was somewhat different. The cells expressing α2 wild-type receptor spread faster than cells expressing α2/α1 chimeric receptor, so that after 30 min on collagen 61.5 ± 6% of adherent α2-transfected cells had spread, whereas only 43 ± 8.3% of adherent α2/α1–transfected cells had spread . At this early time point, mock-transfected cells expressing only α1β1 had just begun to adhere and virtually no spreading was seen. At later time points (i.e., 2 h and later), also the mock-transfected cells spread efficiently on collagen. Since both α2 wild-type and α2/α1 chimeric receptor were expressed at similar levels on the cell surface , the small difference seen in cell spreading may indicate that the cytoplasmic tail of α2 functions in linking the integrin to the cytoskeleton in a manner promoting spreading on collagen. Therefore, we wanted to study the ability of α2 cytoplasmic tail to mediate cytoskeleton-dependent events and assayed the ability of α2- and α2/α1–transfected cells to contract collagen gels. As expected, vector control cells, having endogenous α1β1 but no α2β1, showed very weak contraction during the 72-h experiment (area of the gel 1.8 ± 0.35-fold reduced), whereas wild-type α2β1-expressing cells contract the gel efficiently (area of the gel 4.0 ± 1.0-fold reduced) . The cytoplasmic tail of α2 seemed to be essential for linkage of the integrin to the cytoskeletal machinery since the cells expressing chimeric α2/α1 failed to contract the collagen gel . Overexpression of the wild-type α2 in Saos-2 cells resulted in upregulation of the mRNA levels of both collagen α1(I) (1.4–4.3-fold) and α2(I) (3.3-fold) in response to three-dimensional collagen matrix. To test the generality of this observation, all together three independent single cell clones overexpressing α2β1 were tested (not shown). In contrast, vector control cells showed a downregulation ranging from 0.8 to 2.4-fold for collagen α1(I) mRNA . Interestingly, the chimeric receptor failed to mediate upregulation of the collagen mRNAs in response to three-dimensional collagen matrix as seen with the Northern blot hybridization of total RNA isolated from two single cell clones pooled together. The mRNA levels of collagen α1(I) in cells expressing the chimeric receptor were downregulated by 1.5–1.8-fold in response to collagen compared with the average 3.6-fold upregulation seen in cells expressing the α2 wild-type receptor . Also, expression of collagen α2(I) was downregulated in the α2/α1–transfected cells. These results were again confirmed to be reproducible with RNA from a third independent single cell clone (not shown). Altogether, cells transfected with the chimera responded to collagen identically to the mock-transfected cells. Integrin α1β1 is known to function as a negative regulator of collagen . The transmembrane region of this receptor has been suggested to signal through interaction with caveolin , whereas the possible signaling function of α1 cytoplasmic domain cannot be excluded. In the α2/α1–transfected cells, no further reduction of collagen levels was seen when compared with the mock-transfected cells having endogenous α1β1 integrin, suggesting that the short α1 cytoplasmic tail is not alone sufficient to regulate collagen gene expression. To test this further, we transfected Saos-2 cells with the X2C5PFNeo plasmid coding for α2 subunit with α5 cytoplasmic tail . Again, similarly as both the mock-transfected and the α2/α1–transfected cells, collagen mRNA levels were decreased in response to collagen (not shown). To assess the contribution of increased transcription of collagen α1(I) on this elevation seen in mRNA levels, we performed nuclear run-on experiments. Nuclei were isolated from α2- or α2/α1–transfected cells grown either in monolayer or in three-dimensional collagen gel for 48 h. The rate of the collagen gene transcription was compared with that of GAPDH. A 1.8-fold increase of transcription was seen in α2-transfected clones cultured in three-dimensional collagen when compared with the rate of transcription in cells grown in monolayer . In contrast, cells transfected with the chimeric α2/α1 chain showed a 1.5-fold decrease in transcription rate . Even though the increased transcription rate of collagen α1(I) gene accounted for most of the upregulation seen in α2β1-overexpressing clones, we also wanted to investigate the stability of the mRNAs. We used actinomycin D to block transcription in cell clones grown in monolayer and in collagen gels. Type I collagen mRNAs seem to have relatively long half-lives (>8 h) in Saos-2 cells transfected with wild-type α2, but no obvious difference was seen between the various culture conditions. Both in monolayer and in three-dimensional matrix collagen, mRNA levels were reduced by ∼50% after 8 h treatment with actinomycin D . To identify the downstream components of α2β1-mediated upregulation of collagen gene expression, we tested specific inhibitors at concentrations sufficient to inhibit various signaling kinases . The cells were exposed to inhibitors before seeding them inside collagen. Of the various compounds tested, the p38 inhibitor, SB203580, showed the most potent inhibition of collagen α1(I) gene expression (5.5-fold) in α2β1-overexpressing cells grown in three-dimensional collagen gel . The compounds that showed some inhibition (about twofold) included tyrosine kinase inhibitor herbimycin A, PKG inhibitor KT5823, PKA inhibitor KT5720, and Ras farnesylation inhibitor (R)-(+)-Perillyl alcohol (POH), whereas MEK inhibitor PD98059 had no effect. High concentrations of the PKC inhibitor bisindolylmaleimide resulted in downregulation of collagen mRNA levels (twofold at 5 μM and 4.1-fold at 20 μM concentrations), but this effect was seen in both α2- and mock-transfected cells and was therefore considered to be nonspecific. In addition, phosphatidyl-inositol-3-kinase (PI-3K) inhibitor, wortmannin treatment resulted in a slight reduction of collagen α1(I) mRNA in α2-transfected cells (not shown) and this effect was smaller in vector-transfected cells . The small effects of various inhibitors suggest that corresponding signaling proteins might participate in integrin signaling, but this hypothesis was not studied further. Another specific inhibitor targeting the p38 MAP kinase pathway, SKF86002, was tested to confirm the result obtained with SB203580. Treatment with this compound also resulted in concentration-dependent inhibition of α2β1-mediated upregulation of collagen α1(I) mRNA levels (6.1-fold at 10 μM and 9.5-fold at 20 μM). 20 μM SB203580 was a potent inhibitor of collagen mRNA levels in α2-transfected cells inside collagen , but it had no effect on mRNA levels in mock-transfected cells inside collagen or α2-transfected cells in monolayer , excluding the possibility that the compound could function as a general downregulator of collagen gene expression. The ability of the selective p38 inhibitors, SB203580 and SKF86002, to inhibit α2 cytoplasmic tail–dependent upregulation of collagen mRNA levels lead us to study how p38 activity is regulated in response to collagenous matrix in these cells. We examined the activation of p38 by using a phosphospecific antibody that recognizes phosphorylated p38α and p38β isoforms. The cells expressing wild-type α2 showed a marked activation (fivefold) already at 2 h after seeding the cells inside collagen. The activation gradually decreased during the next 12 h, but remained at levels threefold higher than the 0 h time point . To study whether this activation was due to signaling via the α2β1, we analyzed the levels of phosphorylated p38 in cells expressing chimeric α2/α1 chain. In three separate experiments using different single cell clones, the wild-type α2-expressing cells showed in average 2.3-fold higher levels of active p38 2 h after seeding the cells inside collagen gel than cells expressing chimeric α2/α1 chain. Protein levels of p38 remained constant at all time points, as shown with the control antibody recognizing both activated and inactivated forms of p38β . At a later (24 h) time point, the p38 activation persisted and α2-transfected cells showed a higher level of activation than the cells expressing chimeric α2/α1 chain . To confirm this difference in the levels of active p38 at the 24-h time point, the experiment was repeated five times using four or five parallel samples and two individual single cell clones of both transfections. In all experiments, p38 was activated in response to three-dimensional collagen and, in cells expressing the wild-type α2 chain, the levels were on average 1.9-fold higher (range, 1.3–3.5-fold). The difference in levels of active p38 between the α2 and α2/α1 cells were found to be statistically significant when the results of all five experiments were combined together . Finally, to confirm that this difference is the result of signals dependent on the α2 cytoplasmic tail in response to collagen, we tested the levels of active p38 in the same cell clones when grown in monolayer. The overall levels of active p38 were relatively low and no significant difference between the clones was detected . To date, the p38 MAP kinase group is known to include five isoforms: p38α , p38β , p38β2 , p38γ , and p38δ . To study whether α2β1 cytoplasmic tail could specifically activate some isoform of p38, we overexpressed various forms of flag-tagged p38 kinases in Saos cell stable clones expressing either wild-type α2 or chimeric α2/α1. The transfected cells were seeded inside a collagen gel, after 3 h the cells were collected, flag-tagged p38 was immunoprecipitated, and an in vitro kinase assay was performed. As seen in Fig. 6 E, p38α isoform was activated efficiently in α2-transfected cells (activity 0.47 units; arbitrary units = densitometric units − background), whereas in α2/α1–transfected cells the activity was 0.03. No activation of p38β2 was detected (α2 clone 45 = 0.04 and α2α1 clone 12 = 0.02). p38γ showed high activity in both cell clones (α2 clone 45 = 0.24 and α2/α1 clone 12 = 0.32) and p38δ activity was high in both clones (α2 clone 45 = 0.64 and α2/α1 clone 12 = 0.23). The results were confirmed with two individual α2- or α2/α1–expressing clones . The expression levels of the transiently transfected kinases in various clones were equal as shown by Western blot analysis done by using antibody against the flag-tag. As seen in Fig. 6E and Fig. F , p38α was activated only in α2-transfected cells and no activation of p38β2 was seen. However, equally high activity of p38γ was seen in both α2- and α2/α1–transfected cells and the p38δ activity was higher in α2 cells than in α2/α1 cells. To study which of these activated kinases are relevant to the α2-mediated upregulation of collagen, shown to be inhibited by chemical inhibitors , we tested the effect of these inhibitors in an in vitro kinase assay. α2-transfected Saos-2 cells were transiently transfected with flag-tagged p38 isoforms (α, β2, γ, or δ), the cells were treated with collagen and the kinase activity of each isoform was measured in the presence or absence of the inhibitory compound. In accordance with previously published experimental data and recent structural evidence the inhibitor SB203580 had no effect on the γ and δ isoforms of p38 . Previously, SKF86002 has been shown to inhibit p38α and we show here that it has no effect on the γ and δ isoforms. From these results we can conclude that it is the p38α isoform that is essential in the α2β1 integrin–dependent upregulation of collagen. We have recently shown that seeding dermal fibroblasts inside three-dimensional collagen gels results in activation of ERK1 and 2, JNK/SAPK, and p38 MAPKs . Therefore, we examined also the activation of ERK1 and 2 and JNK/SAPK in both α2- and α2/α1–transfected Saos-2 cells by Western blot analysis of cellular proteins, using phosphospecific antibodies to detect activated forms of these MAP kinases. The levels of activated ERK2 were increased 2 h after seeding the cells inside collagen (3-fold in α2 and 1.5-fold in α2/α1 cells) and they increased further (up to 4-fold in α2 and 7-fold in α2/α1 cells) at 6 h time point. This activation in response to the collagen gel was transient in both clones since no phosphorylated ERK1 or 2 was detected at 12-h time point. Protein levels of ERK2 remained constant at all time points, as shown with the antibody recognizing all forms of ERK2 . Low levels of ERK1 were also detected at 2-, 4-, and 6-h time points. No induction in the levels of phosphorylated JNK/SAPK was seen in these cells in response to three-dimensional collagen. Some activated JNK/SAPK was seen at 0-h time point, immediately after trypsinization but no active protein was detected inside collagen gel. Treatment with anisomycin was used as a positive control for JNK activation. Protein levels of JNK1 remained constant at all time points, as shown with the antibody recognizing all forms of JNK1 . The results with the phosphospecific antibodies were confirmed with a JNK in vitro kinase assay. Endogenous kinase was immunoprecipitated with anti–JNK1 antibody recognizing also JNK2 and 3 and recombinant c-Jun protein was used as a substrate . Ligation of integrins leads to activation of FAK. To check whether FAK would play a role in the activation of p38 in response to three-dimensional collagen, we allowed both α2- and α2/α1–transfected cells to interact with collagen gels for 1, 2, or 3 h. . The polymerization of the collagen gel takes place in 1 h, so shorter time points could not be studied. No phosphorylated FAK was detected in cells treated with collagen, in contrast to cells from both clones adhering to fibronectin . Some phosphorylation of FAK was seen in cells lyzed immediately after trypsinization. The experiment was repeated with similar results by using antibodies recognizing all phosphorylated forms of FAK . In the lower panel of Fig. 9 phosphorylated FAK is the upper band. The lower band seen in all samples could not be recognized by anti–FAK antibody and its identity is unknown. To investigate possible downstream effectors of α2β1 integrin in the activation of p38α, we used dominant negative mutants of the Rho family GTPases and the p38 upstream kinases, MKK3 and MKK4. The effector mutants or an empty vector in control cells were cotransfected with the flag-tagged p38α into the α2-expressing cells and, 36 h after transfection, the cells were exposed to three-dimensional collagen and an in vitro p38 kinase assay was performed. Of the Rho family GTPases Cdc42 seemed essential for α2β1-mediated signaling since dominant negative Cdc42 constantly resulted in an inhibition of p38α activity . Similar inhibition was not seen when the cells were transfected with wild-type Cdc42, used as a control. In three separate experiments, 4 μg/plate dominant negative Rac slightly decreased p38α activity (75 ± 12% of control) and mutant RhoA was only somewhat more effective (70 ± 27% of control). The experiment was also repeated with higher plasmid concentration (8 μg/plate) and p38α activity was unaltered in dominant negative Rac transfected cells (116% of control). Mutant RhoA showed some inhibition (66% of control). Again, dominant negative Cdc42 was the most efficient (17% of control). The dominant negative forms of the MAPK kinases known to function upstream of p38, namely MKK3 and MKK4, both had an inhibitory effect. Dominant negative MKK3 inhibited p38α activity by 90–91% and dominant negative MKK4 by 76–90% . These results indicate that the activity of Cdc42 and the MAPK kinases MKK3 and MKK4 are necessary for the α2β1 integrin-mediated p38α activation. The integrins provide a physical linkage between the cytoskeleton and ECM and they transduce signals initiated by extracellular interactions. Since integrins have no intrinsic kinase activity they need to recruit other proteins, i.e., kinases to trigger signaling . A rapidly increasing number of molecules has been shown to interact with integrins . One obvious site for interaction is the cytoplasmic domain of an integrin subunit and several proteins binding specifically to either the α or the β cytoplasmic tail have been identified. Interactions mediated by the membrane spanning region of integrins have also been shown . These interactions may be used differentially by different integrins establishing the bases for receptor-specific signal transduction. Integrin α subunit–specific interactions with other cellular proteins are of special interest because they may explain the distinct signaling functions of integrin heterodimers sharing a common β1 subunit . The two collagen receptors, α1β1 and α2β1 integrins, have distinct effects on cellular signaling and gene expression . Here, the function of their α cytoplasmic domains was studied by swapping the α1 tail into α2 and expressing the chimeric integrin in cells negative for α2 integrin. The swapping of the α1 and α2 subunit cytoplasmic domains did not affect the localization to focal adhesions or the ability to mediate cell adhesion to collagen. This is in agreement with previously published data where the specific sequence of the α tail seemed less important than the number of residues present. Four to seven residues after the conserved GFFKR sequence were needed to be included for optimal adhesive activity . Here, fast cell spreading and the ability to contract three-dimensional collagen gels were impaired if the cells expressed chimeric α2/α1 instead of the α2 integrin. Previously, α2, α4, and α5 cytoplasmic tails have been shown to be interchangeable with respect to their positive contributions towards cell adhesion , while the ability to mediate collagen gel contraction and cell migration is more restricted to a specific set of α cytoplasmic tails . Collagen gel contraction assays are used to study cell–type I collagen interaction. It is generally thought that the reorganization of collagen fibrils, seen as contraction of floating collagen gels, is dependent on the expression of α2β1 on the cell surface . However, there are more recent reports indicating that α1β1 can have an essential role in the contraction process, especially in smooth muscle cells and liver myofibroblasts , suggesting that α1 cytoplasmic tail, unlike the ones of α4 and α5, could mediate the same interactions as the α2 tail. In contrast to these reports, our results do not support the idea that α1β1 or chimeric α2/α1β1 could mediate collagen gel contraction. This is also the case with α1- and α2/α1–transfected CHO cells (Ivaska, J., and J. Heino, unpublished results). One explanation could be that α1β1 needs an interaction with a certain cytoplasmic structure to be able to mediate contraction, and that this component is only present in a subset of cell types. Synergy between integrin-mediated signaling and signals initiated by growth factors has been well established . Pathways first identified to be activated by mitogens, the MAP kinase cascades, have now been shown to be activated by integrin ligation . Furthermore, integrin specificity in MAP kinase activation has been established. A subset of integrins, namely α1β1, α5β1, α6β4, and αVβ3, has been shown to activate the Ras-ERK pathway via recruitment of the adaptor protein Shc . Other integrins (α2β1, α3β1, and α6β1) were unable to induce ERK activation in these studies . Given the difference between α1β1 and α2β1 integrins in activation of Ras-ERK pathway it is not surprising that ligation of these two receptors have different effects on cellular gene expression. Both α1β1 and α2β1 have been shown to regulate collagen mRNA levels in response to contact with three-dimensional collagen. Integrin α1β1 can function as a negative regulator of collagen synthesis , whereas overexpression of α2β1 in osteosarcoma cells results in an upregulation of collagen mRNA levels . Integrin α2β1 seems to be a positive regulator of MMP-1 and MMP-13 (collagenase-3) expression as well. Further, supporting the differential signaling by these two receptors, α1β1 is not involved in MMP-1 upregulation and it seems to be a less potent upregulator of MMP-13 than α2β1 . Activation of Ras-Raf-ERK pathway by α1β1 integrin may lead to reduced collagen synthesis since Ras-Raf activation has been shown to downregulate type I collagen gene expression . Signaling through α2β1 is less well characterized than α1β1 signaling. MMP-1 upregulation in fibroblasts cultured inside three-dimensional collagen is mediated by PKC-ζ and NFκB, but these pathways have not been directly connected to α2β1 . Three-dimensional collagen mediates PKC-ζ activation in 4 h in CHO cells even though they lack collagen receptor integrins (Ivaska, J., and J. Heino, unpublished results) proposing that the PKC-ζ activation might at least partly be due to a change in cell shape rather than active signaling through collagen binding integrins. Previously, it has been shown that cell contact with either two- or three-dimensional collagen induces activation of ERK1 and ERK2 . We have recently shown the participation of p38 MAPK pathway in induction of MMP-13 expression in response to cell–collagen interaction . Here, we provide several lines of evidence that integrin α2β1 regulates collagen gene transcription by activating p38α in response to collagen, and that this signaling requires the α2 cytoplasmic domain. First, expression of wild-type α2 chain in cells exposed to collagen leads to upregulation of collagen mRNA levels. Chimeric receptor in which the cytoplasmic domain of α2 is replaced with the corresponding sequence in α1, is not able to upregulate collagen synthesis in response to three-dimensional matrix. Second, regulation of collagen gene transcription was shown to require p38 MAPK activity based on the use of two p38 kinase inhibitors, SB203580 and SKF86002. Third, contact with three-dimensional collagen results in only a transient activation of ERK1 and 2, no evident activation of SAPK/JNK, but persistent activation of p38 kinase. Activation of p38 MAPK requires intact α2 subunit since levels of activated p38 were significantly lower in cells expressing chimeric α2/α1 receptor. Finally, using transient transfections of flag-tagged isoforms of p38 and dominant negative signaling proteins, we were able to show that α2 cytoplasmic domain specifically activates the α isoform and has no effect on the p38β2 isoform. We also show that the activity of Cdc42 and the MAPK kinases MKK3 and MKK4 is necessary for the α2β1 integrin-mediated p38α activation. The data presented clearly show that the p38α isoform is essential in the α2β1 integrin–dependent upregulation of collagen expression. The facts to support this areas follows. First, the p38α isoform is activated in response to collagen in the α2-transfected but not the α2/α1–transfected cells. Second, the p38β2 isoform is not activated in these cells. Third, even though p38γ showed high activity in both clones and p38δ was more efficiently activated in the α2-transfected cells, these isoforms cannot be responsible for the upregulation of collagen gene expression since the p38 inhibitors used have no effect on these kinases . Together, these findings demonstrate that the cytoplasmic sequence of α2 integrin subunit regulates the ability of α2β1 integrin to activate p38 kinase in an isoform-specific manner and suggest a novel signaling mechanism for α2β1. An issue that arises from the data is the mechanism by which α2β1 integrin activates the p38 pathway. Integrins are known to activate the Rho family of GTPases. Integrin ligation to the ECM leads to the activation of Cdc42 that subsequently activates Rac; Rho, on the other hand, has been shown to be activated independently of Cdc42 by integrin ligation . Of the GTPases Cdc42 has been shown to activate both the p38 and JNK pathways and Rac1 has been shown to activate p38 . Cdc42 and Rac1 have been shown to induce integrin-mediated cell motility on collagen and invasiveness through collagen gels , suggesting a role for these small GTPases in the inside-out signaling regulating the function of the collagen binding integrins. Here we identify Cdc42 to be important for α2β1-mediated signaling and also show that the MAPK kinases MKK3 and MKK4 may be involved. The signaling molecules downstream of Cdc42 and upstream of the MAPK kinases remain to be clarified in further studies. The various p38 isoforms seem to be differentially activated by upstream MAPKKs: MKK3, MKK4, and MKK6 , which in turn are activated by MAPKKKs . MAPKKKs shown to be effective in the p38 pathway involve ASK and TAK1 (transforming growth factor–activated kinase) but little is known about their upstream effectors. PI-3K has been shown to function in integrin-mediated cell migration and invasion and it may function upstream of Rac1 and Cdc42 . In fact, a lipid product of PI-3K has been shown to interact with Rac1 . A recent study shows that Cdc42 controls integrin-dependent activation of Akt , a kinase whose activity is regulated by PI-3K . Very recently a mechanism was suggested in which PI-3K activates NFκB , a transcription factor that has been shown to be activated in fibroblasts in response to collagen gel . In our experiments, the PI-3K inhibitor, wortmannin, slightly reduced α2β1-mediated upregulation of collagen α1(I) thereby leaving open the possibility that PI-3K may be one of the upstream effectors of the signaling pathway described here. Other candidates for upstream effectors that could mediate α2β1-related activation of p38 include p21 activated kinases (PAKs), the best characterized effectors of Cdc42 and ACKs (activated Cdc42-binding kinases) recently shown to be activated by cell adhesion via integrin β1 . In a study published by Bourdoulous et al. 1998 , alteration in integrin occupancy by fibronectin led to upregulation of ACK and p38 MAPK while a concomitant inhibition of PAK and JNK/SAPK was seen. This is interesting from the point of view of this discussion since similar effects on the MAPKs were seen in the α2-transfected Saos cells in response to collagen. In addition, TAK1 might be an interesting candidate, since it is activated by TGF-β and has been shown to activate both MKK3 and MKK6 . On the other hand, TGF-β has many effects on the cell, one of them being the upregulation of collagen levels . How is integrin signaling then transduced to these molecules, especially to Cdc42? One attractive model for α2β1 signaling would be the phosphorylation of the α2 cytoplasmic domain. Phosphorylation in response to integrin ligation could generate a binding site for an effector kinase inside the cell. However, our preliminary data have failed to convincingly show α2 phosphorylation in response to binding to collagen (Ivaska, J., and J. Heino, unpublished results). It is evident that the two collagen receptors studied here function in close collaboration to regulate cell behavior in response to collagenous matrix. Impaired regulation of collagen turnover may lead to pathological conditions. For example, skin fibroblasts from scleroderma patients show upregulated collagen synthesis and concomitantly reduced expression of α1β1 . A striking example of correlated functions of integrins α1β1 and α2β1 is seen in α1 null mice, in which the absence of α1β1 leads to enhanced collagen synthesis in skin. However, simultaneously collagenase-3 expression is increased, possibly via increased α2β1 ligation, leading to a situation in which the collagen accumulation is seen only if the degradation of collagen is prevented . Previous studies have shown the regulation of specific MAPK pathways by α1β1 integrin and also introduced the dual role of MAPK pathways in the regulation of collagen production . Here, we provide new information which connects α2β1 integrin to the regulation of p38α via Cdc42 and MAPKKs, MKK3 and MKK4, and show how α2β1 functions in the regulation of the delicate balance of collagen accumulation in tissues. | Other | biomedical | en | 0.999997 |
10525545 | Skin punch biopsies (4 mm in diameter) were removed from the trunk of two unrelated patients diagnosed with MD-EBS and transported to the laboratory in transport medium (DMEM containing the following antibiotics: 0.01% (wt/vol) streptomycin, 0.01% (wt/vol) penicillin, and 2.5 U/ml fungisone). The tissue was cut into several pieces and incubated in 0.5% (wt/vol) trypsin, 0.1% (wt/vol) EDTA for 90 min at 37°C to separate the epidermis from the dermis. The epidermal sheets were reincubated in fresh trypsin/EDTA at 37°C for 5 min, pipetted vigorously to encourage basal cell detachment, after which trypsinization was arrested by the addition of DMEM containing 10% FCS, and the resulting cell suspensions were centrifuged. The keratinocyte suspension was plated into two T25 flasks (Greiner) together with 5 × 10 5 irradiated 3T3 feeder cells and keratinocyte medium without EGF. Cells were checked and medium (keratinocyte medium with EGF) changed twice weekly. At 80% confluence, cells were trypsinized, split at 1:5, and replated with fresh 3T3 feeder cells. At passage two, cells were plated in T25 flasks with irradiated 3T3 feeder cells and allowed to grow to 40–50% confluence. The medium was changed to keratinocyte growth medium, a low calcium medium (0.15 mM Ca 2+ ), with 10 ng/ml EGF, 5 μg/ml insulin, 0.5 U/ml hydrocortisone, and 0.4% (vol/vol) bovine pituitary extract (Clonetics Corp.) for 48 h, thus allowing cells to spread out as a monolayer. The human papillomavirus 16 expression plasmid pJ4Ω16 used for the immortalization was a gift from Dr. A. Storey (Molecular Virology Laboratory, ICRF, London, United Kingdom). For transfection, keratinocytes were washed once with OPTI-MEM (GIBCO BRL) and 4 ml OPTI-MEM was added to each flask and incubated at 37°C for 4 h. For each T25 flask, the following solutions were prepared and incubated for 30 min at room temperature before mixing: solution A, 10 μg DNA (human papillomavirus 16 plasmid) in 0.5 ml OPTI-MEM; and solution B, 30 μl Lipofectin in 1.5 ml OPTI-MEM. The two solutions were combined, mixed gently, and incubated for a further 10–15 min. All medium was removed from the flasks and 2 ml of transfection mixture was added. The cells were incubated overnight at 37°C, and the next day 4 ml keratinocyte medium and fresh irradiated 3T3 feeder cells were added. Medium was changed twice a week, and cultures were split at 80% confluence. After transfection, keratinocytes grew for one or two passages and then entered a growth crisis for a period of up to 12 wk. After this crisis, colonies growing from single cells were cloned using cloning rings , and then passaged using the same medium. Two MD-EBS cell lines (PEB-1 and 2) were used in this study. Immortalized normal human keratinocytes (NHK) from foreskin and the immortalized β4-deficient PA-JEB keratinocyte cell line were described previously . Immortalized NHK, PA-JEB, and MD-EBS keratinocytes were grown in keratinocyte serum-free medium (GIBCO BRL) supplemented with 50 μg/ml bovine pituitary extract, 5 ng/ml EGF, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were grown at 37°C in a humidified, 5% CO 2 atmosphere. The MD-EBS and PA-JEB keratinocytes were transiently transfected with cDNA constructs using Lipofectin (GIBCO BRL) according to the manufacturer's procedure. Rabbit polyclonal antisera against α6 and β4 have been described previously . Mouse mAb 58XB4 against β4 was prepared by Dr. A.M. Martínez de Velasco and Mr. D. Kramer in our laboratory. Mouse mAbs 450-11A and 4.3E1 against β4 were kind gifts of Dr. S.J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN) and Dr. E. Engvall (The Burnham Institute, La Jolla, CA), respectively. Mouse mAb 233 against BP180 and mouse mAb 121 against plectin/HD1 were kind gifts of Dr. K. Owaribe (University of Nagoya, Nagoya, Japan). Rabbit polyclonal antiserum against BP180 was kindly provided by Dr. L. Bruckner-Tuderman (University of Münster, Münster, Germany). The rabbit polyclonal antiserum recognizing BP230 was a kind gift from Dr. J.R. Stanley (University of Pennsylvania, Philadelphia, PA). Polyclonal antibodies against plectin were generated by immunizing rabbits with a glutathinone S -transferase (GST) fusion protein containing the NH 2 -terminal domain of plectin (residues 1–339). The antibodies were purified by affinity chromatography on a plectin (1–339)/maltose-binding protein (MBP) Sepharose column. Mouse mAb 6F12, also known as BM-140 , against laminin-5, was a kind gift from Dr. R.E. Burgeson (Cutaneous Biology Research Center, Charlestown, MA). Mouse mAb 7A8 against plectin was purchased from Sigma Chemical Co. Mouse mAbs V9 against vimentin and KL1, recognizing a broad spectrum of keratins, were purchased from Coulter/Immunotech. Mouse mAb SPK-14 against keratin 14 was a kind gift from Dr. D. Ivanyi (Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands). Mouse mAb 12C05 and rabbit polyclonal antiserum HA-11 against the hemagglutinin (HA) epitope (YPYDVPDYA) were purchased from Santa Cruz Biotechnology. Sheep anti–mouse and donkey anti–rabbit HRP-coupled secondary antisera, and donkey anti–rabbit Texas Red conjugated antisera were purchased from Amersham Pharmacia Biotech, and FITC-conjugated goat anti–mouse antiserum from Rockland. MD-EBS keratinocytes were plated in 6-well tissue culture plates and lysed in 80 μl sample buffer. Lysates were boiled for 5 min at 95°C, and 40 μl samples analyzed by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore) and blots were blocked in 2% (wt/vol) baby milk powder in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.01% [vol/vol] Tween 20) for 1 h at 37°C. Subsequently, blots were incubated with primary antisera in 0.2% (wt/vol) baby milk powder in TBST for 1 h at room temperature. After extensive washing, blots were incubated for 1 h at room temperature with secondary HRP-coupled antisera in the same buffer as used for the primary antiserum incubation. Proteins were detected using the chemiluminescence procedure (Amersham Pharmacia Biotech) according to the manufacturer's instructions. MD-EBS and PA-JEB keratinocytes were switched to HAMF12/DMEM (1:3) containing 10% (vol/vol) FCS, 2 mM L-glutamine, 0.4 μg/ml hydrocortisone (Sigma Chemical Co.), and 1 μM isoproterenol (Sigma Chemical Co.) 24 h before the immunolabeling procedure was started. Keratinocytes grown on glass coverslips were washed twice with PBS (pH 7.2), and fixed for 10 min at room temperature in freshly prepared 1% (wt/vol) paraformaldehyde in PBS. Fixed cells were washed twice with PBS and permeabilized in 0.5% (vol/vol) Triton X-100 in PBS for 5 min at room temperature. Cells were rinsed and incubated in 2% (wt/vol) BSA in PBS for 30 min at room temperature, washed with PBS, and incubated with primary antisera or TRITC-conjugated phalloidin in PBS containing 2% BSA for 30 min at 37°C. After washing with PBS, cells were incubated with the secondary antiserum, goat anti–mouse–FITC or donkey anti–rabbit–Texas red diluted 1:100 in PBS containing 2% BSA. After rinsing in PBS, the preparations were mounted in Vectashield (Vector Laboratories Inc.) and viewed under a BioRad MRC-600 confocal laser scanning microscope. All nucleotide and amino acid positions are numbered with the ATG initiation codon at position one. Plasmid inserts were generated by PCR using the proofreading Pwo DNA polymerase (Boehringer Mannheim) and gene-specific sense and antisense primers containing restriction site tags. All plasmid inserts were confirmed by sequence analysis using the T7 Sequencing kit (Amersham Pharmacia Biotech). Plectin-ABD, a clone containing alternative exon 1c, exons 2–8, and almost the complete exon 9 of human epithelial plectin cDNA , and plectin-IFBD (see below) were inserted into pcDNA3HA (a kind gift from Dr. E. Sander, Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands), a derivative of the eukaryotic expression vector pcDNA3 (Invitrogen Corp.) that contains an extra sequence 5′ of the multiple cloning site encoding the HA tag. The yeast galactose metabolism regulatory gene 4 (GAL4) plasmids containing human β4 , human epithelial plectin , and mouse plectin cDNA subclones and full-length cDNA encoding human α skeletal muscle actin , human β cytoplasmic actin , human γ cytoplasmic actin , human keratin 5 , human keratin 8 , human keratin 14 , human keratin 18 , human GFAP , and mouse vimentin are described in Fig. 5 , Fig. 7 , and Fig. 9 . Numbers in superscript correspond to the amino acid residues of subclones encoded within the GAL4 activation domain (AD) or binding domain (BD) fusion proteins. Vectors used were the yeast GAL4 AD or BD expression vectors pACT2 or pAS2-1 (Clontech). The template DNAs used for PCR were full-length cDNAs encoding human β4, human α skeletal muscle actin, mouse vimentin, human plectin (kind gifts from Dr. T. Magin, Institut für Genetik, University of Bonn, Bonn, Germany), human keratins 5, 8, 14, and 18, and cDNA subclones containing bps 1–1018, 2377–3156, and 13054–13725 of human epithelial plectin. Other templates used were EST clones obtained from the IMAGE cDNA clone collection at the Resource Center/Primary Database of the German Human Genome Project (RZPD, Berlin, Germany): 975524 and 1064756 for mouse and 52195 for human plectin 3′ clones; 361338, 364065, and 381924 for human GFAP; 611141 and 613287 for human β cytoplasmic actin; and 41909 and 362511 for human γ cytoplasmic actin. The full-length β4 cDNA construct in the eukaryotic expression vector pRc/CMV (Invitrogen Corp.) has been described previously . The CGG to TGG mutation in codon 1281 of β4, a human patient mutation that results in an R to W amino acid substitution at position 1281 , was introduced into human β4 cDNA by sequence overhang extension PCR. The mutagenesis primers 5′-GACAACCCTAAGAACTGGATGCTGCTTATTG-3′ (sense) and 5′-CAATAAGCAGCATCCAGTTCTTAGGGTTGTC-3′ (antisense), representing positions 3826–3856 of the human β4 coding sequence, were used with normal human β4 cDNA as a template. The resulting DNA fragments were first cloned into the pACT2 vector, verified by DNA sequencing, and used in yeast two-hybrid analysis . For cell transfection experiments, the mutation was subsequently introduced into full-length β4 cDNA in pRc/CMV by exchanging of the appropriate DNA restriction fragments . DNA fragments encoding different fragments of the β4 cytoplasmic domain were isolated from pACT2-β4 plasmids (described above) and cloned into the bacterial GST fusion protein expression vector pRP261, a derivative of the pGEX-3X vector (Amrad Corp. Ltd.) that contains a slightly modified multiple cloning site, for the production of recombinant GST fusion proteins . Plectin-ABD was isolated from pcDNA3HA-plectin ABD (described above) and inserted into the bacterial MBP fusion protein expression vector pMAL-c2X (New England Biolabs Inc.), for the production of MBP fusion proteins . The β4 cDNA expression constructs used for the experiments in Fig. 11 have been described previously . Yeast strain Saccharomyces cerevisiae PJ69-4A (a gift from Dr. P. James, Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI), which contains the genetic markers trp1-901, leu2-3, his3-200, gal4Δ, gal80Δ, LYS2::GAL1-HIS3, GAL2-ADE2 , was used as the host for the two-hybrid assay. It contains two tightly regulated reporter genes, His and Ade, which makes it suitable for the sensitive detection of protein interactions. The use of PJ69-4A was essentially as described in Schaapveld et al. 1998 . In brief, PJ69-4A cells were cotransformed with a pACT2(-derived) as well as a pAS2-1(-derived) plasmid, and aliquots of the same transformation mixture were spread on plates containing SC-LT medium, yeast synthetic complete medium (SC) lacking only the vector markers Leu (for pACT2 and derivatives) and Trp (for pAS2-1 and derivatives), as well as on SC-LTHA plates, lacking Leu, Trp, as well as the interaction markers His and Ade. Plates were scored after 6 and 12 d of growth, and the number of colonies on the SC-LTHA plate compared with that on the SC-LT plate. Positive and negative controls used were as in Schaapveld et al. 1998 . Cotransformation efficiencies (on nonselective SC-LT plates) for all plasmid combinations were always at least 10 4 cfu/μg plasmid DNA, and the difference between the various plasmid combinations tested was never greater than twofold. Cotransformation of yeast PJ69-4A with an empty pAS2-1 and an empty pACT2 vector, with a derived pAS2 plasmid and an empty pACT2 vector, or with an empty pAS2 vector and a derived pACT2 plasmid never resulted in the growth of colonies on selective SC-LTHA plates, showing that none of the GAL4 fusion proteins encoded by the recombinant plasmids used could cause activation of the His and Ade reporter genes by themselves. For pACT2-β4 1457–1752 , –keratin 5, and –keratin 8 constructs, a slight autonomous activation of the reporter genes was found, but this could be repressed by the addition of 2 mM 3-amino-1,2,4-triazole (a His antagonist) to the medium. Escherichia coli strain BL21(DE3), genotype F − ompT gal [dcm] [lon] hsdS B carrying DE3 λ prophage with the T7 RNA polymerase gene (Novagen), was transformed with recombinant pRP261 plasmids. Colonies obtained were used to inoculate Luria-Bertani medium containing 100 μg/ml ampicillin, and cultures were grown overnight at 37°C and 250 rpm. Cultures were then diluted 1:20 in fresh medium, grown to an OD 600 of 0.7 at 30°C and 200 rpm, and induced by the addition of isopropyl β- d -thiogalactopyranoside (IPTG) to 0.4 mM for an additional 3 h. Bacteria were harvested by centrifugation at 4,000 g , resuspended in PBS containing 1 mM EDTA and 1% (vol/vol) Triton X-100, and lysed by sonication. Lysates were cleared by centrifugation for 10 min at 10,000 g and 4°C, and the resulting supernatants were incubated with glutathione agarose beads . Beads with affinity-bound proteins were washed three times with PBS containing 1% (vol/vol) Triton X-100, and equilibrated in 50 mM Tris-HCl (pH 8.0). Bound proteins were eluted in 50 mM Tris-HCl (pH 8.0), containing 10 mM reduced glutathione. Recombinant MBP-plectin ABD fusion protein was expressed and purified as described above, except that amylose resin (800-21; New England Biolabs) was used for the affinity purification, and that equilibration and elution of the resin was in 20 mM Tris-HCl (pH 7.4), 1 mM β-mercaptoethanol without or with 10 mM maltose, respectively. Buffers containing the eluted fusion proteins were exchanged by ultrafiltration using Centricon 10 filters (Amicon; Millipore). Purified recombinant GST fusion proteins containing different fragments of the β4 cytoplasmic domain (0.4 mg/ml) dissolved in actin polymerization buffer (APB) (10 mM Tris-HCl, 2 mM MgCl 2 , 100 mM KCl, 0.5 mM ATP, 0.1 mM β-mercaptoethanol, pH 7.5) or actin-G buffer (AGB) (10 mM Tris-HCl, 0.2 mM CaCl 2 , 0.1 mM β-mercaptoethanol, pH 7.5) were precleared by centrifugation at 100,000 g for 30 minutes at 4°C in a TLA 100.3 rotor (Beckman). Subsequently, protein concentration of the cleared lysates was determined using Bradford protein assay. Bovine α skeletal muscle actin in AGB was polymerized at 4.5 μM for 1 h at room temperature. Samples were diluted to 10–30 μg/ml in the appropriate buffer and spotted, 100 μl per well, on nitrocellulose membrane using a Hoeffer 96 wells dot blot system (Amersham Pharmacia Biotech). Membrane strips were subsequently incubated in blocking buffer (APB or AGB supplemented with 0.2% [wt/vol] heat-inactivated BSA, and 10 μg/ml each of the protease inhibitors aprotinin, leupeptin, and pepstatin). [ 35 S]methionine/cysteine-labeled plectin-ABD protein was obtained by coupled in vitro transcription/translation of pcDNA3HA-plectin ABD DNA using the T n T ® coupled reticulocyte lysate system (Promega). Nonincorporated radiolabeled amino acids were removed from the in vitro translation mixture using Centricon 10 filters. Purified translation mixture (25 μl) was diluted into 3 ml of blocking buffer and incubated for 3 h at room temperature with the nitrocellulose strips. The strips were then washed three times in blocking buffer, air dried, and exposed using Kodak X-Omat AR film. Bovine α skeletal muscle actin (5 μM in AGB) was allowed to polymerize in the presence of MBP-plectin ABD fusion protein (1 μM) by the addition of 0.1 vol of 10× initiation mix (20 mM MgCl 2 , 1 M KCl, 5 mM ATP) for 1 h at room temperature. Polymerized actin filaments complexed with plectin were pelleted by centrifugation for 1 h at 100,000 g and 20°C, and resuspended in APB. Actin–plectin complexes were then incubated with 10 μM of purified, precleared GST-β4 fusion proteins (as described above) for 1 h at room temperature. Actin filaments with bound protein were pelleted as described above, and corresponding amounts of pellet and supernatant were analyzed by SDS-PAGE. Two immortalized keratinocyte cell lines were derived from unrelated MD-EBS patients, and analyzed by Western blot using specific antisera for the different hemidesmosome proteins . Expression of the hemidesmosome proteins was similar in the two MD-EBS cell lines, but different from that in NHK cells because the MD-EBS keratinocytes expressed β4, BP230, and BP180, but not plectin. Next, we analyzed the distribution of hemidesmosome proteins in MD-EBS cells using confocal immunofluorescence microscopy . As expected, no reaction was found with plectin antiserum . The integrin α6β4, as well as BP180 and BP230 were concentrated at cell–substrate contact sites in patch-like structures . This staining pattern is typical for hemidesmosome-like structures in cultured keratinocytes . The extracellular ligand of α6β4, laminin-5, was concentrated underneath the hemidesmosome-like structures . These observations indicate that at least on a laminin-5 matrix, keratinocytes can form hemidesmosomes in the absence of plectin. Plectin contains a highly conserved NH 2 -terminal ABD, and is therefore potentially capable of binding actin; yet F-actin has not been found in association with hemidesmosomes. We speculated that binding of α6β4 to plectin might prevent it from binding to F-actin, possibly by competing with actin for overlapping binding sites on plectin. To examine the subcellular distribution of the NH 2 -terminal ABD of plectin, an expression vector carrying plectin-ABD cDNA, a clone encoding the first 339 amino acid residues of human epithelial plectin encompassing its ABD fused to an HA tag, was transiently transfected in MD-EBS cells . In the majority of cells, the plectin fragment was efficiently expressed, and its staining pattern overlapped with that of phalloidin-stained F-actin . However, in a population of ∼10–20% of the cells, it was also present in basally located hemidesmosome-like clusters not associated with F-actin . Only very few cells exclusively express the plectin fragment in hemidesmosome-like clusters . These data suggest that the NH 2 terminus of plectin is not only capable of associating with F-actin, but also with α6β4-containing hemidesmosome-like structures. The above results prompted us to investigate whether the intact plectin molecule is also capable of associating with F-actin. Therefore, we compared its distribution with that of F-actin in PA-JEB keratinocytes that endogenously express full-length plectin. In addition, we studied the distribution of these two proteins in PA-JEB cells that stably express the integrin α6β4 at their cell surface. In both cell types the staining pattern of plectin with the mAb 121 against plectin/HD1 and an affinity-purified polyclonal antiserum against the plectin-ABD was similar. In the PA-JEB keratinocytes, both antibodies stained plectin diffusely throughout the cytoplasm, with some enrichment of the protein in regions where F-actin was also found to be concentrated . In general, the staining pattern of plectin seen with the mAb 121 appeared more in spots, whereas the staining produced by the polyclonal plectin antiserum was often continuous and clearly associated with cytoskeletal elements . Plectin was only occasionally found to be associated with F-actin at the cell periphery, where microtubules and/or intermediate filaments may be running parallel to F-actin and be linked to it by plectin . Association of plectin with F-actin probably also occurs at other sites in the cell, but it may not be as easily recognized there because the cytoskeletal fibers it links do not run parallel to one another but are intertwined. Furthermore, it should be realized that the distribution of F-actin may be different from that of plectin, because plectin can also cross-link other cytoskeletal systems, i.e., intermediate filament with microtubules . In PA-JEB cells that stably express the integrin α6β4, both antibodies stained hemidesmosome-like structures, which were devoid of F-actin . These results demonstrate that plectin is associated with the cytoskeleton, including F-actin, or with hemidesmosome-like structures, and that the localization of plectin with the latter is dependent on the expression of α6β4. To investigate whether the NH 2 -terminal ABD of plectin can interact directly with the cytoplasmic domain of the β4 integrin subunit, and to determine the binding site on β4, a yeast two-hybrid assay was performed. The plectin-ABD fragment was expressed as a GAL4 BD fusion, together with one of a set of overlapping fragments of the β4 cytoplasmic domain (as GAL4 AD fusions) in the yeast strain PJ69-4A. Interactions were detected by the growth of yeast colonies on selective SC-LTHA plates . A high plating efficiency, indicating that the reporter genes were efficiently expressed as a result of a strong interaction between the plectin-ABD and β4, was observed with the β4 1115–1457 , β4 1115–1382 , and β4 1115–1355 constructs. The site of interaction on the β4 cytoplasmic domain could be mapped to the first and second FNIII repeat and 27 residues of the CS, as present in the β4 1115–1355 construct. The β4 1115–1328 construct with the last 27 amino acids deleted interacted only weakly with the plectin-ABD. The first FNIII repeat itself does not bind plectin-ABD, but is nevertheless essential for this binding, since its deletion in the β4 1217–1328 construct results in a complete abrogation of binding. No interaction could be found with the third or fourth FNIII repeat, alone or when present as a pair. Therefore, the interaction of the NH 2 terminus of plectin with β4 is specific for the first pair of FNIII repeats and 27 amino acids of the CS . Recently, two patients with nonlethal PA-JEB have been described who were either homozygous or heterozygous for a missense mutation, i.e., a single amino acid substitution from R to W at residue 1281 of β4 . Since the R to W mutation was within the binding site for plectin mentioned above, we introduced it into wild-type β4 cDNA to study its effect on the binding of β4 to plectin. This was first tested in a yeast two-hybrid assay . The results show that β4-plectin binding was completely abrogated by this mutation, confirming that the second FNIII repeat of β4 is essential for binding to plectin. We then tested whether the intramolecular interaction within the wild-type β4 cytoplasmic domain, as described previously , was affected in the β4 R1281W mutant. Wild-type and R1281W constructs were expressed in yeast as GAL4 AD fusions together with a GAL4 BD fusion of β4 1457–1752 . Both wild-type and mutant bound strongly to the β4 1457–1752 fusion protein, indicating that intramolecular binding could still occur in the β4 R1281W mutant protein. Next, we tested the effect of the R1281W mutation on the function of β4 in hemidesmosome formation in immortalized keratinocytes isolated from a PA-JEB patient who completely lacked β4 . PA-JEB keratinocytes were transiently transfected with full-length wild-type β4 or β4 R1281W cDNA and analyzed by confocal immunofluorescence microscopy . In transfected PA-JEB cells, both the wild-type β4 and the β4 R1281W protein were found at the basal side of the cell in hemidesmosome-like clusters. Whereas wild-type β4 was colocalized with plectin, BP180, and BP230 in hemidesmosome-like structures , the β4 R1281W protein was only colocalized with BP180 and BP230 . Thus, the data show that plectin can directly bind β4. The recruitment of BP180 into hemidesmosomes does not seem to require plectin; BP180 (and BP230) and plectin can be independently recruited by β4. The cell transfection studies described in Fig. 3 suggest that both F-actin and β4 can bind to the plectin-ABD. Binding of β4 to an NH 2 -terminal part of plectin has been shown previously by Rezniczek et al. 1998 using in vitro binding assays, but this interaction appeared not to require the presence of the plectin-ABD, since its deletion from a smaller plectin fragment did not result in the abrogation of binding to β4. To examine the binding of β4 to the plectin-ABD and the presence of additional binding sites COOH-terminal of the ABD, a set of overlapping fragments of the entire plectin NH 2 terminus was generated. Each of these was expressed in yeast as a GAL4 BD fusion, together with β4 1115–1457 as a GAL4 AD fusion . Binding of β4 1115–1457 to the plectin NH 2 terminus could only be observed for the plectin-ABD fragment that contains residues 1–339 used in the experiments described above. The plectin-ABD clone contains the first 9 exons of the plectin PLEC1 gene, including the variable exon 1c (encoding residues 1–65), which is specific for epithelial plectin and encodes a protein sequence with no known homologies. Exons 2–8 encode the plectin-ABD (amino acid residues 66–302), and exon 9 (residues 303–343) encodes a unique stretch of amino acids. For further mapping of the β4-binding site and for comparing it with that of actin, plectin-ABD subclones were constructed and tested in a yeast two-hybrid assay with β4 1115–1457 or with full-length actin. As shown in Fig. 7 B, the plectin-ABD binds not only β4, but also actin. The sequences encoded by exons 1c and 9 are not involved in binding to β4. Deletion of NH 2 -terminal sequences within the ABD, even of only the first four residues, disrupted binding to β4 completely, suggesting that the first part of the ABD is essential. COOH-terminal deletions showed that deletions extending into the ABD abrogate binding even when all three ABS sequences (residues 72–83, 144–170, and 182–196) were intact, showing that the last part of the ABD is also required. Therefore, the minimal region in plectin required for binding to β4 comprises residues 65–302, i.e., the complete ABD. Investigation of the binding site for actin on the same panel of plectin fragments showed that deletion of the first 35 residues in plectin did not affect its interaction with actin, but that removal of all 64 residues encoded by exon 1c abolished it, in contrast to the binding of β4. This could be due to steric hindrance of the ABD in juxtaposition to the GAL4 BD moiety present in the GAL4-plectin 65–339 fusion protein, since data in the literature do not reveal that amino acids NH 2 -terminal of the ABD are required for binding to actin in other actin-binding proteins . COOH-terminal deletions showed that no residues COOH-terminal of the plectin-ABD are required for binding of actin either. However, in contrast to β4, actin could still bind when COOH-terminal parts of the ABD were deleted up to and including ABS3. Only deletion of ABS2 resulted in complete loss of actin binding. Therefore, ABS1 and ABS2 are sufficient to bind actin. Plectin is versatile in its binding of actin; three different actin isoforms were tested for interaction with plectin 1–339 : α skeletal muscle actin, β cytoplasmic actin, and γ cytoplasmic actin, and all effectively bind plectin (data not shown). In conclusion, the binding sites for β4 and actin both reside in the plectin-ABD. Both sites start at the beginning of the ABD; actin binding only requires ABS1 and ABS2; the binding of β4 also requires ABS3 and more COOH-terminal sequences. β4 and actin do not bind to each other; they do not interact in the yeast two-hybrid assays (data not shown). Together, these results suggest that the binding site for β4 overlaps with that for actin. Therefore, only one of these proteins can bind to a single plectin molecule, binding thus being mutually exclusive. To confirm the yeast two-hybrid results described above, an in vitro binding assay was performed. GST-β4 fusion proteins or F-actin were spotted on nitrocellulose using a dot blot system and the immobilized proteins were overlaid with in vitro translated, radio-labeled plectin-ABD protein . Two different buffers were used in the overlay assay: APB, a high-salt buffer which maintains F-actin in a polymerized state, and AGB, a low-salt buffer in which F-actin can depolymerize to monomeric G-actin. The plectin-ABD strongly bound F-actin, GST-β4 1115–1382 , but not GST-β4 1115–1382* or GST-β4 1457–1752 , which is in accordance with the yeast two-hybrid assay results . No significant binding was found to GST-β4 1115–1328 , although this mutant protein interacted weakly with the plectin-ABD in the yeast two-hybrid assay. These different results may reflect a difference in the sensitivity of the two assays, with the yeast two-hybrid assay being more sensitive than the dot blot assay. Consistent with the absence of binding of the β4 1115–1328 mutant to plectin in the dot blot assay, the mutant protein was also unable to induce a redistribution of plectin in cell transfection experiments . Binding of the plectin-ABD to the GST-β4 1115–1382 fusion protein was stronger in the presence of low salt (AGB). Thus, binding is probably ionic in nature, explaining why charged residues, i.e., R1281, are important for β4–plectin interactions. In contrast, binding of the plectin-ABD to actin was significantly weaker in the presence of low salt. To obtain evidence that β4 and F-actin indeed compete for binding to the plectin-ABD, we performed an in vitro competition assay. F-actin, polymerized in the presence of MBP-plectin-ABD fusion protein, was incubated with a 10-fold molar excess of soluble GST fusion proteins. Then the F-actin complexes were precipitated by centrifugation and the supernatant and pellet were collected separately . In the absence of GST-β4 fusion protein, or in the presence of GST-β4 1115–1328 or GST-β4 1115–1382* , all MBP-plectin protein was found in the pellet together with actin. However, in the presence of GST-β4 1115–1382 , about half of the MBP-plectin protein appeared in the supernatant due to binding to the soluble GST-β4 fusion protein. The results show that β4 can efficiently compete with F-actin for binding to the plectin-ABD. The results presented so far provide evidence for a role of the NH 2 terminus of plectin in the binding of both F-actin and β4. To rule out a role for the COOH terminus of plectin more precisely of its globular repeat domains, we also tested cDNA clones encoding this part of the plectin molecule. Since earlier studies had indicated a role for sequences at the end of the R5 domain in binding to intermediate filament proteins, plectin fragments containing this putative IFBD were also tested for interaction with the keratins 5, 8, 14, and 18, and with vimentin and GFAP. Two plectin cDNA clones were tested in the yeast two-hybrid assay. One encoded most of R5, the complete R6, and the COOH tail, and the other encoded most of R6 and the COOH tail. They were expressed in yeast as GAL4 BD fusion proteins together with GAL4 AD fusion proteins of β4 1115–1457 , β4 1320–1668 , or β4 1457–1752 , or of different intermediate filament proteins . No interaction was found between the plectin fragments and any of the β4 fragments tested. However, specific interaction of the larger plectin fragment, which contained the IFBD at the end of R5, could be detected with different types of intermediate filament proteins. Binding was observed with keratins 14 and 18, but not with keratins 5 or 8, and with both vimentin and GFAP. Thus, the results show that the plectin COOH terminus has no function in the binding to the β4 cytoplasmic domain, but confirm the presence of a distinct intermediate filament protein–binding site in the plectin domain R5, which we have shown to bind the type I and III, but not type II intermediate filament proteins. To investigate whether the plectin-IFBD fragment that interacts with intermediate filament proteins in a yeast two-hybrid assay also associates with intermediate filaments in keratinocytes, we transiently transfected MD-EBS cells with an expression vector containing the plectin-IFBD fused to an HA tag, and analyzed the results by confocal immunofluorescence microscopy . In contrast to normal keratinocytes in vivo, which do not express vimentin but only keratins, the EBS-MD keratinocytes coexpressed vimentin and keratin intermediate filaments. In cells expressing plectin-IFBD, the HA-tagged protein was found to be distributed together with keratins in delicate filamentous structures . Although the expression of plectin-IFBD had no apparent effect on the keratin intermediate filament network, in many cells it induced a collapse of the vimentin network into dense clusters around the nucleus . Colocalization of these two proteins in filamentous structures was only observed in a few cells (data not shown). The dramatic effect of the plectin-IFBD on the integrity of the vimentin intermediate filament network suggests that it interferes with another protein with properties similar to plectin that mediates the anchorage of these filaments at distal or peripheral sites in the cell. It may also indicate that plectin-IFBD binds more strongly to vimentin than to keratins. The α6β4 integrin was always present in basally located hemidesmosome-like clusters, and showed no obvious colocalization with plectin-IFBD . These results suggest that in a normal situation, when the intact plectin protein is present, it helps to provide a scaffold for the vimentin cytoskeleton. To study a possible indirect role for the integrin α6β4 (via plectin) in the organization of the vimentin intermediate filament network, immortalized β4-deficient PA-JEB keratinocytes were transiently transfected with cDNAs encoding wild-type or mutant β4 and used in confocal immunofluorescence microscopy . In nontransfected cells, vimentin is present in rather loose filamentous networks throughout the cytoplasm, but in cells expressing β4 it was also detected in dense basally located clusters colocalized with α6β4 . These results show that expression of β4 leads to a reorganization of the vimentin intermediate filament network. This most likely occurs by binding of β4 to the ABD in the plectin NH 2 terminus, accompanied by binding of vimentin to the IFBD in its COOH terminus. The effect of β4 on the vimentin network would then be indirect and dependent on the binding of β4 to plectin. Further evidence for this was obtained by transfection of one mutant β4 protein, β4 1355 , that can efficiently bind plectin, and one β4 mutant, β4 1328 , that binds plectin in yeast two-hybrid assays, but much less efficiently. In cells transfected with β4 1355 cDNA, the β4 protein fragment was present in basal hemidesmosome-like clusters colocalized with vimentin . The β4 1328 mutant was also found in basal clusters, but it only occasionally colocalized with vimentin . These results show that β4 is capable of organizing the vimentin intermediate filament cytoskeleton. It does so indirectly, via plectin, since only β4 mutants that contained the plectin binding site were capable of organizing vimentin filaments into hemidesmosome-like basal clusters. Since this β4 mutant cannot recruit BP180 or BP230 , the BP proteins do not seem to play a role in this process. We have established immortalized keratinocytes from a human MD-EBS patient. In these cell lines, hemidesmosome-like clusters containing α6β4, BP180, and BP230 (but not plectin) were present at the basal side of the MD-EBS cells at sites of cell–substratum contact, as was the ligand for α6β4 laminin-5. Except for the absence of plectin, these hemidesmosome-like clusters are indistinguishable from hemidesmosomes in NHK cells . FACS ® analysis of the MD-EBS cells gave expression levels of the α6 and β4 subunits that are about half of those in NHK cells (data not shown). In plectin-deficient null-mutant mice, hemidesmosomes were also present, but their number as well as the amount of β4 at the basal side of the epidermal cells was reduced, indicating an important role for plectin in maintaining the integrity of hemidesmosomes . Evidently, normal basal localization of α6β4 into hemidesmosome-like clusters can occur in MD-EBS keratinocytes, but plectin might have a role in retaining α6β4 at the cell surface. It should be noted that hemidesmosome stability and/or the localization of hemidesmosome proteins is probably influenced by mechanical stress in the epidermis of the null-mutant mice. The resistance to mechanical stress of the hemidesmosomes in the MD-EBS keratinocytes has not yet been tested. Expression of a plectin fragment containing the ABD in MD-EBS keratinocytes resulted in two different types of subcellular distribution: colocalization with actin stress fibers or a patch-like organization which is typical for hemidesmosomes. The decoration of actin stress fibers was also found in similar experiments with plectin in fibroblasts and with other proteins of the spectrin family . However, a unique feature of the ABD of plectin, is that in keratinocytes it can also dictate the localization of plectin into hemidesmosomes, together with α6β4. Direct binding of β4 to the plectin-ABD was demonstrated using yeast two-hybrid and dot blot overlay assays. In addition, we showed that β4 can effectively compete plectin-ABD out of an F-actin–plectin complex in an in vitro actin cosedimentation assay. This suggests that competition between β4 and actin for overlapping binding sites on the plectin-ABD is the molecular basis for the relocalization of plectin from actin-containing cytoskeletal complexes (like actin stress fibers or focal contacts) to β4-containing hemidesmosomes at the basal side of keratinocytes when β4 is expressed. It is not yet clear whether these processes are also regulated at a different level, i.e., by protein phosphorylation, or by a regulation of actin filament dynamics via phosphatidylinositol 4,5-bisphosphate (PIP2), for example . Since hemidesmosomes are generally seen as stable adhesion complexes that are less compatible with cell motility than the actin structures, it is clear that plectin recruitment into hemidesmosomes might represent a pivotal factor in adhesion and motility properties of epithelial cells. In a recent study, two distinct plectin-binding sites were identified on the β4 cytoplasmic domain: one that encompasses the first two FNIII repeats and the CS , and another that contains the second pair of FNIII repeats and the COOH terminus . NH 2 -terminal fragments of epithelial rat plectin (both with and without the ABD) were found to interact with both binding sites on β4 in vitro in overlay assays, as well as in vivo by transient transfection of rat kangaroo Ptk2 cells and hemidesmosome-forming 804G rat bladder carcinoma cells, in which β4 and plectin protein fragments were shown to be colocalized mainly in dense perinuclear structures in the cytoplasm . Previously, we have identified a region on the β4 cytoplasmic domain, in the first pair of FNIII repeats and the beginning of the CS that is involved in the formation of a complex with plectin and its recruitment to hemidesmosomes . In this study, we confirmed the presence of the NH 2 -terminal plectin-binding site on the β4 protein, and mapped it to residues 1217–1355. No evidence was found for the more COOH-terminal binding site reported by Rezniczek et al. 1998 . Full-length β4 containing the amino acid mutation R1281W, in marked contrast to wild-type β4 , no longer recruited plectin into basal hemidesmosome-like clusters after being expressed in PA-JEB keratinocytes. This strongly suggests that only the NH 2 -terminal plectin-binding site on β4 is required for the proper localization of plectin into hemidesmosomes. Another difference with the results obtained by Rezniczek et al. 1998 is that we located the binding site for β4 in the plectin-ABD and were not able to demonstrate interaction of β4 with NH 2 -terminal fragments of plectin from which the ABD is absent. Nevertheless, it remains possible that sequences downstream of the ABD, although not able to mediate binding by themselves, could enhance the binding of the ABD to β4. Such a scenario of cooperative binding may proffer an explanation for the finding that in the transfected MD-EBS cells, the plectin-ABD alone is found to be primarily colocalized with F-actin. However, the discrepant results could also stem from the fact that Rezniczek et al. 1998 used denatured proteins for the in vitro overlay experiments, whereas the β4 cytoplasmic domain in vivo is most probably intricately folded . Our concern with the cell transfection experiments presented by these authors is that overexpression of the β4 protein fragments could affect their localization and cause them to aggregate with other cytoplasmic structures, as has been described previously for proteins expressed at high levels . In our studies, the β4 clones used for cell transfections contain the complete extracellular and transmembrane regions, and therefore the expression of β4 at the basal cell surface (the physiological location for α6β4) is limited by the amount of endogenous integrin α6 subunit. Reports of a decreased basal localization of BP180 in epidermal cells of a human MD-EBS patient and of an interaction of the BP180 cytoplasmic domain with the plectin COOH terminus in a yeast two-hybrid experiment suggested a role for plectin in the recruitment of BP180 into hemidesmosomes. Previously, we provided evidence for this by reporting on the basal localization of BP180 in β4-deficient PA-JEB keratinocytes, in which mutant β4 proteins were transiently expressed. Two mutant β4 proteins that could not recruit plectin were also incapable of localizing BP180, whereas their recruitment of BP230 was severely impaired . However, results presented in this paper demonstrate that in MD-EBS keratinocytes, both BP180 and BP230 are colocalized with α6β4 in basal hemidesmosome-like clusters in the absence of plectin. Furthermore, we have found that when expressed in PA-JEB keratinocytes, the β4 R1281W mutant was unable to bind plectin, but could recruit BP180 and BP230 into hemidesmosome-like clusters. Finally, no interaction could be detected between the COOH-terminal fragments of plectin used in this study and the complete BP180 cytoplasmic domain in a yeast two-hybrid assay (data not shown). We reasoned that the two β4 mutant proteins used in our previous study, β4 Δ1219–1319 , in which the complete second FNIII repeat had been deleted, and β4 Δ17 , in which amino acids 1249–1265 of the second FNIII repeat are absent , were incorrectly folded in such a way that the binding site for BP180 on β4 was rendered dysfunctional. There is evidence for an intramolecular folding in vitro of the β4 cytoplasmic domain , in which the COOH-terminal part of the β4 cytoplasmic domain could fold back and bind close to the first pair of FNIII repeats. If so, improper folding of the first pair of FNIII repeats could have an effect on the folding of the second pair of FNIII repeats, where the binding site for BP180 is localized. In the β4 R1281W mutant, this intramolecular association is still intact. We conclude that binding of β4 to plectin and BP180 most probably occurs independently. From our data it is expected that a patient who is homozygous for the R to W mutation at residue 1281 in β4, would have a phenotype similar to that of plectin-deficient patients, i.e., EBS. However, a β4 patient diagnosed to be homozygous for this mutation had a mild form of JEB . Possibly, the R1281W mutation is responsible for the JEB phenotype by preventing the proper expression of α6β4 at the cell surface, thereby compromising the adhesion function of the cell. A role for plectin in facilitating or maintaining high surface levels of α6β4 is suggested, because as mentioned previously, in the two MD-EBS cell lines used in this study a reduction in the levels of α6β4 has been observed. Indeed, the expression of α6β4 in the patient, although clearly detectable, was found to be diminished. In skeletal muscles, plectin is probably involved in connecting the actin filaments to intermediate filaments. These tissues do not express the integrin α6β4, and therefore a muscle phenotype is not expected in a patient homozygous for the R to W mutation. Our results from transfection experiments with the β4 Δ17 and β4 R1281W mutants (this study), which are both mutations identified in patients , show that differences in the severity of the disease in different PA-JEB patients can also result from an impaired function of β4 to recruit BP180 and BP230 into hemidesmosomes. Both mutants are unable to recruit plectin, but only the β4 Δ17 protein had also lost its ability to interact with BP180. Experiments like those described in this paper could be valuable for providing insight into the molecular processes involved in EB disease. We show that COOH-terminal plectin fragments can directly bind to various types of intermediate filament proteins in a yeast two-hybrid assay. Furthermore, upon expression in MD-EBS keratinocytes, the plectin-IFBD fragment is found localized along keratin intermediate filaments and induces a collapse of the vimentin network. This latter finding is of interest because it suggests a specific role for this domain of plectin in stabilizing the vimentin intermediate filament network and its association with hemidesmosomes. The significance of anchoring vimentin to hemidesmosomes (seen in the PA-JEB cells after transfection with β4 cDNA) is not clear, since keratinocytes normally do not express this intermediate filament protein, although it is sometimes seen in rapidly migrating cells. However, it may be more important for cells that are α6β4-positive and express type III intermediate filaments, such as certain types of fibroblasts and endothelial cells. In fact, it has been shown recently that vimentin is associated with the β4 subunit of the α6β4 integrin in endothelial cells . Coexpression of vimentin with keratins is observed in many epithelial tumors, and has been linked to metastatic disease . It is tempting to speculate that in transformed keratinocytes the expression of vimentin may alter the interaction between hemidesmosomes and keratins, and that this may play a role in the development of metastases. In the yeast two-hybrid assay, the binding of plectin to vimentin depends on the presence of the plectin-IFBD (located at the end of domain R5), first identified by Wiche and co-workers , since clones missing this region do not interact with this intermediate filament protein. In addition, plectin binds the type I (or acidic) keratins 14 and 18, and the type III intermediate filament protein GFAP. The type II (or basic) keratins 5 and 8 do not bind, which for the first time demonstrates specificity of the binding of plectin to keratins. In a previous study a mixture of keratins was used, and thus binding to particular keratins could not be distinguished. Since the crucial region in the plectin-IFBD is strongly basic, it is suggested that binding to the acidic type I keratins is ionic in nature. In vivo keratin filaments are exclusively composed of heterodimers, i.e., specific combinations of a type I and a type II monomer. Keratin 5 dimerizes with keratin 14 and keratin 8 with keratin 18. In the yeast two-hybrid assay, only a single keratin was available for interaction with the plectin-IFBD and since keratins cannot form stable homopolymers , plectin most probably is capable of binding monomeric keratin. The fact that plectin associates with intermediate filaments demonstrates that in the formation of keratin dimers the binding site for plectin is not blocked. The association of plectin with intermediate filament proteins can be influenced by specific plectin phosphorylation events. Together, these findings support the idea that plectin-intermediate filament association is a well-regulated event in the organization of the cytoskeleton . In apparent contrast with the findings published by Rezniczek et al. 1998 , which described a binding site for β4 on a COOH-terminal fragment of plectin starting with the last part of domain R5 using in vitro binding assays, we were not able to detect binding of the plectin fragment containing these sequences to the β4 cytoplasmic domain (see previous section for a possible explanation); no interaction was found between GAL4–plectin and GAL4–β4 fusion proteins in a yeast two-hybrid assay and similarly, plectin-IFBD protein was not found to be colocalized with α6β4 in hemidesmosome-like basal clusters upon expression in MD-EBS keratinocytes. Thus, the COOH terminus of plectin appears to be primarily involved in the binding of intermediate filament proteins, rather than of β4. Plectin can bind at least three different actin isoforms with similar efficiencies (data not shown), in contrast to related actin-binding proteins like utrophin, which have different binding efficiencies for various actin isoforms . Actin isoforms are expressed at specific stages of development and only in certain tissues, and can have specific subcellular distributions within one cell . Thus, plectin can act as an actin linker protein in all these tissues and cells. Like plectin, other plakins also show specificity in binding intermediate filament proteins: BP230 binds neurofilament proteins but not vimentin , and desmoplakin (a desmosomal plakin) has a much higher affinity for type II keratins than for type I keratins or vimentin . The different specificities of these plakins for the various intermediate filament proteins suggest slightly divergent functions in the organization of the intermediate filament network. In this respect, it is interesting to note that plectin cannot compensate for the loss of BP230, or vice versa, in human patients or null-mutant mice deficient for one of these proteins . The data presented in this study conclusively prove that the NH 2 terminus of plectin is involved in interactions with actin filaments or with β4 and its COOH terminus in binding to intermediate filament proteins. A similar organization of binding sites has been found for desmoplakin: its NH 2 terminus binds to proteins in the adhesion complex , whereas its COOH terminus interacts with intermediate filament proteins . The assignment of specific and distinct protein-binding sites on the plectin molecule and the study of the regulation of plectin-binding events will allow a more detailed analysis of its function in interlinking adhesion complexes and the intracellular filament networks. | Study | biomedical | en | 0.999996 |
10525546 | Yeast strains used in this study are shown in Table . To construct the Δ apg8 strains, TRP1 , LEU2 , and HIS3 , which was obtained from pJJ288, pJJ252, and pJJ215, respectively, was substituted for the AccI–HpaI fragment containing 98% of the APG8 open reading frame (ORF). TK402, TK404, TK405, TK407, and KVY5 were obtained by selection on appropriate amino acid drop-out medium. Media used in this study were described previously, and SD medium supplemented with 0.5% casamino acid was referred as SD+CA medium . APG8 was cloned according to the method previously reported . pAPG8317 was generated by cloning 2.6 kbp of XbaI–XbaI fragment, including APG8, into pBluescript II KS + . Using pAPG8317 as a template DNA, the SpeI–EcoRI fragment, including the 0.9 kbp of HincII–HincII sequence, was generated by PCR using following primers: APG8F1, 5′-GACTAGTAGGTCTCGCAAGAGAGC-3′; APG8R1, 5′-GGAATTCGAAATCTTGGCTCCGTTG-3′. pTK101 and pTK201 were generated by inserting this SpeI–EcoRI fragment into the yeast centromeric and multicopy vectors, pRS316 and pRS426 , respectively. For construction of 3 × HA (hemagglutinin)-tagged APG8 plasmids, a BamHI site was generated at the 5′ terminus of the APG8 ORF by PCR using APG8F1, APG8R1, and the following primers: 5′-GACATGGGATCCAAGTCTACATTTAAGTCTG-3′ and 5′-AGACTTGGATCCCATGTCTCTAGTAATTAT-3′. The resulting fragment was cloned into pBluescript II KS + , and a BamHI–BamHI fragment containing a 3 × HA sequence generated by PCR was inserted in the BamHI site at the 5′ terminus of the APG8 ORF. The 3 × HA-tagged APG8 plasmids, pTK110 and pTK108, were generated by cloning this SpeI–EcoRI fragment containing the 3 × HA-tagged APG8 gene into yeast centromeric plasmids, pRS314 and pRS316 , respectively. Alkaline phosphatase (ALP) assay was performed as previously described . Cells growing in YEPD medium were treated with nocodazole (Sigma Chemical Co.) for 3 h at a final concentration of 10 μg/ml. Cells were transferred to 0.17% yeast nitrogen base without amino acids and ammonium sulfate supplemented with 1 mM PMSF and 10 μg/ml nocodazole, and further incubated for 4.5 h. The accumulation of autophagic bodies was observed under a microscope. For ALP assay, the nocodazole-treated cells in YEPD were starved in SD(−N) medium containing 10 μg/ml nocodazole for 4.5 h. A peptide including the NH 2 -terminal sequence of Apg8p, MKSTFKSEYPFEKC, was synthesized by a Model 433A peptide synthesizer (PE Applied Biosystems). The peptide was conjugated to Keyhole Limpet Hemocyanin (Sigma Chemical Co.) with sulfosuccinimidyl 4-( p -maleimidophenyl)butyrate (Pierce Chemical Co.) according to the method previously reported . The resulting conjugates were immunized to a rabbit and anti-Apg8p antiserum was obtained. Cell lysates were prepared by breaking cells with the glass beads in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, and the protease inhibitor cocktail™ (Boehringer Mannheim Corp.). Total protein (20 μg) was subjected to SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) membrane (Millipore Corp.). The resulting membrane was incubated with a 1:10,000 dilution of the anti-Apg8p antibody for 1 h, followed by a 1:10,000 dilution of HRP-conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories) for 30 min. Signals were detected by the ECL kit (Nycomed Amersham, Inc.), which was used throughout this study. For the analysis of rapamycin treatment, growing cells were treated with 0.2 μg/ml rapamycin (Sigma Chemical Co.) for 2 h at 30°C in YEPD medium. Lysate preparation, followed by Western blotting, were performed as described above. Cells were suspended in TES solution (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS) and treated with acid phenol at 65°C for 1 h. Aqueous phase was collected and RNA was recovered by ethanol precipitation. mRNA was recovered by Oligotex-dT30 < super > (Takara) according to the manufacturer's protocol, separated on 1.2% agarose containing 2.2 M formaldehyde by electrophoresis, and transferred to NYTRAN membrane (Scleicher & Schuell, Inc.). 32 P-labeled probes for APG8 and ACT1 mRNA were prepared from each ORF fragment using Megaprime DNA labeling system (Nycomed Amersham, Inc.) according to the appended protocol. The mRNA-transferred membrane was incubated with each probe at 42°C overnight, and signals were detected by autoradiography. Subcellular fractionation was performed as previously described by Horazdovsky and Emr 1993 . YW5-1B cells were cultured in YEPD medium at 30°C to 1–2 × 10 7 cells/ml, shifted to SD(−N) medium for 3 h and converted to spheroplasts. Spheroplasting medium was composed of YEP, 1.2 M sorbitol, 20 mM Tris-HCl, pH 7.5, 1% glucose, and 25 μg/10 8 cells of Zymolyase 100T (Seikagaku Kogyo) for growing cells, or 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 1.2 M sorbitol, 20 mM Tris-HCl, pH 7.5, 1% glucose, and 37.5 μg/10 8 cells of Zymolyase 100T for starved cells. Spheroplasts were harvested and lysed with a lysis buffer containing 0.2 M sorbitol, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA and EGTA, 1 mM PMSF, and the protease inhibitor cocktail. Lysates (T) were generated by centrifugation at 500 g for 5 min and then spun at 13,000 g for 15 min to separate to pellet (LSP) and supernatant (LSS). LSS was then centrifuged at 100,000 g for 1 h, and pellet (HSP) and supernatant (HSS) were obtained. LSP and HSP were resuspended in equal volume of the lysis buffer to the original lysates. Equal volume of each sample was subjected to SDS-PAGE, transferred to PVDF membranes, and then incubated with a 1:5,000 dilution of the anti-Apg8p antibody. For the solubilization experiment, lysates were prepared from YW5-1B cells growing or starved for 3 h as described above. Lysates were sonicated and spun at 100,000 g for 1 h to generate pellet. The pellet was suspended in the lysis buffer (without sorbitol) containing 2% Triton X-100, the suspension was chilled on ice for 30 min and centrifuged at 100,000 g for 1 h again to separate supernatant and pellet. Pellet was resuspended in an equal volume of the lysis buffer to the original sample. Proteins recovered in each fraction were precipitated with 10% TCA and resuspended in SDS sample buffer. Equal volume of the samples was subjected to Western blotting as described. Immunofluorescent staining was performed according to the method previously described by Nishikawa et al. 1994 . In this experiment, Δ apg8 and Δ apg8 Δ pep4 cells harboring 3 × HA-tagged APG8 plasmid were used. Spheroplasts prepared from the cells fixed with formaldehyde were laid on the multiwell slide glass (Cel-line Associates, Inc.) coated by polylysine (mol wt >300,000; Sigma Chemical Co.), and permeabilized by treatment with 0.5% Triton X-100 in PBS for 10 min. Permeabilized cells were incubated in PBS containing 1% BSA at room temperature for 10 min and treated with a 1:1,000 dilution of anti-HA mAb, 16B12 (Berkeley Antibody Co., Inc.), in the same buffer at room temperature for 1 h. The cells were then incubated with 10 μg/ml of anti-mouse Ig-fluorescein F(ab′)2 fragment (Boehringer Mannheim Corp.) at room temperature for 1 h and observed under a confocal microscope, LSM 510 (Zeiss). Cells were subjected to rapid freezing and freeze-substitution fixation, and observed as previously reported . For immunoelectron microscopy, ultrathin sections were collected onto formvar-coated nickel grids and blocked in PBS containing 2% BSA at room temperature for 15 min. Incubations were carried out by floating grids on a 20 μl drop of a 1:1,500 dilution of anti-HA mAb, 16B12, at room temperature for 1.5 h. After washing, the grids were incubated for 1 h with 5- or 10-nm gold-conjugated goat anti-mouse IgG (Bio Cell Lab.). The grids were washed several times in PBS followed by several drops of distilled water and fixed with 1% glutaraldehyde for 3 min. The sections were stained with 4% uranyl acetate for 7 min and examined. Cells were starved in SD(−N) medium for 4.5 h and converted to spheroplasts. Spheroplasts were lysed in 20 mM Pipes/KOH, pH 6.8, 0.2 M sorbitol, 0.5 mM PMSF, and the protease inhibitor cocktail. Cleared lysates were generated by centrifugation at 500 g for 5 min, and treated with 100 μg/ml of proteinase K with or without 2% Triton X-100 on ice for 30 min. Equal volume of 20% TCA was added to the lysates to stop the reaction. Precipitant was collected by centrifugation, suspended in SDS sample buffer, and subjected to immunoblotting. To detect the signal of API, 1:5,000 diluted anti-API antibody (gift from D. Klionsky, Section of Microbiology, UC Davis, CA) was used. We reported an apg8-1 mutant defective in the autophagy in yeast . To identify the APG8 , cloning was done as previously described . Subcloning and sequencing revealed that APG8 is identical to the ORF named YBL078c, and encodes a hydrophilic protein of 117 amino acids. During this study, Lang et al. 1998 reported AUT7 as a novel autophagy-responsible gene, which was revealed to be identical to APG8 . We constructed the apg8 null mutant and ascertained morphologically that Apg8p is essential for autophagy, judging from no accumulation of autophagic bodies in the vacuole (data not shown). Furthermore, using the ALP assay system , we confirmed biochemically that Apg8p is essential for autophagy. The cells used for this assay have a truncated form of Pho8p, Pho8Δ60p, in the cytosol as an inactive precursor form. During starvation, Pho8Δ60p is sequestered to the vacuole, depending upon autophagy, where it is processed by the vacuolar enzymes and acquires phosphatase activity. So, we can monitor the progress of the autophagy by the increase of the ALP activity. Wild-type (TN124), Δ apg8 mutant, and the Δ apg8 mutant harboring APG8 on a centromeric plasmid were grown in SD+CA medium until 1–2 × 10 7 cells/ml, and then shifted to SD(−N) medium for three hours. The ALP activity in the cell lysates prepared from each culture was measured . In wild-type cells, the ALP activity increased in response to starvation, whereas in the Δ apg8 cells its elevation was considerably reduced. The autophagic defect in the Δ apg8 mutant was recovered by introducing APG8 on a centromeric plasmid. The result clearly shows that deletion of APG8 causes severe defect in autophagy. A homology search showed that Apg8p and its homologues make a large gene family through yeast to higher eukaryotes . One of the homologues has been reported as rat MAP1-LC3, which is directly bound to microtubule in vitro . Furthermore, Lang et al. 1998 proposed that Apg8p is bound to microtubule via another protein, Aut2p, and functions on the delivery of autophagosomes to the vacuole. We have already cloned 13 APG genes, and realized that AUT2 is allelic to APG4 , and that Apg8p physically interacts with Apg4p. However, Apg4p has an entirely different function from what they proposed (Kirisako, T., and Y. Ohsumi, manuscript in preparation). So far, there is no evidence showing that microtubules play a role in the autophagy in yeast. Nocodazole is a microtubule depolymerizing drug, which affects both spindle and cytoplasmic microtubules . We asked, morphologically and biochemically, whether autophagy proceeds normally in the presence of this drug. Wild-type cells were cultured until 10 7 cells/ml in YEPD medium at 30°C. Nocodazole was added to the culture and it was incubated at 30°C for three hours. After this treatment, >70% of the cells were arrested at G2 stage with a large bud. Then the cells were transferred to starvation medium containing 1 mM PMSF and 10 μg/ml nocodazole, and incubated further at 30°C for 4.5 h. As shown in Fig. 2 A, most large budded cells accumulated the autophagic bodies in their vacuole. TN124 cells were treated the same way, and the ALP activity was measured. Nocodazole treatment did not affect the increase of ALP activity . Furthermore, in a tub2 mutant, autophagic bodies were normally accumulated in the vacuole (data not shown). These results indicate that depolymerization of microtubule does not affect the progress of autophagy. Hence, we concluded that microtubule is not necessary for the autophagy in yeast. We raised an antiserum against Apg8p using a synthetic peptide of the NH 2 -terminal 13 amino acids of Apg8p. Using the anti-Apg8p antibody, we carried out Western blotting of the cell lysate prepared from logarithmically growing cells. In wild-type cells, the anti-Apg8p antibody brought out a single band at ∼15 kD , which is close to the predicted molecular mass of Apg8p (13.6 kD). Intensity of the band was enhanced in the cells harboring APG8 on a multicopy plasmid , whereas no signal was detected in the Δ apg8 cells . The result revealed that Apg8p is expressed in the wild-type cells at growing phase. Since APG8 is required also for the transport of proAPI from the cytosol to the vacuole , Apg8p expressed during vegetative growth should play a role in this pathway. Next, we examined Apg8p levels before and after shift to nitrogen starvation. The wild-type cells growing in YEPD medium were shifted to SD(−N) medium for various periods of time, and the lysates prepared from these cell cultures were subjected to immunoblotting with the anti-Apg8p antibody. As shown in Fig. 3 B, the amount of Apg8p started to increase 30 min after shift to starvation and remained at a high level after two hours. Finally, the amount of Apg8p increased about eightfold in response to starvation . Next, we performed Northern blotting analysis. Total mRNA was prepared from wild-type cells growing in YEPD medium, and the cells shifted to SD(−N) medium for various periods of time. As shown in Fig. 3 C, the amount of APG8 mRNA increased drastically in response to starvation, and reached at a maximum level within 30 min after shift to starvation. After 30 min, the amount gradually decreased, but at six hours, it was still severalfold higher than that of the growing cells. This result indicates that the increase of Apg8p during starvation is regulated at transcription level. Among the 12 APG genes ( APG1 , 4 – 10 , 12–14 , and 16 ) characterized so far, APG8 is the first gene whose expression is enhanced by starvation. Autophagy is known to be induced by inactivation of Tor, a phosphatidylinositol kinase homologue . We investigated the effect of Tor function on the expression of Apg8p. Wild-type cells were treated with rapamycin in YEPD medium for two hours, and the lysates prepared from the cells were subjected to immunoblotting with the anti-Apg8p antibody. As shown in Fig. 3 D, the expression of Apg8p was enhanced in response to the drug. Since rapamycin specifically inhibits the signal transduction mediated by Tor , the transcription of APG8 should be under the control of Tor-mediated signal transduction. In addition, we investigated whether other APG genes are necessary for the increase of Apg8p. Lysates were prepared from the other 14 apg mutant cells before and after shift to starvation and subjected to immunoblotting with the anti-Apg8p antibody. In every other apg mutant, Apg8p increased normally in response to starvation as the wild-type cells (data not shown), indicating that no other APG genes are required for the induction of Apg8p. We carried out subcellular fractionation. Cell lysates were prepared from growing and starved wild-type cells. Cell lysates (T) were centrifuged at 13,000 g for 15 min and the pellet (LSP) fractions were obtained. The resulting supernatant fractions were spun at 100,000 g for one hour to generate supernatant (HSS) and pellet (HSP) fractions, and the distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody. Apg8p was recovered mostly in LSP and HSP fractions under both growing and starvation conditions, although in starved cells it was more detectable in HSS fraction than in growing cells . Next, the cell lysates were vigorously sonicated to exclude lumenal proteins out of organelles or other membrane structures, and was spun at 100,000 g for one hour to generate a pellet fraction. Under both growth conditions, Apg8p mostly remained in the pellet fraction, in contrast CPY, a vacuolar lumenal protein, was completely recovered in supernatant fraction . It suggests that Apg8p is bound to membrane or associated with a pelletable large protein complex. The pellet fractions generated from the sonicated lysates were treated with 2% Triton X-100, incubated for 30 min on ice, and then separated to supernatant and pellet fractions by centrifugation at 100,000 g for another one hour. As shown in Fig. 4 C, 2% Triton X-100 efficiently solubilized Apg8p, indicating that most pelletable Apg8p is bound to some membrane structures under both growing and starvation conditions. To examine intracellular localization of Apg8p, immunofluorescence microscopy was performed. First, we carried out this experiment using the anti-Apg8p antibody, but sufficient signal was not obtained. Then, we constructed a single copy 3 × HA-tagged APG8 plasmid and introduced it into the Δ apg8 mutant. We ascertained that the expression level of the 3 × HA-tagged APG8 gene in the resulting transformant, TK114, was similar to that of authentic APG8 (data not shown), and that the transformant recovered the autophagic activity up to 70% of the wild-type cells . The cells were grown in SD+CA medium until 2 × 10 7 cells/ml, and diluted fourfold in YEPD medium. The cells were grown for two generations, transferred to SD(−N) medium, and incubated for 0, 0.5, and 3 h. The localization of 3 × HA–Apg8p was examined with anti-HA mAb. In the cells growing in YEPD medium (t = 0), the fluorescence image consisted of many tiny dots dispersed throughout the cytoplasm, which was distinct from well known organelles such as ER, Golgi body, nucleus, vacuole, or plasma membrane . In addition, we occasionally observed punctate signals distinct from the tiny dots . 30 min after the shift to the starvation condition, the staining pattern drastically changed; one or two bright, large, punctate signals appeared in the cytoplasm . These large punctate signals always resided in the cytoplasm, just beside the vacuole . The number of these punctate signals was constantly 1–3/cell during starvation. We further examined the localization of Apg8p in the background of Δ pep4 mutant, in which the autophagic bodies accumulate in the vacuole during starvation . TK116 strain was generated by introducing the 3 × HA-tagged APG8 plasmid into the Δ apg8 Δ pep4 . As shown in Fig. 6 A, under growing condition, the fluorescence pattern was observed as tiny dots dispersed in the cytoplasm as TK114 cells. At 30 min after shift to the starvation condition, 1–3 large punctate signals per cell appeared next to the vacuole, also like wild-type cells . At one hour, more punctate signals were observed than wild-type cells, and >50% were detected in the vacuole . The punctate signals in the vacuole gradually increased and completely filled the vacuole at six hours later . The signals coincided with autophagic bodies . As autophagic bodies are derived from autophagosomes, at least some population of Apg8p might be localized on or in the autophagosomes and delivered to the vacuole during starvation. Some of the large punctate signals in the cytoplasm may represent autophagosomes. To obtain further information about the intracellular localization of Apg8p during starvation, immunoelectron microscopic analysis was performed using the Δ pep4 cells, TK116. The logarithmically growing cells were shifted to SD(−N) medium for one, two, or three hours. The localization of 3 × HA-Apg8p in the starved cells was examined with the anti-HA antibody. In the autophagic bodies, density of the gold particles was significantly higher than that in the cytoplasm . This fact was in good agreement with the result obtained from immunofluorescence analysis. Moreover, it was revealed that most of Apg8p was in the lumen, but not on the membrane of autophagic bodies. Next, we examined localization of Apg8p in the cytoplasm of the starved cell. As expected from the result of immunofluorescence microscopy, gold particles were associated with the autophagosomes . Most mature autophagosomes with clear unit membranes and uniform intramembrane space were certainly stained, but not heavily , and, in some cases, the gold particles were mostly detected in their lumen . As shown in Fig. 8C and Fig. D , some autophagosomes were heavily stained with the gold particles. In these autophagosomes, the gold particles were mostly detected along the double membrane. However, their intramembrane space was not homogenous, but partly swollen not like the mature autophagosome. These structures may represent nascent autophagosomes or the latest structures in the autophagosome formation. Besides autophagosomes, we found heavily stained structures with immunogold in the cytoplasm. In Fig. 9 A, the gold particles are located along the surface on a curved membrane sac. Fig. 9 B shows that the gold particles were arranged in a rough circle, and some of them resided along membrane structures, which partly appear as clear double membrane. In Fig. 9 C, a semicircular isolation membrane (large arrow) and its open region (small arrow) were stained with gold particles. The gold particles were apparently more concentrated in the open region rather than on the isolation membrane. In addition, we found that gold particles were concentrated in a limited area close to the vacuole . The electron density in this area was less than that of the cytosol. The image of this Apg8p-enriched area is similar to that of the open region of the semicircular isolation membrane shown in Fig. 9 C. We suppose that all these structures shown in Fig. 9 represent intermediate structures of autophagosome. To elucidate the step at which Apg8p functions in the autophagic pathway, we used ypt7 mutant as a control. Ypt7p, a Rab family protein, is responsible for the fusion events to the vacuole . It was reported that in Δ ypt7 , Cvt vesicles become detectable . We studied by electron microscope, using rapid freezing and freeze-substitution-fixation method, whether Ypt7p is also required for the fusion of autophagosome to the vacuole. As shown in Fig. 10 A, Δ ypt7 cells under starvation had many fragmented vacuoles, and autophagosomes were much more detectable in the cytoplasm than in wild-type cells. Thus, the depletion of YPT7 causes the accumulation of autophagosomes in the cytoplasm. The Δ apg8 cells were examined also by EM. They were mostly normal with a few large vacuoles, but autophagosomes could be hardly detected in their cytoplasm . However, at low frequency, membrane structures, having enclosed a portion of cytosol, were observed only under starvation conditions. Some were indistinguishable from the autophagosome , but others showed more complicated structures, distinct from typical autophagosome, such as the structure having condensed contents or multivesicular structure . Next, Δ apg8 cells having the background of Δ ypt7 were examined. The Δ ypt7 Δ apg8 cells did not accumulate autophagosomes in the cytosol, and at low frequency autophagosome-like structures were detected in their cytoplasm, like Δ apg8 cells (data not shown). We counted the number of autophagosomes and autophagosome-like structures in Δ ypt7 cells and Δ ypt7 Δ apg8 cells. In the sections of 500 cells, we found 269 autophagosomes in Δ ypt7 cells, whereas in Δ ypt7 Δ apg8 cells, only 16 autophagosome-like structures were detected (5.9% of Δ ypt7 cells). The result clearly shows that autophagosomes are not accumulated in Δ apg8 cells. We reported that autophagy is not only responsible for nonselective degradation of cytosolic components, but also highly selective proAPI sequestration to the vacuole during starvation . ProAPI could be a marker as a cargo of the autophagosomes under starvation, just like Cvt vesicles under growing condition . Cell lysate was prepared from starved Δ ypt7 cells and analyzed by immunoblotting with anti-API antibody. As shown in Fig. 10 F, when the lysate was treated with proteinase K, ∼50% of proAPI was resistant to the proteinase, but it was completely digested in the presence of 2% Triton X-100, implying that the proteinase K-resistant proAPI resides in the lumen of autophagosomes. In contrast to Δ ypt7 , proteinase K-resistant proAPI was not detected in Δ apg8 cells . This provides a biochemical evidence that Δ apg8 cells do not accumulate autophagosome. Harding et al. 1995 reported that proteinase K is also completely accessible to proAPI at growing phase in cvt5/apg8 mutant, indicating that Cvt vesicles are not formed in apg8 mutants. During this analysis, we found that proAPI was partially matured in the starved Δ apg8 strain. This partial maturation was not detected in growing Δ apg8 cells (data not shown). It is not a general feature of apg mutants. This suggests that there is some starvation-induced machinery delivering proAPI to the vacuole in apg8 null mutants. As shown in Fig. 10C–E , several kinds of autophagosome-like structures were observed in the starved Δ apg8 cells. Although the frequency of these structures is very low, it is likely that these structures are responsible for the delivery of proAPI to the vacuole under starvation. ProAPI might be selectively sequestered to the vacuole via these membrane structures. However, because the Δ apg8 mutant cannot carry out bulk protein degradation under starvation , the structures must be insufficient for the bulk sequestration of cytoplasmic components to the vacuole. Maturation of proAPI should not reflect the nonselective bulk protein degradation exactly. Lang et al. 1998 stated the accumulation of double membrane structure in aut7/apg8 mutant. Since the EM techniques that they used are quite different from ours, and the preservation of membrane structure is not good, it is hard to directly compare our morphological data with theirs. It is possible that their reported structure is one of autophagosome-like structures reported here. Our morphological and biochemical data demonstrate that Δ apg8 strain never accumulates autophagosomes and strongly indicate that Apg8p plays a crucial role in autophagosome formation. Here, we report characterization of Apg8p, one of the APG gene products essential for autophagy. APG8 turned out to be identical to AUT7 , recently reported . They proposed that Apg8p functions in the delivery of autophagosome to the vacuole along microtubule. However, we found that autophagy proceeds normally, even if the microtubule is depolymerized . This result suggests that microtubule does not play an essential role in the autophagy in yeast. The proposal by Lang et al. 1998 was based upon the physical interactions of Apg4/Aut2p with tubulin and Apg8/Aut7p by in vitro and two-hybrid analyses. But, they did not demonstrate that Apg8p is bound to microtubule via Apg4/Aut2p. We have found that recombinant GST–Apg4p is bound to glutathione-Sepharose column nonspecifically (our unpublished result). It may be due to the acidic nature of Apg4p (predicted isoelectric point is 4.4). One possible interpretation of their result is that Apg4p is bound to tubulin nonspecifically. They also described qualitatively that the Δ apg8 cells accumulate autophagosomes in the cytoplasm. Here, we demonstrated quantitatively that the apg8 null mutant does not accumulate autophagosomes in the cytoplasm. So, we concluded that Apg8p participates in the autophagosome formation. This conclusion is not affected whether Apg4/Aut2p is indeed bound to tubulin. Intermediate structures of autophagosome formation have been poorly characterized in both yeast and mammalian cells because of their dynamic feature and the lack of specific marker of autophagosome. In yeast, only a cup-shape structure was detected as a possible intermediate structure of autophagosome . In this study, we found that most Apg8p is bound to membrane and localized in autophagosomes and autophagic bodies . Apg8p must be a key molecule to identify intermediate structures of autophagosome formation. Actually, we found that Apg8p is localized on premature autophagosomes and isolation membranes . In addition, an Apg8p-enriched region was observed in the cytoplasm close to the vacuole , and a similar image was obtained in the open region of the semicircular isolation membrane . These regions were always electron less-dense, indicating that they contain Apg8p-localized membrane- or lipid-containing structures, possible precursor structures of autophagosomal membrane. Based on our observations, we propose a model for the scheme of autophagosome formation as follows. The isolation membrane is formed by sequential assembly of the precursor structures. As the isolation membrane becomes spherical , its intramembrane space gradually becomes thin and homogenous, and finally a mature autophagosome is formed . Our assembly model is distinct from the one that autophagosome is formed by enclosing the cytosol with preexisting membrane cisterna, such as ER or Golgi body. Autophagosomal membrane shows a unique feature morphologically distinct from well-known organelles . It is observed as a thinner membrane and has a much lower density of intramembrane particles in freeze-fracture images than the membrane of other organelles. These features may be derived from the nature of the precursor structures. The dot structures observed under growing condition may be the same to them and to one of the sources of autophagosomal membrane. Immuno-EM analysis showed that Apg8p was enriched on the cytoplasmic faces of premature autophagosomes and intermediate structures . These data indicate that Apg8p bound to membrane plays its role in the formation of autophagosome. In mature autophagosomes, Apg8p was observed less than in those intermediate structures . In some typical autophagosomes, it was detected more readily in the lumen than on the membrane . In the autophagic bodies, most Apg8p was detected in the lumen . Furthermore, it was scarcely detected on the vacuolar membrane to which the outer membrane of the autophagosome had fused . These results suggest that Apg8p starts to dissociate from the membrane of autophagosome after finishing its role. This may be a reason why more Apg8p was detected in HSS fraction during starvation . Apg8p dissociated from the inner membrane would be entrapped in the lumen of autophagosome and transported to the vacuole, and Apg8p detached from the outer membrane may be recycled for the next autophagosome formation. Since Apg8p is not an integral membrane protein, it would be able to attach and detach from membrane. We found that some portion of Apg8p is tightly bound to membrane (data not shown). However, it is still unclear how Apg8p interacts with membrane structures, particularly autophagosomal membrane. The interaction of Apg8p with membrane is now under investigation. The apg8 null mutation severely impairs autophagosome formation, leading to the defect in bulk nonselective protein transport and degradation . However, a small amount of mature API was detected during starvation. ProAPI may be transported to the vacuole via structures rarely observed in Δ apg8 cells . Among them, there are the structures indistinguishable from autophagosome , suggesting that a small number of autophagosomes are built up in the absence of Apg8p. Thus, Apg8p may modulate the efficiency of autophagosome formation. In addition, we found the starvation-induced structures showing abnormal morphology in the Δ apg8 cells . Alternatively, Apg8p might regulate the morphogenesis of autophagosome. In any case, Apg8p would play an important role in the assembly of the precursor structures into autophagosomal membrane. The Cvt pathway proceeds with topologically the same membrane dynamics to the autophagy . Proteinase K is accessible to proAPI without detergent in the growing cvt5/apg8 mutant cells , indicating that Apg8p also is required for the formation of Cvt vesicle. Since Apg8p is localized on autophagosomes, it would be reasonable to speculate that it is localized on the Cvt vesicles also. In the growing cells, small punctate signals of 3 × HA–Apg8p may represent the Cvt vesicles . Between the two pathways, the most apparent difference is the size of vesicles. The autophagosomes are 300–900-nm in diam, while the Cvt vesicles are 140–160-nm . Surface area of autophagosomal membrane is calculated at ∼16-fold of Cvt vesicle, on average. Therefore, the autophagosome formation would require more Apg8p than the Cvt vesicle formation. Moreover, a significant amount of Apg8p is transported to the vacuole upon autophagy during starvation. These may be the reasons why Apg8p increases during starvation. However, it is still an open question whether the increase of Apg8p is actually necessary for the autophagy. At least, it is clear that the increase of Apg8p is not sufficient for the induction of autophagy, because overexpression of Apg8p did not induce autophagy under growing condition (data not shown). In addition, we showed that Apg8p is transcriptionally upregulated by inactivation of Tor signaling cascade. We found STRE-like sequences in the promoter region of APG8 . STREs are found in the genes induced in response to various stresses, such as CTT1 , encoding cytosolic catalase T, and play roles as positive control element . Marchler et al. 1993 reported the mutation in the STREs of CTT1 (CTT1-23), which causes loss of induction activity. The corresponding mutation was introduced in the STRE-like sequences of APG8 . However, it led to the increase of Apg8p at growing phase instead of the loss of the induction during starvation (data not shown), suggesting that a negative regulator is bound to the cis-elements to repress the expression under nutrient rich condition. Apg8p is the first identified molecule that is localized on the autophagy-related membrane structures. Now it becomes a useful marker for further analysis on the whole process of membrane dynamics in autophagy. Primary structures of Apg8p are highly conserved among homologues in other organisms . We anticipate that some Apg8p homologues may function in the process of formation of autophagosome in higher eukaryotes. Further study of Apg8p will provide a breakthrough for elucidating the molecular mechanism of autophagy. | Study | biomedical | en | 0.999998 |
10525547 | The mouse LCK gene was inserted upstream from the enhanced green fluorescent protein (EGFP) gene (J. Pines, Wellcome/CRC Institute, Cambridge, UK) and subcloned into the pcDNA3 expression plasmid (Invitrogen Corp.). Site-directed mutagenesis (Transformer System, CLONTECH Laboratories) was used to remove the LCK termination and GFP initiation codons. The pcDNA3-LCK-GFP construct produced an LCK-GFP fusion protein with a six amino acid glycine-rich linker region. The ZAP-70(SH2) 2 -GFP construct, containing the two SH2 domains of ZAP-70 fused to the NH 2 terminus of EGFP , was a kind gift from L. Samelson (National Institutes of Health, Bethesda, MD). E6.1 Jurkat T cells and derivative cell lines were cultured in RPMI medium supplemented with 5% FCS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Transient transfection of DNA constructs was by electroporation, using 10 μg DNA and 10 7 cells washed twice with serum-free RPMI and resuspended in 250 μl (Bio-Rad gene pulser, 960 μF, 250 V). The cells were then cultured overnight in 5% FCS/RPMI before analysis. For all fluorescence microscopy experiments, cells were washed in serum-free RPMI and attached to TESPA-coated coverslips (3-aminopropyltriethoxy silane; Sigma Chemical Co.) by incubation on ice for 45 min (5 × 10 5 cells/slip). CT-B–rhodamine conjugate (List Biological Laboratories) was used to label endogenous glycosphingolipids, at 10 μg/ml in PBS with 0.1% BSA. Lipid raft aggregation, or patching, was induced after CT-B labeling as described by Fra et al. 1994 , by incubating the cells with anti–CT-B antibody (1/250 in PBS/0.1% BSA; Calbiochem-Novabiochem Corp.) for 30 min on ice, and then 20 min at 37°C. For fixation, cells were then treated with 3% paraformaldehyde in PBS for 30 min at room temperature. Patching of the TCR was achieved in the same way, using 10 μg/ml anti-CD3 mAb (UCHT1) followed by 5 μg/ml anti-mouse Texas red conjugate (Nycomed Amersham, Inc.). BODIPY FL-labeled C 5 -ganglioside GM1 (150 nM in PBS; Molecular Probes, Inc.) was incorporated into live cells attached on coverslips by incubation for 30 min on ice. The cells were then fixed directly or patched with CT-B–rhodamine and anti–CT-B before fixation, as above. No cross-talk was detected between BODIPY and rhodamine fluorescence channels. For immunofluorescence staining, cells were attached to coverslips and fixed as described, and then blocked with 2% BSA/PBS for 10 min. When intracellular staining was required, blocking was preceded by permeabilization with 0.1% NP-40/PBS for 5 min. Cells were then incubated with primary antibody (1–10 μg/ml in 2% BSA/PBS) for 30 min, washed in PBS, followed by incubation with fluorescein-conjugated secondary antibodies (Nycomed Amersham, Inc.) for 15 min. The primary antibodies used were: LCK1 polyclonal antiserum or LCK 3A5 mAb (Santa Cruz), UCHT1 (anti-CD3 mAb; P. Beverley, Jenner Institute, Compton, Berkshire, UK), anti-LAT antiserum (L. Samelson, National Institutes of Health, Bethesda, MD), antidecay accelerating factor (anti-DAF) mAb IIH6 (M.E. Medof, University Hospital of Cleveland, OH), anti-CD59- and anti-CD45-fluorescein-conjugated mAbs (Serotec Ltd.), antitransferrin receptor (anti-TfR) mAb (Serotec Ltd.), 4G10 antiphosphotyrosine (anti-PTyr) mAb (B. Druker, Oregon University, Portland, OR), and polyclonal anti-PTyr antibody (Upstate Biotechnology Inc.). Where indicated, the TCR was also stained using UCHT1 biotin-conjugated mAb (Sigma Chemical Co.) followed by streptavidin Texas red conjugate (Calbiochem-Novabiochem Corp.). Confocal microscopy was performed with a Leica TCS SP confocal microscope with 63 and 100× objective lenses, using laser excitation at 488 and 568 nm. The widths of fluorescein/GFP and rhodamine/Texas red emission channels were set such that bleed through across channels was negligible. For stimulation by CT-B patching, 10 6 cells/100 μl were treated in microfuge tubes using the same conditions described for immunofluorescence, using unconjugated CT-B (Calbiochem-Novabiochem Corp.). Where indicated, the equivalent number of cells were stimulated with 2 μg/ml OKT3 anti-CD3 antibody (Fab′) 2 fragments for 5 min at 37°C, or with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.) for 10 min at 37°C. After treatment, the cells were washed in cold PBS and harvested in lysis buffer at 10 6 cells/100 μl (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 10% glycerol, 1% Triton X-100, 20 mM NaF, 10 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and 5 μg/ml each of chymostatin, leupeptin, and pepstatin). After 5 min on ice, cell debris was removed by centrifugation. Cleared lysates were assayed for protein content (Bio-Rad protein assay reagent) and samples, normalized for total protein, were analyzed by SDS-PAGE and transferred to nitrocellulose. Blots were probed with appropriate antibodies and detected with HRP-conjugated secondary antibodies and chemiluminescence (New England Nuclear Life Sciences). Antibodies used for Western blots were: 4G10 anti-PTyr mAb, ZAP-4 anti-ZAP-70 antiserum , anti-LAT antiserum (M. Turner, Babraham Institute, Cambridge, UK), anti-ERK-2 antiserum (C. Marshall, ICR, London, UK), and antisera specific for activated ERK1/2 (PhosphoPlus p44/42 antibody, New England Biolabs). ZAP-4 was also used for immunoprecipitation of ZAP-70 from lysates, by coupling to protein A–Sepharose (Pharmacia Biotech, Inc.) with dimethylpimelimidate and incubation with 250 μl of lysate at 4°C for 2 h. Similarly, GST-Grb2 pulldown experiments were performed by incubating cell lysates with 5 μg of GST fusion protein that was immobilized by binding to glutathione-Sepharose (Pharmacia Biotech, Inc.). Precipitated protein was recovered by centrifugation, washed three times in lysis buffer, and analyzed by SDS-PAGE and Western blotting. RPMI-washed cells were incubated with 2 μM Indo 1-AM (Molecular Probes, Inc.) at 5 × 10 6 cells/ml for 30 min at 37°C, washed in RPMI, and kept on ice. CT-B patching of cells was as described. Ca 2+ flux was monitored at 37°C using an LS50 Perkin-Elmer luminescence spectrometer, with excitation at 355 nm and emission measured at 480 and 405 nm, representing free versus Indo-1-associated intracellular Ca 2+ , respectively, to give an absorbance ratio. To confirm Indo-1 loading, ionomycin was added to a sample of each set of cells used and the Ca 2+ flux monitored. 10 7 cells/point were transfected with 10 μg of pBR322-3XNFAT-Luc vector (G. Crabtree, Stanford University, CA), as described above, and incubated for 2 h at 37°C. The cells were then stimulated as indicated with CT-B patching or with anti-CD3 antibodies, as described above. After culturing overnight, control and treated cells were lysed in 120 μl of cell culture lysis reagent (Promega) for 10 min on ice, and centrifuged. 50 μl of the cleared supernatant was assayed for luciferase using the Promega luciferase assay kit with a Clinilumat (Berthold) luminometer. All treatments were performed in duplicate and the results shown are mean ± SEM. To localize LCK without the need to permeabilize cells with detergent, which could disrupt membrane organization, a construct was generated in which GFP was fused onto the COOH terminus of full length LCK . When expressed in LCK-deficient JCam-1.6 cells , the LCK-GFP fusion protein was able to phosphorylate the TCR ζ chain after CD3 cross-linking and reconstitute the ability of the TCR to induce nuclear factor of activated T cells (NFAT), indicating that it retained wild-type LCK signaling function (data not shown). Sucrose gradient centrifugation of Triton X-100 solubilized cell lysates prepared from transiently transfected E6.1 Jurkat T cells also revealed that a significant percentage of LCK-GFP partitioned into the low density raft fraction (data not shown), similar to the endogenous protein . Transient transfection of LCK-GFP into Jurkat T cells revealed a predominantly homogenous localization at the plasma membrane , with some intracellular staining probably representing late endosomes, similar to the distribution of the endogenous protein detected by immunofluorescence . To study the distribution of LCK-GFP with respect to lipid rafts, Jurkat T cells were stained with rhodamine-labeled CT-B. This reagent binds to glycosphingolipids, with a strong affinity for GM1 and lower affinity for other gangliosides , and therefore, can be used as a marker for lipid rafts, which are enriched in glycosphingolipids , although nonraft GM1 will also be detected. Staining of live Jurkat T cells with CT-B–rhodamine demonstrated a homogeneous distribution of GM1 at the plasma membrane , similar to the distribution of LCK-GFP. This suggested that raft microdomains may be too small to visualize by light microscopy, which has a resolution of ∼200 nm, consistent with a recent study that estimated lipid rafts are <70 nm in diameter . The homogeneous distribution of CT-B was detected despite the fact that it binds GM1 pentavalently and could therefore potentially cause GM1 aggregation. However, when the CT-B was cross-linked with anti–CT-B antibody, staining became concentrated to distinct patches within the membrane. Significantly, a substantial fraction of transfected LCK-GFP was associated with the CT-B–stained patches , both in fixed (F) and live (L) cells, although the extent of colocalization varied between cells, as shown. Colocalization of endogenous LCK with CT-B patches was also confirmed by immunofluorescence . These results indicate that LCK is preferentially localized to glycosphingolipid-rich domains within unpermeabilized plasma membrane, supporting recent experiments using permeabilized cells . GPI-linked receptors are strongly associated with lipid rafts isolated biochemically by virtue of their Triton X-100 insolubility and low density , whilst other membrane proteins, such as the transferrin receptor (TfR) and the tyrosine phosphatase CD45, do not copurify . To compare CT-B patches with known biochemical properties of lipid rafts, Jurkat T cells were stained for the GPI-linked proteins CD59 and DAF (CD55), and for CD45 and the TfR, in both CT-B–patched and control cells. Consistent with biochemical analyses, CD59 and DAF (data not shown) were both substantially concentrated in CT-B patches, compared with a more uniform distribution in unpatched cells. The linker molecule LAT, which is essential for TCR signaling and copurifies with lipid rafts isolated biochemically from T cells , also colocalized with CT-B patches . The TfR and CD45, however, remained uniformly distributed after CT-B patching. Indeed, CD45 even appeared to be partially excluded in some cells . To confirm the ability of CT-B to cross-link the bulk of cell surface GM1, Jurkat T cells were labeled with GM1-BODIPY and the cells were then treated with CT-B plus anti–CT-B antibody. Unpatched GM1-BODIPY showed an even distribution at the plasma membrane. However, after CT-B/anti–CT-B treatment, almost all detectable label was restricted to patches . Since lipid rafts are characteristically detergent-resistant, the association of LCK and GPI-linked receptors with the CT-B patches was determined after extraction by Triton X-100. Immunofluorescence showed that LCK-GFP, CD59 , and DAF (data not shown) concentrated in the CT-B patches were insensitive to detergent extraction. However, the TfR, which was not concentrated in CT-B patches, was effectively removed. Also, LCK-GFP that was not in patches appeared to be more sensitive to detergent treatment, whereas colocalization of LCK-GFP with lipid patches typically became more pronounced after Triton X-100 extraction . Depletion of cellular cholesterol impairs the ability of GPI-anchored proteins to associate with the detergent-insoluble membrane raft fraction . To examine whether there was a similar requirement for the association of GPI receptors and LCK with CT-B–induced patches, Jurkat T cells, prepatched with CT-B, were treated with 10 mM methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol . This treatment alone did not disrupt CT-B–induced patches or their association with LCK, CD59 , or DAF (data not shown), although CD59 staining was somewhat reduced. However, MβCD treatment did cause the CT-B patches to become detergent-sensitive, as patches of colocalized proteins were severely disrupted (LCK) or completely lost (CD59) after extraction with Triton X-100 in MβCD-treated cells, but not control cells. Together, therefore, the data in this section indicate that the CT-B–induced patches correspond to aggregated lipid rafts and are consistent with a previous study analyzing lipid domain structure in BHK and Jurkat T cells using cross-linked CT-B . TCR association with lipid rafts is not detectable in our experiments using detergent insolubility and sucrose gradient ultracentrifugation (Kabouridis, P., T. Magee, and S. Ley, unpublished observations). However, as stated in the introduction, detergent extraction may disrupt the association of proteins that interact weakly with lipid rafts. To examine the possible association of the TCR with lipid rafts without using detergent, Jurkat T cells were patched with cross-linked CT-B and, after fixing, stained with an anti-CD3 mAb. The TCR was found to clearly colocalize with the CT-B–induced patches, whereas TCR staining was more evenly distributed when cells were fixed before CT-B staining . Colocalization was evident within 2 min after CT-B cross-linking (data not shown). Triton X-100 treatment of CT-B–patched cells resulted in an almost complete loss of CD3 staining from CT-B patches , unlike the other CT-B colocalizing proteins , suggesting a weak association of the TCR with lipid rafts. However, cross-linking of the receptor with anti-CD3 mAb before extraction stabilized the association of the receptor with lipid patches in the presence of detergent. In contrast, cross-linking of the TfR had no effect on TfR detergent sensitivity . Cross-linking of the TCR with anti-CD3 mAb stimulates the rapid tyrosine phosphorylation of intracellular proteins and activation of downstream signaling pathways . To examine whether TCR signal transduction involves its association with lipid rafts, Jurkat T cells were incubated with anti-CD3 mAb and cross-linked with anti-Ig antibody. This treatment induced patching of the TCR at the cell surface. The TCR patches were colocalized with both LCK-GFP and CD59, but not with CD45 , indicating that CD3 cross-linking stimulates a similar aggregation of proteins, as seen in CT-B patches. In the converse experiment, antibody-mediated cross-linking of CD59 also resulted in copatching of CD3. Cross-linking of CD45 with antibodies, which also induced its patching at the plasma membrane, had no effect on lipid rafts, as indicated by LCK-GFP distribution (data not shown). Thus, anti-CD3 patching induces aggregation of lipid rafts which concentrates the TCR with LCK, but not the protein tyrosine phosphatase CD45. To investigate the specificity of the LCK-TCR coaggregation, we analyzed JCam-1.6 T cells that lack endogenous LCK, but have been transfected to express LCK fused to the extracellular domain of CD16 and the transmembrane domain of CD7 . This LCK chimera is excluded from rafts isolated biochemically and does not properly reconstitute TCR signaling in JCam-1.6 cells . Staining for LCK in 16:7:LCK-expressing cells after patching of the TCR showed that the LCK chimera did not colocalize with the patches , unlike LCK-GFP or endogenous LCK stained in normal Jurkat T cells (not shown). This indicates that colocalization of LCK with CD3 patches is dependent on the correct targeting of LCK to lipid rafts. TCR ligation stimulates tyrosine phosphorylation at the cell cortex, coincident with the inner face of the plasma membrane . To investigate if TCR cross-linked patches contained tyrosine-phosphorylated proteins, Jurkat T cells were patched with anti-CD3 mAb and, after fixing, stained with anti-PTyr antiserum. The TCR-induced patches were strongly stained for PTyr, compared with unstimulated cells . Significantly, aggregation of lipid rafts by CT-B cross-linking also stimulated strong PTyr staining coincident with the patches , and this was visible as early as 1 min after cross-linking (data not shown). CT-B–induced tyrosine phosphorylation was dependent on LCK, as no increase in PTyr staining was induced by raft aggregation in JCam-1.6 cells lacking LCK expression . However, TCR association with aggregated lipid rafts was clearly not dependent on LCK, since CD3 staining colocalized with CT-B patches in JCam-1.6 cells . Thus, aggregated lipid rafts, induced by CD3 or CT-B cross-linking, appear to be sites of PTK signaling. CD3 cross-linking induces phosphorylation of ITAMs of the TCR by LCK and subsequent recruitment of ZAP-70 PTK . These signaling events may be monitored in intact cells using a chimeric ZAP-70 double SH2 protein linked to GFP, which translocates from the cytoplasm to the plasma membrane upon binding to phospho-ITAMs . To investigate whether aggregated lipid rafts were the sites of phosphorylation of the TCR, Jurkat T cells were transiently transfected with the ZAP-70(SH2) 2 -GFP construct. The cells were then patched with anti-CD3 mAb, anti–CT-B, or anti-TfR mAbs and fixed. Both CD3 and CT-B patching induced ZAP-70(SH2) 2 -GFP to relocalize to the plasma membrane and become concentrated in the lipid raft patches . In contrast, cross-linking of the TfR had no effect on ZAP-70(SH2) 2 -GFP distribution, compared with control cells . The TCR, therefore, appears to become tyrosine phosphorylated specifically in the environment of aggregated lipid rafts. The stimulation of tyrosine phosphorylation and association of ZAP-70(SH2) 2 -GFP induced by cross-linking either the TCR or CT-B–labeled rafts (above) suggested that lipid raft aggregation might be sufficient to stimulate TCR signaling. To investigate this further, lysates were prepared from cells after CD3- or CT-B–cross-linking. Western blotting with anti-PTyr antibody revealed that CT-B cross-linking triggered tyrosine phosphorylation of a similar set of proteins to those stimulated by anti-CD3 mAb . Analysis of ZAP-70 immunoprecipitates from cell lysates also showed that cross-linked CT-B, but not treatment with CT-B alone, induced ZAP-70 tyrosine phosphorylation and association with the phosphorylated TCR ζ chain . CT-B cross-linking also stimulated phosphorylation of the LAT linker protein, as measured by the induced association of LAT with a GST-Grb2 fusion protein and detection with PTyr antibody . These are also proximal signaling events triggered by TCR ligation . The ability of CT-B cross-linking to induce other signaling pathways downstream of the TCR was also investigated. CT-B cross-linking was found to induce a rapid increase in intracellular-free Ca 2+ , although less efficiently than stimulation with anti-CD3 mAb . Secondly, CT-B cross-linking potently induced activation of the ERK MAP kinases, as measured by Western blotting with an antiphospho-ERK antibody, to a similar level as anti-CD3 mAb . CT-B–induced ERK activation was detectable within 1 min of cross-linking, similar to that observed with CD3 cross-linking (data not shown). Activation of the Ras-ERK pathway and of Ca 2+ fluxing are together required to stimulate the T cell-specific transcription factor NFAT, which regulates interleukin-2 expression . Therefore, NFAT activity was assayed in Jurkat T cells transiently transfected with a construct containing the luciferase reporter gene under the control of three copies of the NFAT regulatory element and cultured for 24 h. CT-B cross-linking clearly stimulated the production of NFAT, although less efficiently than that achieved with anti-CD3 mAb . Thus, aggregation of lipid rafts by CT-B cross-linking activates signaling pathways similar to those induced by TCR ligation. A panel of somatic mutants of the Jurkat T cell line was used to determine the genetic requirements for signaling induced by cross-linked CT-B, using ERK phosphorylation as a readout assay. This analysis revealed that cells deficient in expression of the TCR/CD3 complex , CD45 , LCK , and ZAP-70 displayed poor activation of ERK after CT-B cross-linking, compared with parental Jurkat cells . Anti-CD3 mAb-induced activation of ERK was also inhibited in these cell lines . Similarly, these gene products were necessary for activation of both Ca 2+ fluxing and NFAT production after CT-B patching (data not shown). Control experiments demonstrated that cross-linked CT-B induced patching of lipid rafts in all of the cell lines tested and, with the exception of the TCR-negative Jurkat T cells, these patches were associated with the TCR (data not shown). Similarly, in JCam2 cells, which are deficient in LAT expression , no activation of ERK was detected after CT-B or CD3 cross-linking, in contrast to PMA stimulation . However, both CT-B and CD3 cross-linking increased ERK activity in LAT-reconstituted JCam2 cells . Thus, the genetic requirements for signaling induced by aggegation of lipid rafts with cross-linked CT-B are similar to those for the TCR. The CD16:CD7:LCK transmembrane chimera (16:7:LCK) does not target correctly to rafts isolated biochemically or to TCR-patched rafts in intact cells . Furthermore, cells expressing this chimera are deficient in signaling in response to TCR cross-linking . Therefore, to determine if CT-B–induced signaling is dependent on targeting of LCK to rafts, Ca 2+ flux in cells expressing the 16:7:LCK chimera was investigated in response to CT-B patching. Unlike normal Jurkat cells, no Ca 2+ flux was observed after addition of anti–CT-B cross-linking antibody . Likewise, aggregation of the LCK chimera with anti-CD16 antibody had no effect. However, when the CT-B patches were cocross-linked with the LCK chimera using anti-Ig antibodies, a transient flux was induced . This indicates that not only is LCK expression required for signaling stimulated by lipid raft aggregation , but its correct targeting to these lipid rafts is also necessary. This study provides several lines of evidence that membrane patches formed by cross-linking CT-B correspond to regions of aggregated lipid rafts in intact cells, and that in T cells these membrane subdomains are enriched in key signaling molecules and represent active sites of signaling. Thus, CT-B–labeled membrane patches displayed characteristics consistent with biochemically isolated lipid rafts, including colocalization with the GPI-linked proteins CD59 and CD55, and also the T cell signaling proteins LCK and LAT , all of which copurify with lipid rafts biochemically. In addition, LCK-GFP colocalized with CT-B patches in nonpermeabilized cells, ruling out the possibility of artifacts from detergent treatment, and confirming results with permeabilized cells . In contrast, nonraft associated proteins, such as the TfR and CD45, did not colocalize with CT-B patches. Furthermore, whereas TfR staining was lost after Triton X-100 extraction, LCK and CD59 association with CT-B patches was resistant to Triton X-100 , indicative of their raft association. However, pretreatment of cells with MβCD to deplete membrane cholesterol rendered these proteins Triton X-100-extractable, similar to the MβCD-induced release of these proteins from the raft fraction isolated biochemically from lymphocyte cells . This is also consistent with effects of cholesterol depletion in disrupting the clustered distribution of GPI-linked proteins and the interaction of the IgE receptor and the Src-family kinase LYN with rafts . While GM1 is concentrated in lipid rafts isolated biochemically , staining has also revealed colocalization with caveolae , which are small plasma membrane invaginations found in cells expressing caveolin proteins that share the nonionic detergent-insolubility and low density characteristics of lipid rafts . Caveolins have been proposed to function as scaffolding proteins for signaling molecules and could determine their localization . However, no expression of the three caveolin family members is detectable in Jurkat T cells , or other lymphocyte cell lines . Also, Jurkat T cells contain no caveolae structures, even with exogenous expression of caveolin-1 at levels similar to MDCK cells that contain caveolae (P. Janes, S. Ley, and A. Magee, unpublished data). Therefore, the localization of T cell signaling proteins to lipid rafts appears to be independent of caveolins and caveolae. Our results showing association of the TCR with CT-B cross-linked lipid rafts contrasts with biochemical studies analyzing lipid raft composition and highlights the limitations of using detergent insolubility as the only criterion to monitor the association of a particular protein with lipid rafts. Although CD3 staining was clearly concentrated in CT-B patches, this association was completely lost after Triton X-100 extraction. Similarly, the association of VSV-G with lipid rafts is not preserved after Triton X-100 extraction . It is likely that only proteins that are strongly associated with lipid rafts are Triton X-100 insoluble, whereas weakly associated proteins are extracted. The contradictory results using detergent insolubility to determine whether the TCR is associated with lipid rafts may be explained by such a weak association. The TCR also colocalized with CT-B cross-linked lipid raft patches in LCK-deficient JCam-1.6 cells and in Jurkat cells pretreated with the Src family kinase inhibitor PP1 (data not shown), indicating that the association of the TCR with lipid rafts does not require activity of Src PTKs which are essential for TCR signaling . These observations suggest that the receptor may be constitutively associated with lipid rafts, rather than being actively recruited to these structures. TCR aggregation induced by CT-B patching may, therefore, simply occur as a result of coalescence of lipid microdomains with which the TCR is already associated. The reported increased association of the TCR with detergent-insoluble lipid rafts detected after CD3 cross-linking may reflect stabilization of this association by antibody cross-linking, as demonstrated in Fig. 4 B, rather than movement into rafts. Signal transduction via the TCR is initiated after antibody cross-linking, suggesting that signaling is triggered by receptor oligomerization. Accordingly, TCR signaling is triggered by oligomers of soluble MHC molecules bound to cognate peptide, but not by monomers . However, the precise mechanism by which oligomerization triggers TCR signaling is unclear. The results in this study suggest that TCR oligomerization may be important in driving the formation of aggregates of lipid rafts with which it associates. Thus, TCR cross-linking and CT-B–mediated lipid raft aggregation caused a similar redistribution of raft-associated proteins including LCK and the TCR, but not the protein tyrosine phosphatase CD45. Raft aggregation may therefore facilitate tyrosine phosphorylation of TCR ITAMs by increasing the concentration of the TCR and LCK in close proximity, whilst excluding CD45. Consistent with this hypothesis, both a general increase in tyrosine phosphorylation and specific tyrosine phosphorylation of TCR ITAMs occurred in aggregated lipid rafts after either TCR or CT-B patching. Furthermore, CT-B cross-linking induced a similar pattern of protein tyrosine phosphorylation and stimulated the same signaling pathways as the TCR , with the same genetic requirements . Previous work also supports this view, since CT-B–induced calcium flux in Jurkat T cells has been shown to be markedly reduced in cells lacking the TCR β chain . Redistribution of CT-B–labeled GM1 was not detectable after anti-CD3 mAb cross-linking (data not shown), consistent with a previous study . This may reflect the presence of a significant fraction of GM1 outside of rafts . Our results suggest that the raft-associated proteins investigated above (e.g., CD59) may be better markers for these domains, and that TCR patching alone is sufficient to drive lipid raft aggregation, although this is likely to be enhanced by coaggregation with other lipid raft-associated proteins. Consistent with this, Viola et al. 1999 found that stimulation of T cells with low anti-CD3 mAb concentration was enhanced by simultaneous treatment with CT-B or anti-CD59 antibody, suggesting that cross-linking of rafts provided costimulation. Furthermore, cross-linking CD3 with the coreceptor CD28, which increases T cell activation, also enhanced raft aggregation, as indicated by a visible redistribution of CT-B–labeled GM1 . In the present study, lipid raft aggregation induced by CT-B cross-linking was found to activate TCR signaling less efficiently than direct cross-linking of the TCR by anti-CD3 mAb in some assays , possibly due to dilution of the rafts by nonraft-associated GM1. However, lipid raft aggregation is likely to facilitate T cell stimulation induced by TCR ligation, both alone and in concert with coreceptor ligation. The observation that LCK is enriched in patched lipid rafts with the TCR, but not CD45, led us to investigate the importance of specific targeting of LCK to these rafts using a transmembrane LCK chimera (16:7:LCK). This chimera is not associated with rafts isolated biochemically and does not properly reconstitute signaling in LCK-deficient cells in response to TCR cross-linking . The 16:7:LCK chimera did not localize to TCR-patched rafts , and was unable to induce Ca 2+ -signaling by CT-B–mediated raft aggregation , until forced to interact with CT-B–labeled rafts by antibody cocross-linking. These results show that specific targeting of LCK to rafts is essential for raft-mediated TCR signaling. This targeting is likely to be driven by acylation of LCK, which is necessary for its signaling function , since the 10 amino acid LCK NH 2 terminus containing the three acylation sites is sufficient to target GFP to lipid rafts isolated biochemically (Janes, P., C. Jackson, S. Ley, and T. Magee, unpublished data). Similar results have been reported for other acylated proteins, including G protein α-subunits . The activity of LCK in rafts purified biochemically is markedly reduced compared with LCK in the nonraft fraction . This is presumed to be due to inaccessibility of raft-associated LCK to CD45, which dephosphorylates the negative regulatory tyrosine 505 of LCK . However, in our study, tyrosine phosphorylation of the TCR, which is mediated by LCK , occurred in raft patches identified by confocal microscopy, and PTyr stimulation was dependent on LCK expression . Therefore, LCK appeared to be active in lipid raft patches in which it was concentrated, despite lack of colocalized CD45. A possible explanation for this paradox is that active LCK is more sensitive to detergent extraction, and is removed from rafts isolated biochemically, similar to the TCR. If LCK is active in rafts, this suggests either that LCK retains activity for a time after exclusion from CD45, or that CD45 may not be absolutely required for LCK activity. In support of the latter hypothesis, LCK activity is substantially increased in T cell lines and thymocytes that lack CD45, despite hyperphosphorylation of Tyr 505 . Similarly, in B cells, the fraction of the Src-family PTK LYN that is associated with the B cell receptor displays increased activity in the absence of CD45 expression . In these experiments, both the positive and negative regulatory tyrosines of each kinase were hyperphosphorylated, suggesting that CD45 dephosphorylates the positive regulatory site in addition to the negative regulatory site, thereby inhibiting kinase activity. In addition, CD45 may also dephosphorylate LCK substrates and/or the TCR ζ chain , resulting in reduced signaling. Indeed, when CD45 and the TCR are cocross-linked, TCR signaling is strongly downregulated . Therefore, exclusion of CD45 from the TCR and LCK, facilitated by lipid raft aggregation, could potentially stimulate tyrosine phosphorylation and T cell activation. Interestingly, it has been suggested that T cell activation by antigen-presenting cells (APCs) may involve formation of zones of CD45 exclusion, in which TCR tyrosine phosphorylation may be facilitated . This prediction was based on steric considerations from the predicted size of the CD45 extracellular domain, compared with the TCR/MHC complex . Certainly, there is segregation of molecules within APC-T cell contact sites, with TCRs becoming concentrated centrally within a ring of LFA-1/ICAM-1 coreceptors, termed supramolecular activation clusters, or SMACs , which is consistent with size exclusion . Our data are in keeping with this model of T cell activation, suggesting that differential affinity for lipid rafts may result in a similar segregation of the TCR and CD45 after stimulation with anti-CD3 mAb, and this may facilitate steric segregation in APC-T cell interactions. However, whereas SMAC formation occurs over 30–60 min and is apparently dependent on cytoskeletal rearrangement , the events described here probably correspond to more immediate signaling responses. Thus, the coaggregation of the TCR with CT-B–labeled lipid rafts, and the stimulation of tyrosine phosphorylation after CD3 or CT-B cross-linking were visible within one to two minutes, as was stimulation of ERK activity. Also, disruption of actin polymerization with cytochalasin D does not inhibit CT-B–induced copatching of CD3 (Janes, P., S. Ley, and A. Magee, unpublished results), or PTyr stimulation by cross-linking of CT-B or CD3 , whereas actin accumulation to CT-B patches is kinase-dependent . In future studies, it will be important to determine whether lipid rafts are also involved in the formation of organized zones of signaling proteins at contacts between T cells and APCs. Ligation of cell-surface GPI-anchored proteins triggers transmembrane signal transduction in T cells . However, as GPI-linked proteins are associated only with the outer leaflet of the lipid bilayer, the mechanism of signaling has remained obscure. It is possible that these molecules interact with transmembrane proteins which transmit a signal, or, alternatively, GPI-linked protein association with lipid rafts, which contain Src-family kinases required for their signaling, may be important . The results of this study suggest that both explanations may be correct. Thus, CD59 and DAF were present in lipid raft patches with the TCR and LCK, and antibody cross-linking of CD59 caused coaggregation of the TCR. This suggests that ligation of GPI receptors may trigger TCR signaling by aggregating lipid rafts. Consistent with this hypothesis, induction of lymphokine production by GPI-linked molecules requires expression of the TCR at the cell surface . Thus, the mechanism of signaling after antibody stimulation of the TCR or GPI-linked receptors in T cells may be very similar. Aggregation of the IgE receptor Fc∈R1 activates the associated Src-family kinase LYN, initiating a signaling cascade that culminates in degranulation . Clustering of Fc∈R1 results in its association with patches enriched in GM1 and its copurification with detergent-insoluble lipid rafts . Furthermore, only the receptor that copurifies with lipid rafts serves as a substrate for LYN . Activation of PTK signaling by Fc∈R1, therefore, appears to involve its association with aggregated lipid rafts, similar to the TCR. Many hematopoietic cell receptors signal via Src kinase-mediated phosphorylation of conserved tyrosine residues in their cytoplasmic domains . It is possible that these receptors also utilize lipid rafts to initiate transmembrane signal transduction. In conclusion, this study demonstrates that stimulation of Jurkat T cells by cross-linking the TCR induces aggregation of lipid rafts in which the TCR and LCK, but not CD45, are concentrated, producing an environment where tyrosine phosphorylation is favored and signaling is triggered. Accordingly, TCR stimulation can be mimicked by direct aggregation of rafts with CT-B cross-linking, which induces a similar redistribution of molecules and similar signaling events. In future studies, it will be important to characterize the structural features of the TCR, which allow it to associate with rafts and would be expected to be critical for signal transduction. | Study | biomedical | en | 0.999996 |
10527326 | Past theoretical mechanisms have failed to explain the observed, postoperative hypothermia in laparoscopic patients. Mismatch of tissue and inflation gas thermal capacities rule out any substantial global tissue temperature reductions during laparoscopic procedures. Overlooked, however, is the possibility that severe hypothermia is due to local super-cooling of tissue caused by evaporation from tissue surfaces of peritoneal fluid water into the dry jet of insufflation gas. This mechanism is examined analytically and experimentally and is found to be real and significant. Patient hypothermia during laparoscopic surgery is widely reported in recent literature. 1 – 6 While this clinical condition and its undesirable consequences to the patient are well understood, the exact cause of the additional hypothermia due to laparoscopy is not clear. It is reported that the heat capacity effects of the CO 2 insufflation gas are not sufficient to cause physiologically significant, bulk tissue cooling. 7 , 8 The question remains, therefore, What is the cause of a patient's additional hypothermic response observed during laparoscopic surgery? At least one possible causative factor could be severe cooling of local tissue surfaces through the evaporation of peritoneal fluid by the jet of dry insufflation gas (usually CO 2 ) impinging on the epithelial surface while the gas is being introduced to the peritoneal cavity through a trocar or other device, such as a Veress needle. This study was designed to explore the possibility that such a mechanism could result in substantial cooling of surface tissue during the initial abdominal insufflation and/or subsequent insufflation during the laparoscopic procedure. A theoretical analysis was completed of the evaporative cooling effects caused by the simultaneous tissue-conductive, and gas-convective, jet heat and mass transport that reasonably could be expected to occur during typical insufflation. Laboratory measurements of the temperature transient occurring when a dry CO 2 gas jet impinged on simulated and animal tissue material was obtained to verify the results of these theoretical calculations. The experimental studies included measurement of the effects of procedure variables: insufflation gas flow rates; height's of the gas jet; tissue cooling-area size; type of gas entry port; tissue type; and, most importantly, the thermal/humidity condition of the insufflation gas stream. Table 1 summarizes the range of these variables for which theoretical and experimental results were obtained in this work. Evaporation jet cooling of the epithelial tissue occurs when the end of a Veress needle or trocar is positioned close to a tissue surface, as shown in Figure 1 . The insufflation gas exits from the end of the Veress needle or trocar in a free vertical jet. When the gas jet reaches the epithelial surface , the gas flow is redirected horizontally and flows radially away from the centerline of the entry port on the impingement surface (the stagnation point). Because of a very rapid flow transition on the epithelial surface, high heat and mass transfer rates are generated in the area of the stagnation point with the maximum effect occurring at the stagnation point. When the insufflation gas is dry, as it is in all laparoscopic procedures, a large evaporation-driving force is generated in the resulting gas phase boundary layer, with velocity profile transitions, in very high evaporation rates around the stagnation point. The energy needed to evaporate fluid from peritoneal tissue surfaces is from jet evaporation and cools the surface at a rapid rate. How fast, how long, and how much tissue will be cooled and to what temperature is a complex function of the gas flow rate (F 1/m), the height of the gas injection device above the tissue (H mm), the dimension of the gas injection device (D, the diameter of the circular entrance port, or W, the width of the annular jet from a trocar), the size of the cooled tissue area (measured by r, the distance from the stagnation point for the circular jet, or x, the distance from the center of slot width W of an annular jet), the magnitude of the tissue's thermal parameters of thermal conductivity and diffusivity and, most important, the relative humidity of the insufflation gas. The theoretical analysis and these results are the focus of this study. Experimental verification of the theoretical results were obtained through in vitro measurements of jet cooling of synthetic and animal tissue surfaces at various flow and geometry conditions known to influence the cooling rates. The rapid tissue cooling rates were measured with single and four thermocouple (t/c) configurations that detected tissue surface temperature at the stagnation point and with the four t/c configuration, at progressively larger distances (r) from the stagnation point . Temperatures were collected by computer data acquisition at rates of 5 to 15 points per second and stored for analysis and display. Great care was taken during the experimental measurements with the apparatus seen in Figure 1 to assure that the surrounding conditions of temperature (T ∞ ) and humidity simulated conditions that are experienced in the human abdomen. Much theoretical and experimental work has been done on heat and mass transfer into and from circular and slot gas jet streams. 9 – 11 The mass transfer elements of these studies, however, have been limited to sublimation of a solid into a gas jet; and gas jet evaporative mass transfer of a liquid surface has been studied very little. For this study, we have used the well known and reliable heat-mass transfer analog for convective flow, making it relatively simple to circumvent this lack of direct experimental correlations. Referring to Figure 1 , the impingement surface of our model is wet with peritoneal fluid and is cooled by latent evaporative heat flux into the jet of dry CO 2 insufflation gas. The heat transfer flux due to this evaporative mass transfer is characterized by the equation, where h e is the evaporative heat transfer coefficient, p s , the vapor pressure of the surface fluid (a function of surface temperature), and ø is the jet gas's relative humidity (assumed zero for this analysis). As has been discussed, the heat and mass transfer coefficients needed in Equation 1 vary considerably from a maximum at the stagnation point to decreasingly lower values at positions r (or x for a slot jet) away from the circular jet center line. The integral mean values for the transfer coefficients in these regions in terms of r and x; the diameter, D, of the circular jet (or W, the width of the slot jet); and the height of the nozzle, H, from the impingement surface have previously been determined and is called the Martin's Nusselt number. 9 From the Martin's Nusselt number correlations we obtained mean (with r or x) heat transfer coefficients, h e . The classic heat-mass transfer analog is in the form where Sh is the Sherwood number, Nu = Nusselt number, Sc = Schmidt number, and Pr is the Prandtl number. The constant n is empirically determined from the specific flow configuration. For round and slot jet flows, Martin determined this constant to be n = 0.42. Equation 2 , with Sc = 0.49, Pr = 0.75 for diffusion of water through CO 2 , permitted the calculation of the mass transfer coefficients h D . The evaporative heat transfer coefficient, h e of Equation 1 , was determined by the simple conversion of the mass transfer coefficient, h D , from concentration units to partial pressure units, with assumptions of ideal gases and the boundary layer mean temperatures and the known values of water's heat of vaporization. This led to the following simple relationship: Thus, with Martin's correlations for Nusselt number as a function of entry port type (gas jet stream), size, orientation, flow conditions, and height, H, above the impingement surface, and with Equations 1 – 3 described above, it is possible to determine the mean (between the stagnation point and the position r or x) heat flux due to evaporation for any set of flow and entry port configuration conditions. These values then become the critical boundary conditions for the unsteady state, tissue-conduction problem. The speed that peritoneal tissue will be cooled by evaporative jet cooling is governed by the jet's evaporative surface heat flux, J h,e , the size of the jet, and the conduction characteristics of the tissue. To simplify the analysis, it was assumed that the very high heat fluxes caused by the evaporative gas jet would completely dominate tissue internal heating due to metabolism and perfusion near the tissue surface. Additionally, it was assumed that the tissue dimensions would be large in comparison to that of the gas jet, for example, very large conduction pathways. Under these conditions, the general transient heat conduction differential equation reduces to where, for our model, the reduced temperature, theta, is defined as and the boundary conditions for evaporative surface cooling, with c = J e /k where k is the surface tissue's thermal conductivity, are as follows: This classic transient-conduction problem has been solved by many. The solution for the general case, as well as the special situation of x = 0, or the impingement surface temperature of our model can be expressed as 12 where, for our jet evaporation system, c = J h,e /k and Solutions to Equation 5 are complicated by the fact that the value for c = J h,e /k is itself a nonlinear function of surface temperature, T, through J h,e and its dependence on the vapor pressure term p s which, in turn, is a nonlinear function of surface temperature. This results in an Equation 5 that is transcendental in T and must be solved numerically by iterative, non-constant interval, finite-difference techniques. Figure 2 shows the results of an Equation 5 calculation for the surface tissue temperature transient, caused by evaporative gas jet cooling of a circular area of 0.79 sq. cm around the impingement point. The data of this figure are calculated for a Veress needle height over the tissue of 5 mm and for insufflation gas flows of 1 to 11 liters per minute. A typical set of surface temperature cooling curves for evaporative jet cooling via a trocar gas injection device is shown in Figure 3 . As with the Veress needle theoretical results, the effect of insufflation gas flow rate is indicated parametrically for CO 2 flows from 1 to 11 liters per minute. Experimental measurements have been made to verify the results of the theoretical analysis of laparoscopic evaporative gas jet cooling. These measurements were carried out in two phases. The first (Phase 1) was a series of tests using a single thermocouple (t/c) measurement, at the stagnation point = T1, of the temperature transient detected in 3 mm thick sections of synthetic materials; cellular polyurethane (cp) and woven rayonpolyester (wr). Data were collected at a rate of five points a second for Phase 1 measurements. A second series of tests (Phase 2) consisted of measurements using cellular polyurethane and woven rayon, and ∼3 mm sections of animal tissue consisting of turkey or ham. A four thermocouple array, with the first t/c (T1), was located at the stagnation point, and the remaining t/cs (T2, T3, & T4) were spaced on a radial line 1, 2 and 4 mm from the first thermocouple, respectively. Phase 2 temperature data were collected simultaneously from each t/c at a rate of approximately 15 points per second for each thermocouple. The t/cs for this series, as were those of Phase 1, were small (0.2 mm diameter) sensors, with fast response times (time constant □ 0.6 sec), and positioned from below, at or just slightly below (∼ 0.2 mm) the impingement surface. A 1 mm, inside diameter, Veress needle and a standard 10 mm trocar were used as the insufflation gas injection ports. These were positioned 2 to 5 mm above the tissue surfaces, and gas flows were varied between two and eight liters per minute. The experimental jet/impingement surface configuration of Figure 1 was totally enclosed in a transparent container to provide an isolated environment that reproduced the gas space, temperature, and humidity conditions that the gas injection device and impingement surface experience during a laparoscopic procedure, (CO 2 , 37°C and □ =100%). During all tests the “tissue” surface was irrigated with a temperature controlled sterile saline drip at 37°C. Figure 4 shows the results of Phase 1 experimental tests of evaporative jet cooling at the stagnation point on a wet cellular polyurethane impingement surface at 37°C. The results of a typical Phase 2 experimental test are seen in Figure 5 , which shows the surface temperature cooling transient for ham tissue when evaporatively cooled by a 1 mm circular jet at approximately 6 liters per minute flow rate. These data are typical of the measurements obtained at the flow rates and jet height tests of this study. Figure 6 shows typical impingement surface cooling transients resulting from an approximately 6 liters per minute CO 2 flow rate from a 10 mm trocar. The effects of changes in insufflation gas flow rate and gas port height above the impingement surface on tissue temperature decrease with the initiation of evaporative surface cooling. Figure 7 shows the results of these experiments. Here, the average of replicate runs of temperature measurements of thermocouple TC1 (the impingement point temperature), measured five seconds after initiation of the insufflation gas flow, is plotted against various gas flows. The parameters of Figure 7 represent lines of constant height, H, of the tip of the injector above the tissue surface To confirm that the rapid temperature drops demonstrated in the tissue tests of this study were caused primarily by evaporation of tissue-surface liquid, a series of experiments were carried out in which the insufflation gas was both heated (to 37°C) and humidified (to >95 relative humidity) prior to its introduction to the insufflation gas injection port. A typical result of these tests is shown in Figure 8 , where thermocouples TC1 through TC4 measured the tissue surface temperatures at various distances from the impingement point for approximately eight seconds after initiation of the gas flow rate of approximately 6 liters per minute. The results of the analytical modeling of evaporative jet cooling of tissue surfaces gives dramatic indications of rapid and significant cooling of these surfaces, as seen in Figures 2 and 3 . These results predicted that, for circular jets, tissue surface temperatures could drop to under 20°C in less than three seconds after initiation of insufflation gas flow and cool a tissue area of more than 0.75 square centimeter. Our modeling of this cooling effect showed serious tissue temperature drops for a wide range of delivery port types, which resulted from jet streaming of gas and, importantly, for jet heights (H) up to 1 cm. Comparison of Figures 2 and 3 demonstrates that the decrease of surface temperature is relatively independent of type of gas injection device, although the Veress needle injector caused a somewhat faster tissue-cooling rate (≈ 1.2°C/s) than the 10 mm trocar. Results, however, from the trocar analysis, predicted that considerably more tissue surface area would experience the cooling effect (≈ 1.9 cm 2 ). Phase 1 experimental testing showed very large cooling rates of −30°C/s in the first second of gas flow; larger, in fact, than cooling rates predicted by the analytical model. The temperature for these tests tended to reach an equilibrium level within two to five seconds of the initiation of the evaporative gas jet. This effect most certainly is due to the jet dispersing and evaporating the fluid in the region of the t/c. Under these conditions, the slower evaporation rate could be offset by internal heat conduction to the tissue surface. Phase 2 results confirm the theoretical prediction of the degree of evaporative cooling on wet tissue both qualitatively and quantitatively. At insufflation flow rates above 51/m, significant areas of “tissue” (> 2 cm 2 ) were cooled to temperatures less than 18°C within six seconds of initiating gas flow. These cooling effects, as can be seen in Figure 7 , were relatively independent of the height, H, of the gas port site above the tissue surface. That these cooling effects are caused by evaporation of surface fluid into jets of dry CO 2 is demonstrated by the results of Figure 8 , where pre-heated and humidified insufflation gas jet streams with flow rates over 5 1/m caused less than 1°C of local surface cooling. In some cases, local tissue was heated approximately 1°C. These small temperature rises or drops experienced with the introduction of heated/humidified insufflation gas were caused by small temperature differences (positive or negative □ 1°C) between the temperature of the incoming gas and the temperature of the injection device. The degree of tissue cooling, with either the Veress needle or the trocar gas injector were found, in Phase 2 experiments, to be independent of the types of tissue tested. The differences in maximum cooling rate and/or the minimum temperature reached by the tissue in the first five to seven seconds of evaporative jet cooling were not statistically significant for the different types of tissue tested. The crossing of TC1 and TC2 curves after two seconds of evaporative cooling as shown in Figure 7 , most likely is due to drying around the stagnation point. Tissue heat conduction from the sub-surface regions toward the stagnation point (SP) would heat this region after most of the resident liquid is evaporated and its cooling effect decreased. The analytical modeling and experimental testing of this study show that substantial evaporative cooling occurs to extensive local regions of wet surface tissue when dry insufflation gas flows against its surface. Cooling rates of −20°C/s and local tissue temperature reductions of 20°C were measured. These findings suggest that local neurological reaction and tissue damage are possible from such hypothermic events and that this can be overcome. 5 This study demonstrates and is confirmation of theory validating experimental and clinical findings. Clinical observations and improvement of peri- and postoperative patient hypothermia by heating and humidifying the gas during laparoscopy has been previously demonstrated. 13 This study shows further that the effects of insufflation gas jet evaporative cooling during laparoscopy are completely eliminated by humidifying the insufflation gas stream. | Study | biomedical | en | 0.999997 |
10527327 | Benign cystic teratomas, or dermoid cysts, are germ cell tumors of the ovary that account for 20-25% of all ovarian tumors and are bilateral in 10-15% of cases. 1 They have a low incidence of malignancy, reported as 1-3%. 2 , 3 The majority of dermoid cysts are asymptomatic and are often discovered incidentally upon pelvic exam. The potential for complications such as torsion, spontaneous rupture, risk of chemical peritonitis, and malignancy usually makes surgical treatment necessary upon diagnosis. Traditional therapy for a dermoid cyst has been cystectomy or oophorectomy via laparotomy. The laparoscopic approach has become increasingly accepted since 1989. 4 Because most patients with cystic teratomas are of reproductive age, a conservative approach is ideal; laparoscopy may minimize adhesion formation and thus decrease the chance of compromising fertility. Since our first publication, an increasing number of surgeons are reporting varied approaches. 4 – 16 In this study, we evaluate the safety and efficacy of laparoscopic management of dermoid cysts based on our more than ten years' experience. Eighty-one patients with a preoperative diagnosis of unilateral or bilateral dermoid cysts between March 1988 and August 1998, for a total of 93 dermoid cysts, were included in the study. Sixty-one operations were performed at the Center for Special Pelvic Surgery (Atlanta, Georgia) and 20 at Stanford University Medical Center (Stanford, California). Patient charts were reviewed for demographic data, chief complaint, past history, preoperative investigation, operative techniques (including evidence of tumor spillage, method of specimen removal, blood loss, and other procedures), operative time, complications, duration of hospital stay, pathology report, and postoperative follow-up. Because this was a retrospective study, it was not necessary to obtain Institutional Review Board (IRB) approval. All patients had preoperative transvaginal sonography. All operations were performed under general anesthesia with endotracheal intubation. After pneumoperitoneum was achieved, diagnostic laparoscopy was used to thoroughly evaluate the pelvis and upper abdomen. First, peritoneal washing was obtained. Then, ovaries were closely examined for potential gross malignancy. Frozen section was employed for any suspicious lesions on a routine basis to rule out malignancy. Laparoscopic removal of dermoid cysts was performed as described previously. 4 Briefly, a cleavage plane was created between the cyst and stroma of ovary with hydrodissection, and the cyst was enucleated. We tried to minimize spillage in all cases. If spillage did occur, copious irrigation of the abdominal cavity with approximately 10-12 liters of Ringer's was used to wash out cyst contents. In the earlier cases, we used two different removal techniques. In the first technique, the cyst contents were aspirated through a 12-guage laparoscopic needle suction irrigator probe and spillage avoided as much as possible. The emptied cyst was shelled out from the ovary and removed through a trocar sleeve. In the second technique, the cyst was enucleated and then removed through a posterior colpotomy. Since 1995, however, in most cases, an impermeable sack (Endobag: Ethicon, Somerville, NJ) was used for removal. Using this method, the cyst was placed in an impermeable bag and then aspirated in the bag and removed through a 12 mm suprapubic trocar. After removal, electrocautery, low power CO 2 laser, or a few sutures were often used to invert and approximate the ovarian edges. Suturing was minimized in all cases to reduce postoperative adhesion formation. However, when cyst was large, intraluminal, 40 PDS was utilized to approximate the edges. Conversion to laparotomy was not necessary in any of the operations. Eighty-one patients underwent surgery for laparoscopic removal of dermoid cysts. The mean patient age was 35.4 years (range 16-60), and the mean parity was 0.70 (range 0-4). The chief complaint was pelvic pain in 40 patients, abnormal uterine bleeding and pain in 11 patients, and only abnormal uterine bleeding in three patients. Twenty-seven patients were asymptomatic. All but one patient was preoperatively diagnosed by either pelvic exam or transvaginal ultrasound. The one patient not preoperatively diagnosed with a dermoid cyst had one discovered coincidentally with bilateral salpingo-oophorectomy performed due to family history of ovarian cancer. Two of the patients presenting with pain had cyst torsion and were treated with unilateral salpingo-oophorectomy. Three patients had recurrent dermoid cysts after previous surgery at another center. Two with contralateral recurrence had cystectomy. The other patient had bilateral cysts, only one of which was recurrent; she had salpingo-oophorectomy for the recurring dermoids and cystectomy for the new cyst. Three pregnant patients were diagnosed with a dermoid cyst during prenatal examination, two at 16 weeks and one at 12 weeks. Cyst removal was performed via aspiration and enucleation followed by removal through a trocar in the first two patients, and via enucleation and removal within an endobag in the third. These three patients have since delivered without complication. Sixty-nine patients had unilateral cysts (85.2%), and 12 patients had bilateral cysts (14.8%). Varying procedures were performed based on patient age and history. Seventy of the cysts were removed by cystectomy (75%), 14 by salpingo-oophorectomy (15%), and 9 by salpingo-oophorectomy with hysterectomy (10%). Of the 14 cysts removed via salpingo-oophorectomy, 11 were unilateral, and 3 bilateral. The indications for unilateral salpingo-oophorectomy were possible malignancy due to age (>40) in 8 of 11 patients, torsion in 2, and recurrence in 1. The indications for bilateral salpingo-oophorectomy were postmenopausal status for all three patients. Two wished to preserve their uterus; the other had prior hysterectomy. The indications for hysterectomy were endometriosis (8 cases) or postmenopausal mass (1 case). Of the cysts treated via either cystectomy or salpingo-oophorectomy, 53 were removed through the trocar sleeve without an endobag, and 22 were removed within an endobag. Aspiration and morcellation was almost always used inside the impermeable sack to facilitate cyst removal through the trocar sleeve. Nine cysts were removed through a posterior colpotomy. The other nine cysts were removed concomitantly with hysterectomy. The spillage rate per patient was 48% (39/81 spillages), but the total spillage rate was 42% (39/93 spillages) for the cysts removed. Spillage rates also varied with removal method: 60% (32) for aspiration and removal through a trocar sleeve without an endobag, 44% (4) for colpotomy removal, and 13.6% (3) for removal within an endobag. No spillages were recorded when removal was concomitant with hysterectomy ( Table 1 ) . Spillage was not correlated with cyst size: mean ± SD cyst diameter was 4.6 ± 2.1 cm and 4.3 ± 2.3 cm for spilled and unspilled cysts, respectively (p > .05). Concomitant surgical procedures consisted of treatment of endometriosis (32), myomectomy (8), treatment of non-dermoid cyst (7), adhesiolysis (2), enterocele repair (2), appendectomy (1), presacral neurectomy (1), bowel resection (1), bilateral tubal reanastomosis (1), and bilateral salpingo neostomy (1). Mean cyst diameter, blood loss and operative time were 4.5 (range 1-12) cm, 84 ± 76 mL, and 130 ± 53 minutes, respectively. Operative time for the 17 patients who had no additional procedures beyond dermoid cyst removal (either by cystectomy or unilateral salpingo-oophorectomy) was 103 ± 30 minutes. There were no intraoperative complications. Pathological diagnosis identified mature cystic teratomas in all cases. Overall mean hospital stay was 0.98 (range 0-11) days. Excluding the patient with a bowel resection, who stayed for 11 days, mean stay was only 0.83 (range 0-5) days. All these patients had a hospital stay of less than two days, except for the two cyst torsion cases. Postoperative follow-up mean was 23.8 (range 0.5-84) months; 12 patients were lost to long-term follow-up. During this period, there was one postoperative complication of an incisional infection at the umbilical trocar site, occurring ten days after surgery. The infection resolved after antibiotic treatment. One patient experienced a recurrent dermoid cyst, treated with salpingo-oophorectomy. Three patients developed non-dermoid ovarian cysts during the follow-up. Two other patients had subsequent surgery unrelated to cyst development: one a hysterectomy, the other a bilateral salpingo-oophorectomy. Seven patients became pregnant during the follow-up period. Spillage of cyst contents, potentially leading to complications such as chemical peritonitis or spread of malignancy, is the most important risk in laparoscopic management of dermoid cysts. Spillage rates in laparoscopy are 15-100%, 4 – 16 compared to only 4-13% via laparotomy. 11 – 13 Excluding case reports, a review of the literature reveals a total of 14 studies, including the present study, documenting 470 laparoscopic dermoid cystectomies. Spillage occurred in 310 cases (66%). Major postoperative complications were seen in only one case, 11 with chronic granulomatous peritonitis occurring nine months postoperatively. Taking these results into consideration with the literature, we can conclude that the rate of clinical chemical peritonitis following spillage from laparoscopic dermoid cystectomy is 0.2% ( Table 2 ) . In this series, our spillage rate was 48% per patient, with no cases of clinical chemical peritonitis following surgery. No cases of chemical peritonitis following intraoperative spillage during laparotomy have been recently reported. 11 – 13 It is possible to aspirate the cyst after placing it intact inside a laparoscopic bag. Our increased use of this technique has significantly reduced the potential for spillage. In this study, the laparoscopic spillage rate for removal within an endobag was 13.6%, a rate comparable with laparotomy. 11 – 13 When spillage occurs despite precautions, it is of prime importance to copiously irrigate the abdominal cavity with Ringer's lactate to effectively wash out cyst contents. Another criticism of laparoscopy has been potential implantation of cystic components into the abdominal wall while removing the cyst through the operative channel. There is one case report 17 of implantation leading to fistulization of the bladder and rectum. Careful cyst removal via the trocar sleeve without contacting the abdominal wall obviates this potential complication. A survey of 156 members of the American Society of Gynecologic Oncologists 18 revealed 42 cases of laparoscopic removal of ovarian tumors subsequently found to be malignant. In our follow-up, we did not find any cases of subsequent malignancy. Although it has been reported that malignant ovarian cyst rupture may not affect the prognosis for ovarian cancer, 19 , 20 spread of malignancy is still a potential problem for laparoscopic management. Spillage should be avoided as much as possible through careful aspiration or use of an impermeable sack. Postoperative chemotherapy may be used in case of unexpected rupture of a malignant cyst. Questions concerning diagnosis of dermoid cysts still exist. Ultrasound, especially transvaginal, may assist in diagnosis. However, malignancy is still difficult to exclude with sonography, because dermoid cysts and malignant tumors of the ovary may both have mixed solid components. Tumor markers such as CA125 are generally helpful but are not enough to diagnose malignancy every time. 21 For this reason, frozen section of suspicious lesions and excrescencies may be necessary. One of the advantages of laparoscopic management of dermoid cysts is decreased adhesion formation. In our previous study, 4 second-look laparoscopy revealed no adhesions in three patients who had spillage, whereas the one patient without spillage had mild periovarian adhesions. In a study by Chapron et al, 9 10 of 56 patients with dermoid cystectomies had second-look laparoscopy. Eight of these ten patients had intraperitoneal spillage, but only two had adhesions upon second-look laparoscopy. Lin 13 also reported one patient who had second-look laparoscopy after 12 months. Despite spillage during the previous operative course, the patient had minimal adhesions. None of these three studies revealed evidence of granulamatous implants. Our laparoscopic operative times are comparable with other laparotomy reports. Christoforoni 11 reported operative times for laparotomy of 92 ± 11 minutes. In our study, mean operative time excluding other procedures was 103 ± 30 minutes, a value approaching laparotomy times. As has previously been established, hospital stay, blood loss, patient morbidity, and cosmetic results may be significantly better with laparoscopy compared to laparotomy. To our knowledge, there are only a few other studies documenting a laparoscopic dermoid cystectomy in a pregnant patient. 22 – 23 In this study, we report three cases of dermoid cyst removal in a pregnant patient. In two of the cases, there was spillage due to enlarged cyst size and technical difficulties. Postoperative period and delivery was without complication in all three cases. Based upon our more than ten years' experience in a total of 90 patients and 102 dermoid cysts, including our first publication, 4 we believe that laparoscopic management of dermoid cysts may be a safe and beneficial procedure when performed by an experienced surgeon. | Other | biomedical | en | 0.999997 |
10527328 | Hysterectomy is the fourth most common in-patient operation in the United States. About two-thirds of all hysterectomies are still performed abdominally. Over the last ten years, gynecologists have been obtaining the necessary skills to perform laparoscopically assisted vaginal hysterectomies (LAVH) or total laparoscopic hysterectomies (TLH) in order to convert an abdominal procedure into a laparoscopic/vaginal procedure. Similar to vaginal hysterectomy, LAVH and TLH have a shorter hospital stay, less painful recovery, and a quicker return to normal activity relative to an abdominal procedure. 1 – 3 We have attempted to adopt techniques which assist in making LAVH and TLH easier, more cost-effective, expeditious, and safer to perform. Since May 1998, we have been performing total laparoscopic hysterectomy using the harmonic scalpel as the sole coagulation and cutting device, including transection of the uterine vessels and opening of the vagina. The harmonic scalpel has provided a cost-reduction by limiting instrumentation and increasing speed of the procedure. This one instrument is used to achieve hemostasis and transect tissue. It provides a high degree of safety by heating tissue to form a protein coagulum, which gives excellent hemostasis at a significantly lower temperature than standard electrosurgery, thus decreasing the risk of thermal injury to surrounding tissue. We began performing total laparoscopic hysterectomies to maximize vaginal length, preserve the entire uterosacral ligaments for better long-term vaginal support, and decrease operating room time relative to LAVH. Twenty-six TLH cases were attempted between May and December 1998. Instrumentation used to facilitate this TLH procedure included the Laparosonic Coagulating Shears (LCS) attachment to the harmonic scalpel (Ethicon, Cincinnati, OH). The 10 cm LCS was used during the first half of our series, after which we converted to the 5 cm LCS. This allowed us to use two secondary 5 mm ports in addition to the umbilical 10 mm port. In addition, we used the Rumi system uterine manipulator, Koh Cup Vaginal Fornices Delineator, Colpo-Pneumo Occluder (Cooper Surgical, Shelton, CT). 4 Twenty-two cases of total laparoscopic hysterectomy using the harmonic scalpel were performed with no major complications. Four cases that began as TLH were converted to LAVH secondary to large fibroids, making dissection of the uterine vessels, mobilization of the uterus, or opening of the vagina difficult laparoscopically. No increased morbidity was noted in comparison to LAVH, vaginal hysterectomy, or total abdominal hysterectomy performed within the same time period at the same institution. Two patients' status post-TLH were treated with oral antibiotics for mild vaginal cuff cellulitis with rapid resolution. Average hospital stay was 1.8 days for TLH, 1.9 days for LAVH, 2.3 days for vaginal hysterectomy, and 2.8 days for total abdominal hysterectomy. We have been very satisfied using the LCS attachment to the harmonic scalpel as our main instrumentation in performing TLH. It is safer than standard electrosurgery. By working at significantly lower temperatures (100 C° versus 300 to 400 C°), there is less lateral thermal spread with the harmonic scalpel. The decreased thermal damage to surrounding tissue (relative to electrosurgery) lowers the risk of inadvertent injury to the ureters and bladder. By decreasing the amount of necrosing tissue, the risk of a fistula formation should be theoretically reduced, and there should be less postoperative pain. Harmonic scalpel is cost-effective relative to the use of staple devices. Staple devices cost an average of $1,600 per case versus $325 for harmonic scalpel. Although the harmonic scalpel is more expensive than a reusable bipolar device (by $325 per case), it saves money by decreasing operative time by an average of 15 to 30 minutes. This is facilitated by decreasing the need for instrument changes and using the bottom of the active harmonic scalpel blade as a cutting device to facilitate colpotomy. Initially, we had concerns regarding transection of the uterine artery with the harmonic scalpel. We knew from related experience that it could be used to effectively coagulate vessels within the infundibulopelvic ligament as well as gastric arteries. For approximately six months prior to beginning our TLH procedure, we routinely transected the uterine arteries at the time of LAVH. This gave us the opportunity to reassess the uterine pedicles laparoscopically after the procedure was essentially completed. There was no delayed bleeding noted at the uterine pedicles. Approximately 20 cases were performed in this fashion. During our TLH series, we have had no postoperative bleeding problems secondary to using the harmonic scalpel. The use of bipolar electrocautery has become rarely necessary. Techniques that we have found helpful include careful dissection and skeletonization of the uterine artery and taking enough time during coagulation for hemostasis to occur prior to transection of the artery. This can easily be mastered with laboratory training and by performing the first few cases with an experienced surgeon. Vaginal hysterectomy is still our preferred procedure, but TLH or LAVH can be used as a substitute for the majority of abdominal hysterectomies. Using the harmonic scalpel has facilitated the technical performance of TLH and can safely be used to perform this procedure. | Other | biomedical | en | 0.999997 |
10527329 | Patients with abdominal pain have been a great challenge for the general surgeon. In many cases, even after the history, physical examination, laboratory tests and image exams, the diagnosis cannot be concluded. In referral centers, a laparoscopic approach, has been the “gold standard” for many elective procedures, and has been used for abdominal emergencies, traumatic as well as non-traumatic. Surgeons can use videolaparoscopy in cases of acute abdomen for 1) diagnosis only (ie, patients with abdominal pain due to endometriosis), 2) diagnosis and treatment (ie, female patients with abdominal pain secondary to appendicitis), 3) treatment only (ie, patients with acute cholecystitis), and 4) indicating the best place to do the incision in cases where conversion to laparotomy is absolutely necessary. Between January 1992 and December 1996, 462 charts of patients who were admitted to the emergency room of the Sao Rafael Hospital (SRH), with non-traumatic acute abdomen, were reviewed, retrospectively. The SRH has its own policy to automatically generate a series of laboratory tests for patients with abdominal pain. These tests include a complete blood count (CBC), liver functions tests, serum electrolytes, serum creatinine and blood urea, amylase, urinalysis, B-hCG. Plain abdominal X-ray, ultrasonography and/or CT scan were required, according to the main complaint associated with a physical exam. Chest radiography and electrocardiogram (ECG) were done to rule out extra-abdominal causes of pain. Videolaparoscopy was done in all 462 patients to deter-mine the diagnosis and/or treat the patients. All patients were hemodynamically stable. Preoperative care included six hours fasting, when possible; relief of symptoms of pain, nausea, vomiting, fever, dehydration, electrolyte disturbance, and blood transfusion when indicated. In all cases, the procedure was done under general anesthesia. The patient's position varied according to the presumptive diagnosis as well as to the surgeon's choice. Open access techniques to establish pneumoperitoneum were used in cases where patients had abdominal distention, umbilical hernia or previous abdominal surgery near the umbilicus. In the remaining cases, a closed method utilizing the Veress needle to establish pneumoperitoneum was used. Abdominal pressure was kept under 15 mm Hg to avoid the negative effects of excessive pneumoperitoneum. The first trocar was placed in umbilicus, and other trocars were placed as required by the disease pathology. Prophylactic antibiotic was given in all cases – usually a first- or second-generation cephalosporine. Continued antibiotic therapy was used when indicated. Patients with non-traumatic acute abdomen were classified into seven groups according to etiology ( Table 1 ) . The vast majority of the patients had inflammatory acute abdomen (82.03%). Acute appendicitis was the most frequent cause, followed by acute cholecystitis; others causes are listed in the Table 2 , as well as the rate of con-version to traditional laparotomy. Endometriosis is a frequent cause of abdominal pain. In these cases, abdominal and pelvic fluid was aspirated and sent to pathology. When endometriotic tissues were found, they were cauterized, and the patients were followed by a gynecologist. All five of the patients with acute pancreatitits and three cases of enteritis were submitted to videolaparoscopy without a definite preoperative diagnosis. Conservative treatment was adopted for all of these cases. Two hundred and ten patients with appendicitis under-went a definitive laparoscopic procedure with a success rate of 97.14%. Three trocars were used; the camera was introduced into the umbilical trocar (10 mm), the second trocar was inserted into the left iliac fosse (10 mm), and the third above the pubis (5 mm). The mesoappendix was cauterized with bipolar electrical current, or secured with titanium clips. The appendiceal base was tied with two endo-loops. In some cases, it was necessary to remove the appendix using an endo-bag to avoid contact with the abdominal wall and thereby reducing the rate of wound infection. There was no exclusion criteria for choosing the laparoscopic approach for acute appendicitis. The appendix was graded according to the evolution from edema to necrosis; the rate of conversion was very low. In patients with pelvic peritonitis due to pelvic inflammatory disease, an aspiration/washing procedure was performed to clean the abdominal cavity. In one case, it was necessary to perform bilateral salpingectomy, due to an intense salpingitis. In only one case was conversion done (unilateral oophorectomy), due to dense adhesions. Three patients who were submitted to videolaparoscopic cholecystectomy had bile peritonitis postoperatively. In each case, laparoscopy was re-performed and large amounts of bile were found in the abdominal cavity. The cavity was washed and the bile aspirated; a closed drained system was placed after peritoneal toilet. The source of the bile leak was found in only one case. (A small bile duct in the hepatic parenchyma. This was secured with laparoscopic suture.) Abdominal hemorrhage occurred in 51 female patients due to gynecologic complications. Rupture of an ovarian cyst was the most frequent cause (21 cases), followed by ectopic pregnancy (17 cases). In all of these cases, diagnosis was obtained by ultrasonography. In 13 cases, a small amount of blood was found in the Pouch of Douglas ( Table 5 ) . There was no associated pathology. In six patients, vascular occlusion or semi-occlusion was the cause of acute abdomen: ischemic colitis in one case, mesenteric ischemia in two cases, meso ovarian torsion in two cases and fallopian tube torsion in one case. Conversion to open procedure was done in cases of mesenteric ischemia in which segmental enterectomies were required. In one of the two cases of meso ovarian torsion (where extensive adhesions were found), open oophorectomy was performed ( Table 6 ) . In hollow viscera perforation (8 cases), six cases were due to diverticulitis, with five cases presenting localized peritonitis treated by aspiration. One patient with sigmoid necrosis, perforation and a large amount of liquid in the abdomen required laparotomy guided by laparoscopy with a sigmoid resection and terminal colostomy (Hartmann procedure). The other two cases of hollow viscera perforation were a jejunal perforation caused by lymphoma. They were treated by laparotomy followed by enterectomy. One case of perforated duodenal ulcer was treated by laparoscopic suture and aspiration/washing ( Table 7 ) . Bowel obstruction occurred in 15 patients: nine had adhesions, which were treated by laparoscopic adhesiolysis with no conversion; two had internal hernia, also treated by laparoscopy; and four patients had bowel obstruction due to colon cancer, which were treated by laparotomy with colectomy ( Table 8 ) . For this group of patients, who had an established diagnosis, laparoscopy was done for therapeutic purposes. The group was composed of 113 cases of acute cholecystitis and 13 cases of intra-abdominal abscess. The patients with clinical, laboratory and ultrasonographic findings of acute cholecystitis were submitted to laparoscopic cholecystectomy. In addition, all cases had patho-logical confirmation of acute cholecystitis. The European laparoscopic technique modified by Prof. Enrico Croce was used. For patients with a high suspicion of biliary duct stones, endoscopic cholangiography was done with papilotomy as required, and intraoperative cholangiography was done in nine patients with cystic and bile duct dilatation. There were complications in 7.96% of these patients with no mortality. The conversion rate was 12.4%; significantly higher than in the patients with symptomatic chronic cholecystitis treated by laparoscopic cholecystectomy in São Rafael Hospital. All degrees of acute cholecystitis were treated by laparoscopic cholecystectomy. Twelve patients with abdominal abscess were treated by the laparoscopic approach, which was successful in ten cases. One case of pelvic abscess with difficult access and dense adhesions was treated by laparotomy. One case of iatrogenic duodenal lesion (thermal injury during laparoscopic cholescystectomy), with abscess, was treated by laparotomy and suture. These were patients in whom laparoscopy helped achieve a diagnosis and indicated the best location to perform the incision: six patients with appendicitis, two with abdominal abscess, one with pelvic inflammatory disease and one with enteritis. Different regional incidences of pathologies that can cause acute abdomen make comparison among these cases difficult. Even with an expert surgeon and advanced diagnostic tools, it can yet be difficult to make an accurate preoperative diagnosis in many patients, which can delay proper treatment or lead to an unnecessary exploratory laparotomy. 1 Patients with an acute abdomen diagnosis and who undergo laparotomy have a negative rate of exploration as high as 22%. Some studies propose the use of laparoscopy to evaluate acute abdomen as it has high diagnostic accuracy, is associated with a low rate of negative laparotomy, and has low mortality and morbidity. 2 – 6 In this study, group I (diagnosis group) was composed of 18 patients with a variety of diagnoses such as pancreatitis and endometriosis in which an accurate diagnosis, provided by laparoscopy, avoided an unnecessary laparotomy. Group II (diagnosis and treatment group) had the most patients in whom laparoscopy defined the diagnosis for the acute abdominal condition and provided surgical treatment by laparoscopic means, including appendicitis in which the proper diagnosis followed by laparoscopic appendicectomy reaches a high success rate with a very low conversion rate (2.86%). It can be compared with several studies. 7 – 9 In reference to pelvic inflammatory disease, an important subgroup of patients benefited from the laparoscopic approach, especially premenopausal women (in whom differential diagnosis with gynecological conditions can be difficult) except in one case where the patient was submitted to open oophorectomy due to dense adhesions. 10 In a special subgroup formed of three patients with biliary peritonitis, laparoscopic cholecystectomy proved to be an apporiate method for both diagnosis and treatment. Laparoscopy proved to be effective as a diagnostic and therapeutic method in women with abdominal hemorrhage, thus avoiding the open procedure. 11 , 12 In the sub-group with vascular occlusions, laparoscopy provided an accurate diagnosis and made it possible to avoid laparotomies in patients with ischemic colitis and adenexal torsion (except in one case of mesovarian torsion), requiring the open procedure in mesenteric ischemic cases. 13 In the cases of perforation of hollow viscera from duodenum to colon, diagnosis and minimal-access surgery were benefits of laparoscopy in five patients with diverticulitis and in one patient with perforated duodenal ulcer, thereby avoiding the open procedure. 14 , 15 Patients with bowel obstruction (the last subgroup), a condition that until recently was not an indication for laparoscopy, were treated by laparoscopy. All of these cases were caused by adhesions or internal hernias. Laparotomy and colectomy were required for the four cases of bowel obstruction due to colonic cancer, as noted by other authors. 16 In group III (treatment group), patients with acute cholecystitis and intra-abdominal abscess were treated by laparoscopy. At the beginning of their experiences with laparoscopic cholecystectomy, many authors excluded acute cholecystitis from their series. Today, a laparoscopic approach has been accepted by many major referal centers. 17 , 18 This series shows a high-resolution rate with a 12.4% conversion to open procedure. In patients with intra-abdominal abscess where the percutaneous approach was not achieved or indicated by radiology, the laparoscopic approach was used as an option of treatment as in other series. 18 , 19 Group IV (guiding incision) showed the benefits of a laparoscopic diagnosis in selecting the best site to perform the incision. 20 This series has shown that in the majority of cases (99.35%) using a laparoscopic approach the surgeon was able to diagnose the cause of non-traumatic acute abdominal pain and, when used for therapeutic purpose, could treat 93.07% of the cases, with an overall conversion rate of 6.92%. | Other | biomedical | en | 0.999997 |
10527330 | Morbid obesity is defined as being 100 pounds (45 kg) or more over the ideal body weight according to the Metropolitan Life Insurance Company height and weight tables. According to the National Institutes of Health Consensus Conference in 1996, surgery remains the only effective treatment for morbid obesity. 1 Indications for surgical treatment of morbid obesity are patients with a body mass index (BMI) greater than 40 kg/m 2 or, alternatively, a BMI between 35-40 kg/m 2 with weight-related serious comorbidity. 2 There are a variety of operations for morbid obesity. These include jejunal-ileal bypass, biliopancreatic diversion, vertical banded gastroplasty, and Roux-en-Y gastric bypass. Payne and co-workers originally introduced jejunoileal bypass. This operation was abandoned due to severe long-term complications such as cirrhosis and liver failure. Biliopancreatic diversion is a malabsorptive procedure described by Scopinaro and co-workers in Genoa, Italy. 3 In the United States, the two most frequently performed operations for morbid obesity are the vertical banded gastroplasty and the Roux-en-Y gastric bypass. 4 The Roux-en-Y gastric bypass is a complex operation that can be associated with high morbidity, especially wound-related complications. Advances in laparoscopic optics, surgical techniques and instrumentation have made it possible to perform the gastric bypass operation using the laparoscopic approach. In this article, we present our technique of the laparoscopic Rouxen-Y gastric bypass for patients with morbid obesity. We performed a retrospective chart review of four initial cases of laparoscopic Roux-en-Y gastric bypass performed at the University of California Davis Medical Center. Charts were reviewed for demographic data, operative results, morbidity and mortality. The patient is placed in a supine position. The surgeon stands on the patient's right side, and the assistant surgeon stands on the patient's left side. Carbon dioxide pneumoperitoneum is created using the Veress needle technique placed at the left midclavicular line slightly above the umbilicus. The intra-abdominal pressure is maintained at 15-17 mm Hg. Five abdominal ports are introduced on the anterior abdominal wall . A 45° angle telescope is used routinely to provide better visualization of the gastroesophageal junction. The patient is placed in a steep reverse Trendelenburg position with a foot plate at the end of the table to help secure the patient. The left lobe of the liver is retracted using a self-retaining liver retractor (Diamond-Flex retractor, Genzyme Surgical Product, Tucker, GA). Our initial dissection is on the hepatogastric ligament of the proximal stomach to enter the lesser sac. A 15-30 ml gastric pouch is created by dividing the gastric cardia approximately 1 cm below the gastroesophageal junction. The greater omentum and transverse colon are retracted to the upper abdomen. The ligament of Treitz is identified, and a window in the transverse colon mesentery is created to the left of the middle colic vessels. A retrocolic, retrogastric tunnel is made for the Roux-limb. The jejunum is transected 30 cm distal to the ligament of Treitz to create the Roux-limb . Two applications of the 2.5 mm Endo-GIA vascular stapler (USSC , Norwalk, CT) are applied to divide the jejunal mesentery. A Penrose drain is sutured to the stapled end of the Roux limb. The Roux-limb is measured 75 cm in length for patients with a BMI up to 50 kg/m 2 or 150 cm for patients with a BMI greater than 50 kg/m 2 . A stapled, functional end-to-side jejuno-jejunostomy is created using two applications of the Endo-GIA stapler. The remaining enterotomy site is stapled closed. Care must be taken to avoid narrowing the jejunal lumen when closing the enterotomy. The Penrose drain attached to the Roux limb is tunneled along the retrocolic and retrogastric path and positioned near the transected stomach. Flexible upper endoscopy is performed, and a snare grasper is passed through the gastric pouch into the peritoneal cavity . A loop wire is placed into the abdominal cavity, grasped by the snare, pulled into the gastric pouch and then pulled out through the mouth. The loop wire is attached to an anvil of the 21 mm CEEA Stapler (USSC). The anvil is pulled down through the stapled gastric pouch. The CEEA stapler is placed through the end of the Roux limb to create a functional end-to-side gastrojejunostomy anastomosis . The anastomosis is reinforced with interrupted sutures. The open end of the Roux limb is closed using the Endo GIA stapler (USSC). The gastrojejunostomy site is inspected endoscopically and tested for leaks. All ports larger than 5 mm are closed with 0-Vicryl suture using the Endoclose device (USSC). The patient is extubated in the operating suite prior to being transferred to the recovery room. The patient is transferred to the floor from the recovery room. A gastrograffin swallow study is performed on postoperative day 2. Clear liquid is started on the evening of postoperative day 2 if there was no leak on the gastrograffin swallow. The patient is discharged on postoperative day 3 and scheduled for an office visit on postoperative day 7. Four patients (3 females and 1 male) underwent laparoscopic Roux-en-Y gastric bypass from August 1998 to September 1998. The average age was 53 years. The mean BMI was 51.2 kg/m 2 . There were no intraoperative emergencies or difficulties requiring conversion to laparotomy. The mean operative time was 381.3 + 119.7 minutes. The estimated blood loss was 275 + 64.5 ml. One of four patients was observed in the ICU for one night. The mean length of hospital stay was four days. There were no postoperative complications. Mason and colleagues described the gastric bypass in 1969 based on observation that patients who underwent subtotal gastrectomy with Billroth II reconstruction lose a significant amount of weight after the operation. The original gastric bypass operation was later modified by Griffen and colleagues, who substituted the loop gastrojejunostomy for a Roux-en-Y gastrojejunostomy. Gastric bypass can be associated with significant morbidity. Complications related to Roux-en-Y gastric bypass include intraoperative splenic injury, anastomotic leaks, deep venous thrombosis, pulmonary embolism, wound infection, incisional hernia, and respiratory complications. 5 In an attempt to minimize the postoperative complications, decrease length of hospitalization, and enhance the recovery period, multiple authors have reported their initial results of laparoscopic approaches to the treatment of morbid obesity. Belachew and colleagues reported on 350 patients who underwent an adjustable silicone gastric banding procedure. 6 Short-term results of their first 75 patients undergoing laparoscopic gastric bypass (LGB) demonstrated that weight loss and reversal of comorbidities with laparoscopic Roux-en-Y gastric bypass were comparable to the open approach. Wittgrove was the first to report the laparoscopic Roux-en-Y gastric bypass. 7 Our laparoscopic Roux-en-Y gastric bypass operation is a modification of the technique developed by Wittgrove and refined by Schauer and colleagues at the University of Pittsburgh. Important aspects of this operation include 1) the use of the Ultrasonic dissector for entering the lesser sac on the gastric lesser curvature; 2) creation of a 15-20 ml pouch by dividing the stomach 1 cm below the gastroesophageal junction; 3) the Roux limb must be routed retrocolic and retrogastric to adequately reach the transected gastric pouch; and 4) the end-to-side gastroenterostomy anastomosis is performed with the anvil placed transorally and pulled down to the gastric pouch. Laparoscopic Roux-en-Y gastric bypass is a complex but feasible operation requiring appropriate instrumentation and advanced laparoscopic skills. This technique deserves further clinical evaluation to determine the long-term complication and weight loss potential as compared to the conventional open approach. | Other | biomedical | en | 0.999997 |
10527331 | Laparoscopic and thoracoscopic techniques developed recently have provided a new dimension for performing surgical correction of functional disorders of the esophagus. These new approaches have changed the attitudes of physicians and patients in regard to the treatment of esophageal dysfunction, but have also increased the risk of superficial and inappropriate choices of candidates for surgery. A precise diagnosis must be performed before any therapy. Therapeutic success depends on the confirmation of the esophageal disease as the cause of the symptoms, on understanding the basic cause of dysfunction and on identifying the patient to be treated surgically. 1 The surgeon must perform the right procedure for the right disease on the right patient. This work shows a retrospective study involving surgical treatment of gastroesophageal reflux disease (GERD) laparoscopically. The purpose is to establish the value of the systematic use of esophageal manometry and 24-hour pH monitoring in selecting surgical patients and in performing the functional evaluation of the esophagus during the pre and postoperative period. In order to do so, we analyze and compare the results of the two periods. The addition of esophageal manometry and 24-hour pH monitoring to the tests performed in order to select surgical cases of GERD began in September 1995. In June 1997, 60 patients had undergone a partial videolaparoscopic fundoplication at 270 degrees (Lind procedure). 2 Upper digestive endoscopy, contrast radiography and functional evaluation were used in order to select the patients systematically. In the group of patients submitted to surgery, 40 (66.6%) were male and 20 (33.3%) were female. The mean age was 45 years-old, with ages ranging from 12 years to 79 years. The indication for surgery was based on symptoms refractory to medical treatment in 58 cases (96.6%) and paraesophageal hernia in 2 cases (3.3%). All presented hiatal hernia at radiography and some degree of endoscopic esophagitis. According to the modified classification of Savary-Miller, 3 had grade I esophagitis (1.8%), 26 grade II (43.3%), 24 grade III (40.0%) and 7 grade IV (11.6%). Esophageal manometry was performed using a perfusion system and computer program for automated digital analysis. The following information resulting from manometry was evaluated in this study: mean pressure of the lower esophageal sphincter (normal ranging from 8 mm Hg to 26 mm Hg) with DeMeester method 3 and the presence of any degree of hypomotility in the body of the esophagus. Dysmotility was measured using the parameters of amplitude, duration and progression (peristaltism or simultaneity) of the swallowing contractions. The 24-hour pH monitoring was measured with a single sensor located 5 cm above the lower esophageal sphincter (LES) . The results were evaluated by the DeMeester score, 4 taking as a maximum limit of normality the 95 percentile, whose value is 14.72. The said score includes the variables shown in Table 1 . The postoperative measures were obtained 45 days after fundoplication. Before surgery, LES proved hypotonic in 41 patients (68.3%) and normal in 19 (31.67%). The pressure of the LES was on the average 9.2 mm Hg during the preoperative period and 15.2 mm Hg during the postoperative period, with a calculated rise of 6.0 mm Hg. This rise in pressure, however, was higher in the group of hypotonic patients and significantly lower in the group of patients with normal pressure of the LES ( Table 2 ) . Hypomotility of the body of the esophagus appeared in 14 patients (23.3%). After surgical correction, the 46 cases of normal motility (76.6%) remained normal, except for 2 patients (4.3%) who had an increased amplitude of the peristaltic contractions. Among the 14 cases of hypomotility, 4 patients (28.5%) had a postoperative improvement expressed in the increased amplitude and duration of the contractions that had been reduced or were absent in the lower third of the esophagus. Six patients (10%) presented early dysphagia with a resolution in less than two months. A total of 51 cases (85.0%) had pathological acid reflux found by pH monitoring before surgery. There was a combination of normal pH and esophagitis in nine cases (15%). The DeMeester score of the group of patients in the postoperative period was 31.4. After the procedure, it went down to an average of 3.2. Forty-eight patients (80.0%) did not present any reflux episodes at any time during the 24-hour pH monitoring during the postoperative period. The choice of patients with GERD who will benefit most from surgical correction depends on a careful evaluation covering several aspects: the presence of symptoms, degree of esophagitis, size and type of hiatal hernia, esophageal motility, LES pressure, presence and intensity of reflux and response to medications. These aspects, to be taken into account when deciding to submit the patient to surgery, are obtained, respectively, from the anamnesis, endoscopy, contrast radiography, esophageal manometry, 24-hour pH monitoring and therapeutic test with acid secretion inhibitors. All the data together will allow the definition of the best treatment. Using isolated data cannot help select the best case for laparoscopic fundoplication, and adequate selection is one of reason for the good results shown in specialized literature. 5 It is essential to know whether the patient has relief from his or her symptoms while using medication to predict symptom regression after making a valve, which will prevent gastroesophageal reflux. 6 A mechanical defect in the LES can be found in 60% to 70% of the patients with GERD. 7 The mean pressure of LES below the normal values is predictive of a bad prognosis for drug therapy 8 and good prognosis for surgical treatment. 9 Multifactorial analysis performed in selecting our cases and understanding the pathophysiology of gastroesophageal reflux are essential in order to best deal with medical situations that are sometimes confusing, such as when we find normal pressures in the LES. Measuring sphincter pressure using what is called “volume vector” is considered one of the most effective ways of detecting mechanical deficiency, as well as the only form of achieving postoperative control of the surgical correction. 3 However, the transient relaxation of the LES, besides being one of the most important areas of study in esophageal physiology, is currently considered to be closely related to GERD. 10 In 1964, McNally et al 11 presented a study that discussed the mechanism of eructation based on the transient relaxations of the LES, caused not by swallowing but by the distension of the stomach with air. Curiously, the relationship between this phenomenon and GERD was only suggested later, explaining the association of GERD and normal LES pressure at stationary manometry. Most studies on transient relaxations show that the participation of this mechanism in GERD is on the order of 63 to 74%. 12 , 13 Considering the comments on LES function, we see that marked hypotonia leads to a high probability of dependence on prolonged medical control and, consequently, surgery. On the other hand, the certain finding of normal mean pressure in the LES does not rule against a fundoplication. Making an antireflux valve will naturally raise the pressure of a hypotonic LES, but this is not its main function. Fundoplication should aim to prevent the transient relaxations not induced by swallowing, maintaining a pressure close to the normal average of 13.8 mm Hg. 14 The main objective of manometry of the esophageal body during the preoperative period is to detect changes in motility, which will prevent fundoplication or require some adaptation of the technique to be used. Some authors advocate rendering the type of valve adequate to the function of the esophageal body, suggesting partial fundoplications when hypomotility is present. 15 Around 7% of the patients with GERD present some degree of primary disease of motility 16 and, besides this, 20% present an acquired or secondary disorder, like those caused by medication, trauma or lesion due to reflux itself. 17 Esophageal manometry easily diagnoses “nutcracker” esophagus, diffuse spasm and achalasia. Improvement is expected in the cases of hypomotility caused by pathological reflux itself, predominantly in the lower third of the organ, as long as fibrosis has not yet been established. Ottigmon et al, 18 after a 28-month follow-up, showed that dysphagia was present in 39% of the patients submitted to surgery using Nissen's technique, and in 13% of the Toupet group (partial valve). There was no change in esophageal motility in the Nissen group, whereas, in the Toupet group, a rise in peristaltic velocity occurred from 3.2 cm/s before surgery to 4.4 cm/s after the procedure. In our present paper, improved peristalsis occurred in 28.5% of the patients with deficiency. The DeMeester scoring system for 24-hour pH monitoring, according to authors Johnson and DeMeester, supplies a 90.3% sensitivity and 90.0% specificity to diagnose GERD. 19 However, pH monitoring is not useful in diagnosing reflux esophagitis and, therefore, does not replace the endoscopic study. In patients with a clinical suspicion of GERD, in whom no esophagitis is detected, the indication for esophageal pH monitoring is essential and irreplaceable in order to perform the diagnosis of the clinical form of “pathological reflux without esophagitis.” 20 When the diagnosis of esophagitis is established endoscopically in patients with suggestive symptoms, pH monitoring could be considered unnecessary. 21 There was a significant percentage (around 25%) of the patients who, despite presenting esophagitis, had a normal pH in a single 24-hour period studied. 22 , 23 The result of the pH monitoring by itself also does not define the therapy to be applied, even though Jamieson et al 24 mention prolonged 24-hour pH monitoring as the most sensitive and specific examination for the diagnosis of GERD. In our study, the nine patients (15.0%) who had a normal pH showed erosive esophagitis at endoscopy, symptoms refractory to medical treatment, and a large hiatal hernia. It is essential to understand, however, that this exam makes it possible to assess the evolution of reflux with a given surgical technique even with some limitations, controlling the postoperative result at a recent or late stage. Its function is also to diagnose absence of reflux in patients who still present some symptom after the fundoplication. The goals of preoperative evaluation are to confirm the disease, find the difficulties in clinical management, relate the disease to symptoms (typical or atypical) and make sure of the functional condition of the esophagus, which will allow it to adapt to a valve. The goals of postoperative evaluation are to verify raised LES pressure in the hypotonic cases, evaluate the presence of residual relaxation at swallowing, locate the LES and prove the absence of gastroesophageal reflux. The results of esophageal manometries and 24-hour pH monitoring before and after antireflux surgery show that these methods are effective in revealing the level of functional modification established by the corrective procedure and also in helping select the surgical cases, using objective data. The findings of the present study were similar to those obtained at centers that use the same technology. The comparisons reinforce the fact that a standard profile should be sought in evaluation and preoperative control and another standard profile should be expected in postoperative follow-up. | Study | biomedical | en | 0.999995 |
10527332 | From a therapeutic point of view, the introduction of highly effective drugs for the care of peptic ulcer disease has radically changed the situation in just a few years. The discovery of anti-H2 receptors in the mid-1970s, 1 and, subsequently, of proton pump inhibitors, prokinectics and, finally, the advent of combined medical therapies capable of abolishing acidity and, above all, of eradicating Helicobacter Pylori, has meant that classical surgery was set aside in favor of medical treatment. In recent years, only the complications of peptic ulcer disease, perforation, hemorrhage and stenoses have been treated surgically, as it is obvious that the uncomplicated form of the disease can be completely dealt with by medical management. However, the advent of minimally invasive surgery has somewhat modified this situation. The following are the most commonly performed vagotomy techniques: left thoracoscopic bilateral truncal vagotomy, superselective vagotomy, Hill-Barker's anterior superselective and posterior truncal vagotomy (the technique we prefer), 2 , 3 Taylor's posterior truncal vagotomy and anterior seromyotomy 4 and Gomez-Ferrer's operation (mechanical section-suture of the anterior gastric wall). Each of these operations has proved to be easy to perform with minimally invasive access and, in various cases, with an almost complete lack of intraoperative complications. Total truncal vagotomy, be it thoracoscopic or laparoscopic, must be prescribed for patients with alvus, which tends to be associated with diarrhea, and bulbar substenosis. On the contrary, thoracoscopic access, in our opinion, is the best choice for cases if ulcer relapse or if jejunal ulcers should appear after surgery. We perform a thoracoscopic truncal vagotomy in those patients who have undergone a previous gastric drainage procedure or an incomplete abdominal vagotomy. 5 , 6 A “functional” operation like vagotomy is ideal from a practical and conceptual point of view to be used with minimally invasive thoracoscopic or laparoscopic access. It is also a valid alternative to medical therapy with a lasting and effective therapeutic effect. Recently, in some third world countries, the laparoscopic operation has been replaced by medical therapy, which is certainly more expensive but can be discontinued when the patient is symptom free. It can reasonably be said that candidates for minimally invasive surgery for duodenal ulcer are as follows: All patients, especially young patients, with duodenal ulcer (including juxtapyloric ulcers) that require regular medical therapy for a prolonged time (over several decades); All patients who have ulcer relapses due to an anomalous intake of medical therapy in relation to method and time; All patients with a low compliance to chronic medical therapy; All patients who, in spite of a correct medical therapy, have ulcer disease complicated by substenosis, bleeding, and who need high doses of drugs. A final aspect, which is certainly less important but must not be overlooked, is the economic comparison in terms of cost-benefit between protracted medical treatment and minimally invasive surgery, which is decidedly in favor of the latter. With regard to cure of duodenal ulcer, the choice between medical and surgical therapy is, therefore, extremely precise, with a net preference for medical therapy in patients with a short clinical history and prompt therapeutic response. 7 From October 1991 to October 1998, we submitted 38 patients (31 males and 7 females) with an average age of 51 years (range 22-78 years) to vagotomy with minimally invasive access: 23 Hill-Barkers, 2 Taylors, 9 thoracoscopic truncal vagotomies and 4 laparoscopic truncal vagotomies, one of which also underwent a HeineckeMiculicz's pyloroplasty plus laparoscopic cholecystectomy because of a stenotic duodenal ulcer and cholecystitis. Of the patients submitted to surgical operations using the thoracoscopic vagotomy, five had ulcers of the neostoma, three had hemorrhagic gastritis of the gastric neostoma, and one had incomplete abdominal vagotomy. Of the patients submitted to the Hill-Barker's technique, eight were resistant to medical therapy, 11 decided not to continue with long-term medical therapy, three took medical therapy on an inconsistent basis, and one with long-lasting ulcer disease requested vagotomy in association with laparoscopic cholecystectomy. In 22 patients, a bleeding complication preceded surgery. In all patients, the diagnosis was confirmed by esophagogastroduodenoscopy and biopsy with histological confirmation that the lesion was benign. Preoperative gastric acid testing with pentagastrin stimulation (6 meg/Kg) was performed. When possible, an active ulcer was managed with medical therapy before vagotomy. After surgery, if an active ulcer was present, drug therapy was continued until the ulcer had healed. Endoscopic and clinical reviews were made eight weeks after the operation, after which 15 patients underwent a gastric acid test with insulin stimulation (0.2 U/Kg) to assess the completeness of vagotomy (Hollander's test). The basal and stimulated postoperative values were compared to the preoperative test. Patients were further reviewed 6, 12 and 24 months after the operation and thereafter only if needed. These patients are still interviewed at regular intervals over the telephone. The operation is carried out under general anaesthesia with selective endotracheal intubation to obtain collapse of the left lung and aided mechanical ventilation. Before the operation, we position a nasogastric probe, which is removed 24 hours later, in order to prevent possible gastric distension and also to make it easier to identify the esophagus during the operation. We avoid positioning a urinary bladder catheter and always administer a shortterm antibiotic therapy. The patient is placed on his or her right side, as in a left thoracotomy, with right arm outstretched to maintain an infusion line. The left arm is suspended and placed cranially to raise the apex of the scapula as much as possible and to help increase the space between the ribs. The operating table is split at a level of the apex of the scapula, putting the lower half of the body in a downward sloping position, thereby increasing both the seperation of the intercostal spaces and lowering of the diaphragm. The operating surgeon is positioned on the left side of the patient together with an assistant, who operates the camera and stands to the right of the operating surgeon. The instrumentalist and instrument table are also on the left-hand side of the patient, near the patient's feet. The videolaparoscopic equipment is placed on the opposite side, in front of the operators . Once the left lung has been excluded, a 10 mm thoracic trocar is positioned in correspondence with the sixth intercostal space in the posterior axillary line, through which we insert a laparoscope with a visual angle of 30 degrees operated by the assistant's left hand (the 30 degree scope makes it possible to explore all areas of the thoracic cavity better than with a frontal optic). After exploring the thoracic cavity, again under visual endoscopic control, two 5 mm trocars are inserted in the median axillary line in correspondence with the fourth (right hand of the operator) and of the eighth intercostal space (operator's left hand). Sometimes a fourth 5 mm trocar may be needed in the anterior axillary line, fifth intercostal space, through which a retractor or irrigator-aspirator can be inserted. The irrigator-aspirator, held in the assistant's right hand, can help retract the lower left pulmonary lobe and help keep the operator's field clear of bleeding or smoke . The operation begins with a longitudinal incision of the mediastinal pleura, after sectioning, if necessary, the lower triangular ligament of the lung. The incision is in correspondence with the third distal of the esophagus in a restricted rear space from the aorta, descending frontally from the left inferior pulmonary lobe, above from the left ventricle of the heart and below from the esophageal hiatus and the left cupula of the diaphragm. The esophagus is isolated by means of blunt dissection with the aid of atraumatic forceps inserted in the trocar in the seventh intercostal space (operator's left hand) and with an ultra-sound monopolar hamulus or with a bipolar forceps inserted in the trocar in the fifth intercostal space (operator's right hand). The anterior or left vagus rami are easily identified in the periesophageal tissue, appearing as a whitish trunk the nerve is isolated and loaded on the hamulus; it is coagulated and sectioned above and below, removing a section of about 1 cm, which is sent for a histological analysis . There are two ways to isolate the posterior or right vagus nerve: either by lifting the esophagus and shifting it to the right after it has been secured in the operator's forceps, and dissecting in this space with the retractor operated with the right hand of the assistant, or by lowering the esophagus and shifting it toward the left in order to gain access to its postero-lateral surface . Once the truncus vagalis posterior has been isolated, we can proceed with its coagulation and sectioning in the same manner as with the left. A search is also made for possible accessory or anomalous distribution rami that need to be sectioned. After securing hemostasis in the dissected space, a 5 mm drain is positioned in the pleural cavity through the trocar in the eighth intercostal space, which is made waterproof while proceeding with the suturing of the other cutaneous incisions. The anaesthetist reexpands the collapsed lung. After making sure there is no air leak, the drain is removed and the last site is sutured. The operation terminates with infiltration of the intercostal spaces with a long-lasting anaesthetic such as bupivacaine. In our experience, the average duration of the surgery is 30 minutes, and the patient can be discharged on day two since there is no parietal trauma. The average duration of the operation by thoracoscopy was 30 minutes (range 20-40 minutes) with return to normal feeding in 1 day, and return home on day 3 (range 2-5 days). The patients were followed for 3-54 months. Twenty-two patients (91.3%) out of the 23 submitted to anterior superselective and posterior truncal vagotomy, and the patients submitted to thoracoscopic vagotomy, were pain and ulcer free without medical therapy. One patient (4.3%) was lost to follow-up. One ulcer recurred seven months after the operation. This was the first patient upon whom the operation was performed and whose acid test showed an incomplete vagotomy. After the recurrence, a thoracoscopic truncal vagotomy was performed with good results, as the patient was symptom free four months after the operation and “Hollander negative.” We had no mortality and no intraoperative or postoperative complications. In the thoracoscopic series, we had one patient who had epigastric fullness and impaired gastric emptying three months after the operation. Satisfaction during follow-up is 100% Visik HI. On the basis of what has been published, we can say that truncal vagotomies are associated with a need for pyloric dilation in 10%, 5% result in left phrenic paralysis and 5% have chronic diarrhea. Superselectiye vagotomies are associated with a pathological gastroesophageal reflux in 12%, 4% experience gastroparesis and 4% have an ulcer relapse. Posterior truncal vagotomy and seromyotomy according to Taylor is associated with 3% diarrhea, 3% bezoar, 1% reflux disease and 4% relapse. 8 – 12 Another complication with vagotomy is the sensation of post-prandial gastric fullness, which at times forces patients to eat small, frequent meals, as is the case with those who have undergone gastric resection; this complication was observed in 47% of the patients after truncal vagotomy and in 30% of the patients after proximal selective vagotomy. 11 Truncal vagotomy also causes a decided increase in gallbladder volume with atonicity, which is unlike the other types of vagotomies in which the vagal hepato-biliar rami are preserved. This stasis leads to an increase in the incidence of cholelithiasis, which is estimated to be between 18 and 23%. 13 , 14 Finally, the interruption of the celiac vagal ramus, causes loss of muscular tone in the right colon and in the glandular intestine, with paretic dilation and a slowdown of the ileac transit, is the fundamental cause of diarrhea. 15 Just as left thoracoscopic vagotomy represents the most effective treatment for ulcers of the neostoma in patients who have undergone gastric resection (for example, hemorrhagic gastritis of the gastric neostoma), so does Hill-Barker's superselective anterior and truncal posterior vagotomy performed with laparoscopic access represent, in our opinion, the best choice in other cases of ulcer diseases. 3 The advent of minimally invasive surgery has made it possible not only to surgically correct peptic disease without major operative trauma and release patients from the need for chronic medical therapy, but also to obtain functional results that are similar to traditional surgical results. In just a few weeks, medical therapy cures over 95% of the patients; however, when the medications are suspended, the rate of relapse varies from 10% after one months' therapy to 65% after 18 months' therapy. Vagotomy performed thoracoscopically is simple, quick, and effective, and the magnification of an excellent laparoscope (even better if equipped with digital image processes capable of increasing the contrasts between the various structures) provides a definite visualization and section of the vagal fibers. | Review | biomedical | en | 0.999998 |
10527333 | The isolated finding of a retroperitoneal mass (RM) often represents a diagnostic challenge. Even when possible, an image-guided biopsy frequently provides an inadequate specimen for diagnostic purposes. Open retroperitoneal exploration is often the only option capable of obtaining sufficient tissue for diagnosis; however, this necessitates a major operation. With increasing experience in laparoscopic retroperitoneal surgery, 1 , 2 the use of laparoscopy for exploration of an indeterminate retroperitoneal mass may provide a minimally invasive alternative to open exploration. Herein, we report our experience with four laparoscopic explorations for RM and compare our results with four contemporary open explorations for RM. From July 1995 to January 1998, four consecutive patients, aged 50 to 62 years old, with computed tomo-graphic findings of a retroperitoneal mass underwent laparoscopic exploration by one surgeon (RVC). Another four consecutive patients underwent open exploration by other surgeons at the same hospital. The medical records of these patients were reviewed. Preoperative evaluation included computed tomography of the abdomen and chest radiography. In each case, the only finding was a retroperitoneal mass . In the laparoscopic group, all patients had either preoperative biopsy of the mass or a biopsy of an enlarged peripheral lymph node. For laparoscopic exploration, all patients underwent placement of a ureteral stent and Foley catheter. The patient was then turned from a supine to a full lateral position. A pneumoperitoneum was created with a Veress needle inserted 3 cm above and medial to the anterior superior iliac spine; a 12 mm port was placed. Additional 12 mm ports were placed in the mid-clavicular line subcostally and just above and lateral to the umbilicus. The colon was mobilized medially by incising the line of Toldt. Another 5 mm port was placed in the posterior axillary line subcostally for placement of a 5 mm retractor. The colonic mesentery was further separated from Gerota's fascia; the mass was identified and either an incisional or excisional biopsy was done. In patients undergoing open exploration, all lesions were approached transperitoneally by a midline incision. The colon was mobilized medially. The mass was excised in three patients and biopsied in one patient. Total surgery time included the time for stent placement and the laparoscopic surgery. Blood loss was assessed by the anesthetist's estimation and by comparing preoperative and postoperative hematocrit. Also, we recorded the complications, time to ambulation, time for resumption of a regular diet, analgesic use, the hospital stay and the hospital charges. A definitive diagnosis was obtained for all patients after the exploration either by incisional or excisional biopsy . The age, sex, past medical history and preoperative investigation results are summarized in Table 1 . Preoperative biopsy was performed in six patients; only two findings correlated with the final pathologic report. Of note, the tumors were smaller in the laparoscopic group (3 of 4 < 5 cm) while two of the four lesions in the open group were 10 cm ( Table 1 ) . Postoperative complications were observed in one of the laparoscopic explorations and in three of the open explorations. There was no operative mortality ( Table 2 ) . The only complication in the laparoscopic group was a major complication: proximal, small bowel obstruction due to incarceration of bowel into a 12 mm port site. This occurred despite closing the fascia of the 12 mm incisions with a single 1-0 absorbable suture. The patient underwent laparoscopic reduction and repair of the hernia on postoperative day 4. In the open group, there was one major complication (urine extravasation), as well as two minor complications (a urinary tract infection and a subcutaneous hematoma). The blood loss (90 vs 440 ml), hematocrit drop (5.1 vs 7.8 %), time to resumption of regular diet (3 vs 5 days), amount of morphine sulfate equivalents required (128 mg vs 161 mg), time to ambulation (2.3 vs 6 days) and hospital stay (4.8 vs 6 days) were each less in the laparoscopy patients ( Table 3 ) . The operation time was longer for the laparoscopic procedure (7.8 vs 4.3 hours); the laparoscopic time included the time to place the ureteral stent and to reposition the patient. Due to the prolonged operation time, the laparoscopic procedure was about $5000 more costly than the open approach ( Table 3 ) . Retroperitoneal tumors may either arise from solid organs (eg, kidney, pancreas and adrenal) or from non-specific tissues that traverse the retroperitoneal space (eg, lymphatic tissue, muscle, nerve, fat and connective tissue). These lesions may be benign, malignant or inflammatory in nature ( Table 4 ) . Computed tomography (CT) and magnetic resonance imaging (MRI) can provide information on the location, anatomy and extent of the mass, but are otherwise largely nondiagnostic. 3 , 4 Indeed, in all instances, the determination of appropriate therapy depends upon obtaining an adequate tissue sample for histologic diagnosis. In this respect, image-guided percutaneous biopsy can be used, 5 but it suffers from a low diagnostic yield due to the small amount of tissue obtained and because an inflammatory infiltrate may have an appearance similar to a malignancy. Indeed, preoperative image-guided biopsies were either incorrect or inadequate in four of our six cases. Accordingly, surgical exploration with adequate tissue sampling is frequently necessary to establish a definitive diagnosis. 6 For some malignant and benign tumors of the retroperitoneum, an excisional biopsy may be both diagnostic and curative. 3 , 7 , 8 Laparoscopic exploration potentially can provide a minimally invasive means to obtain adequate tissue for histologic diagnosis without the need for a major midline abdominal or flank incision. All of our patients who underwent laparoscopic exploration tolerated the procedure well and were able to ambulate and resume a full diet within five days. The postoperative pain was minimal, and the hospital stay was brief (average 4.8 days). In the laparoscopic cases, two patients had an excisional biopsy, and two patients had an incisional biopsy. In all four cases, a definitive diagnosis was made, and no further surgical intervention was necessary. In comparison with open exploration, the laparoscopic approach was equally as effective, yielding a definitive diagnosis in all four cases. However, due to longer operative time, the laparoscopic procedure was more costly and, hence, less efficient than the open approach. With regard to morbidity, patient recovery and hospital stay, laparoscopic exploration was more favorable. In summary, we believe that laparoscopic exploration for a retroperitoneal mass of undetermined origin is a viable alternative to open exploration. The laparoscopic approach is as effective, albeit less efficient, than an open procedure; however, the laparoscopic approach provided benefits with regard to patient morbidity and convalescence. As urologic surgeons become more experienced with laparoscopic techniques and with the advent of more efficient nondisposable instrumentation, we anticipate that the operative time and cost for more complex laparoscopic procedures, such as retroperitoneal exploration, will decrease. Nonetheless, our initial experience with laparoscopic retroperitoneal exploration is favorable, and we are now offering this approach as first-line therapy in these patients. | Other | biomedical | en | 0.999997 |
10527334 | Abdominal pain is one of the most common complaints in children. 1 In most cases, the pain resolves with conservative treatment. 2 , 3 Some children continue to have abdominal pain, and despite all investigations, no cause for the pain is found. These children are classified as having the syndrome of chronic recurrent abdominal pain. 1 , 3 – 5 After extensive investigations fail to clarify the etiology of the pain, these children are often referred to the surgeon, in hope that a surgical intervention will alleviate the symptoms. Previously, laparotomy and appendectomy have been recommended. 1 , 2 The exploration was usually done through a right lower quadrant McBurney incision; this approach provided limited exposure of the peritoneal cavity. 2 , 4 With the advent of new minimally invasive surgical techniques, diagnostic laparoscopy has become a very important tool in the management of children with chronic recurrent abdominal pain. 3 , 6 , 7 The purpose of the present study was to evaluate our experience with diagnostic laparoscopy in the management of 13 children with chronic recurrent abdominal pain. From 1994 to 1997, 13 children with chronic recurrent abdominal pain were subjected to diagnostic laparoscopy. Ages varied from 10 to 17 years (mean, 13 years). There were six males and seven females. The duration of abdominal pain varied from 3 weeks to 12 months (mean, 2 months). In all children, this pain was debilitating and severe enough to warrant repeated visits to the pediatrician, emergency room visits, or hospital admission, as well as absence from school. Diagnostic investigations included abdominal ultrasound in 11 patients, computerized tomography (CT) of the abdomen in seven, upper gastrointestinal series in five, small bowel follow-through in three, contrast enema in two, Meckel's scan in one, upper gastrointestinal endoscopy in six, and colonoscopy in three. In all patients, extensive hematologic studies were done. None of these investigations provided data that contributed to the diagnosis. Services consulted included the gastrointestinal service in 11 patients, gynecology in three, psychiatry in three and neurology in one. Table 1 summarizes patient characteristics, laparoscopic/histological findings, and outcomes. Laparoscopic findings included cecal adhesions in five patients, inflammatory bowel disease in two, large mesenteric lymph nodes in two, kink in the appendix in two, Fallopian tube cysts in two, torsion of an ovarian cyst in one and salpingitis in one. Laparoscopic appendectomy was done in all patients. Histological examination of the appendix demonstrated acute appendicitis in two patients, periappendicitis in two, congested appendix in two, and eosinophilic infiltrates in one. Histologically normal appendix was reported in six patients. An appendicalith was found in three patients. There were no reports of chronic appendicitis. All patients recovered uneventfully. There were no operative complications. One patient presented three months following laparoscopy with incomplete small bowel obstruction, which resolved with conservative management. There were no other postoperative complications. The abdominal pain completely resolved in ten patients following laparoscopy. Three patients returned with abdominal pain. A patient presented eight months later with right upper quadrant abdominal pain. She was found to have gallbladder dyskinesia. 8 Her pain resolved following laparoscopic cholecystectomy. She was well for three months, but then her pain recurred in the epigastric area and right upper quadrant. Endoscopic retrograde cholangiopancreatography demonstrated high biliary pressure and partial pancreas divisum. She was treated with sphincterotomy. Although she had significant improvement, her pain has not yet completely resolved. A second patient presented six months later with left-sided abdominal pain. A second laparoscopy revealed intestinal and splenic adhesions. Her pain resolved following laparoscopic lysis of adhesions. A third patient presented six months following laparoscopy with lower abdominal pain and fever, secondary to pelvic inflammatory disease. Her symptoms resolved following antibiotic treatment. The two patients with inflammatory bowel disease improved with medical management. The patient with salpingitis responded to antibiotic therapy. Preoperative hospital stay varied from 1 day to 16 days (median, 4 days). Postoperative hospitalization ranged from 2 to 5 days (median, 3 days). Except for the patient with biliary dyskinesia, all other patients are now symptom free. Chronic recurrent abdominal pain is a common problem in children and adolescents. The cause of the pain is seldom found by clinical, laboratory, and imaging studies. It has been estimated that no organic cause for the pain is found in over 90% of children. 1 , 9 , 10 The best approach in children with chronic recurrent abdominal pain must include a careful and detailed clinical history and physical examination, as well as judiciously applied laboratory and imaging studies. 1 , 5 Abdominal ultrasound is usually the first investigation ordered. This test was done in 11 patients in the present study. It did not reveal any abnormalities, even in a 14-year-old girl with intermittent torsion of a large ovarian cyst. Generally, ultrasound has been recommended as one of the initial examinations, mainly because it is noninvasive and can exclude serious abdominal pathology. In a recent study in 57 children with recurrent abdominal pain, 5 the findings of abdominal ultrasound were normal in 56 (98%), three of whom subsequently had appendectomy. The lack of positive findings by sonography has been confirmed by other investigators. 3 , 5 , 6 The role of ultrasound continues to be that of reassurance to parents and treating physicians, as it is useful in excluding important pathology amenable to ultrasound detection. CT of the abdomen was done in seven patients in the present study. It also did not reveal any abnormalities. The role of this more invasive and expensive investigation in the management of children with chronic recurrent abdominal pain remains to be evaluated, since it contributes very little to diagnostic efforts in the patient with normal findings on abdominal ultrasound. We feel that upper gastrointestinal endoscopy is important when ulcer disease or Helicobacter pylori is suspected as the cause of abdominal pain. Colonoscopy was done in three patients with suspected inflammatory bowel disease. However, it did not contribute to the diagnosis. In the past, laparotomy through a limited right lower quadrant McBurney incision and appendectomy have been recommended. 2 , 4 , 11 This approach has provided good results for some investigators, with resolution of symptoms in the majority of patients, despite the fact that the appendix was histologically normal in most patients cured by appendectomy. 1 , 3 , 4 , 11 This finding has led some authors to postulate a placebo effect of appendectomy. 4 In 10 of 13 cases in the present series, the pain completely resolved following laparoscopy and appendectomy. Two patients returned with pain and required further treatment. Six patients had a histologically normal appendix, of whom only one patient had no other significant findings, except for large mesenteric lymph nodes; another patient had inflammatory bowel disease, and the remaining four had fecaliths and inspissated fecal material in the lumen of the appendix. The presence of inspissated fecal material in the appendiceal lumen has been associated with chronic abdominal pain in children. 2 Cecal adhesions were observed in five patients. This finding could represent previous cecal or appendiceal inflammation. Interestingly, of the two patients with histologically confirmed acute appendicitis, one had a 3-month history of abdominal pain, and the other a 3-week history. Of the three patients who returned with abdominal pain, one had biliary dyskinesia 8 and, although she has significantly improved following cholecystectomy and sphincterotomy, her pain has not completely resolved. The second patient returned with abdominal pain secondary to adhesions, and her pain resolved following laparoscopic lysis of adhesions. The symptoms in a third patient who returned six months later with lower abdominal pain and fever secondary to pelvic inflammatory disease, resolved with antibiotic therapy. Overall, laparoscopic findings identifying factors associated with abdominal pain were observed in 12 of 13 patients in the present study. In a recent report, positive findings were encountered by laparoscopy in 73% of children with chronic recurrent abdominal pain. 3 The laparoscopic findings and results in our small group of patients validate the conclusions of other investigators that chronic recurrent abdominal pain has an organic basis in a significant number of patients. 3 With the advent of modern techniques and equipment, diagnostic laparoscopy has become an important tool in the management of children with chronic recurrent abdominal pain. When laparoscopy is undertaken, it should be performed methodically and thoroughly. Careful examination of the whole peritoneal cavity must be done, including running of the small bowel and examination of the Fallopian tubes, uterus and ovaries. When a significant finding such as acute appendicitis or salpingitis is encountered, the procedure can be terminated after appropriate action has been taken. Since the safety of laparoscopic appendectomy has been well established, 12 laparoscopic appendectomy should be done when no other significant cause of abdominal pain has been identified, even if the appendix looks normal. A careful and detailed clinical history and physical examination remain the most valuable tools in the management of children with chronic recurrent abdominal pain. The judicious use of imaging studies and laboratory investigations can contribute to eliminating some serious causes of the pain. As we gain more experience and confidence with diagnostic laparoscopy, this procedure is becoming a powerful tool in the management of these children. The early use of diagnostic laparoscopy may avoid unnecessary and expensive tests as well as prolonged hospitalization. It can also alleviate the uncertainty and anxiety in families and patients alike. Based on our experience and results, we advocate the early use of diagnostic laparoscopy in children with chronic recurrent abdominal pain after reasonable investigations have not helped to establish an accurate diagnosis, and conservative management has failed to alleviate this pain. | Other | biomedical | en | 0.999997 |
10527335 | Previous reports indicate that the number of patients undergoing cholecystectomy has increased since the introduction of laparoscopic cholecystectomy (LC). 1 – 3 Few studies have specifically looked at the incidence of chronic acalculous biliary disease, preoperative evaluation, or the patient demographics in these cases. The purpose of this study was to evaluate a relatively geographically isolated population of patients undergoing elective cholecystectomy without cholelithiasis over the period of time in which open cholecystectomy (OC) was supplanted by the laparoscopic approach with respect to changing preoperative evaluation, time of original symptom development to operation, and postoperative course and symptomatology. Some comparisons to patients with calculous biliary disease also are identified and discussed. This retrospective study consisted of all patients who underwent cholecystectomy for chronic acalculous cholecystitis in Saginaw, Michigan between June 1, 1985 and June 30, 1995. The latter date was selected to allow for at least 18 months of postoperative follow-up. The first LC was performed on June 14, 1990. Patients were identified by reviewing all charts assigned ICD-9 codes for cholecystectomy (51.22, 51.23) and those who had associated 575.X codes for disease not associated with cholelithiasis (defined as 574.X). Of 719 patients with acalculous cholecystitis, 143 were excluded due to acute acalculous disease. Of the remaining 576 patients , 180 patients were excluded in the LC era and 95 from the OC era due to 1) calculus disease identified preoperatively and not confirmed intraoperatively or at pathologic review, and 2) acalculous disease preoperatively found to have calculi at operation or on pathologic analysis. The remaining 301 patients were reviewed for presenting symptomatology, patient characteristics, preoperative diagnostics, as well as postoperative symptomatology, evaluation, diagnoses, and operations performed after cholecystectomy. All subsequent hospital admissions and emergency room visits were reviewed through May 1997. Office records were available for review for 158 cases, 25 from the OC era and 133 from the LC era. Statistical analysis was performed using the SPSS statistical program utilizing Fischer's exact test, Chi-square, and paired t-test as appropriate. All means were expressed with the standard deviation and significance was considered when p<0.05. During the decade from June 1985 to June 1995, 7181 cholecystectomies were performed. Of this total, 6462 patients had cholelithiasis; the remainder had diagnoses of acute or chronic acalculous cholecystitis or biliary dyskinesia. During the study period, 211 of 3247 (4.2%) cholecystectomies were performed for chronic acalculous cholecystitis, an increase from 90 of 3934 (2.3%) prior to the introduction of laparoscopy (p<0.05). Of the 301 patients with chronic acalculous cholecystitis, 211 (70%) underwent cholecystectomy since the advent of laparoscopy. All patients presented with abdominal pain; other symptoms included episodic vomiting, bloating, frequent eructation, fatty food intolerance, diarrhea, and dyspepsia. The mean age of patients with acalculous disease was 42.2+/−11.6 in the OC era and 46.5+/−14.5 in the LC era (range 14-82 years, p<0.05). In both eras, the mean age was significantly younger than those with stones . The vast majority of all patients undergoing cholecystectomy for all diagnoses were white women, 72.1% in patients with acalculous disease—a statistically significant increase from 65.1% in patients with cholelithiasis (p<0.05). Similarly, there was a statistically significant increase in the percentage of white women with chronic acalculous disease from 64.7% in the OC era to 75.7% in the LC era (p<0.05). Patients in the LC and OC eras did not differ in preoperative body mass index, number of medications, or number of positive laboratory tests. Non-steroidal anti-inflammatory medications (NSAID) and omeprazole were used more commonly in patients during the LC era; whereas, antacids were more common in the OC era (p<0.05). There were no other significant differences in the other 13 medications studied between the groups of patients. Preoperative diagnostic evaluation included ultrasound (US), upper gastrointestinal series (UGI), biliary scintigraphy (HIDA), cholecystokinin-stimulated HIDA (CCKHIDA), computed tomography (CT), oral cholecystography, (OCG), barium enema (BE), endoscopic retrograde cholangiopancreatography (ERCP), esophagogastroduodenoscopy (EGD), colonoscopy, intravenous pyelogram (IVP), and plain radiographs (XRAY). The number of tests performed significantly decreased from 4.7+/−2.4 in the first half of the decade to 3.2+/−1.8 in the LC era . Similarly, individual tests decreased except hepatobiliary scintigraphy, which increased due to the usage of CCK-HIDA, and US, the most common test performed, at a frequency of 94% remained unchanged through the decade . The average CCK-HIDA ejection fraction was 17.3%+/−12.1% (range 0-81%, normal considered >35%). Of patients having a CCK-HIDA, 44.9% had reproduction of symptoms. Positive US findings occurred in 27.9% of patients, predominantly sludge (8.6%), thickened gallbladder wall (8.3%), and polyps (10.3%), without statistical variance throughout the study period. On microscopic reports, chronic and subacute pathology was diagnosed more commonly in the LC era (p<0.05). Normal pathology did not change statistically between the time periods and was 23% . Sixty-four patients (22%) continued to have postoperative symptoms, and this did not significantly change during the decade of study. Twenty-two (7.3%) patients had additional diagnoses within one year. Of these, 8 patients (2.7%) underwent another abdominal operation within one year for continued symptoms. No statistically significant difference existed between OC and LC groups in the incidence of subsequent operations performed within one year of cholecystectomy. Symptomatology was attributed to a diverse array of nonbiliary diagnoses: peptic ulcer disease (n=1), myofascial syndrome (n=2), gastroesophageal reflux (n=3), cardiac disease with atypical angina (n=1), chronic pancreatitis including pancreas divisum (n=1), pancreatic cancer (n=1), and Meckle's diverticulum (n=1). In this series of patients from a relatively isolated geographic community, the prevalence of cholecystectomy for chronic acalculous cholecystitis almost doubled during the decade studied—an increase sustained and statistically significant since the introduction of laparoscopy. Patients who underwent LC for chronic acalculous disease had on average three preoperative tests prior to cholecystectomy as compared to nearly five per patient in the OC era. In the vast majority of the laparoscopy cases, these tests included an ultrasound for gallbladder structure and the CCK-HIDA for gallbladder function. Only one additional test was performed not specific for biliary pathology prior to operation. The CCK-HIDA scan has been available in Saginaw since May 1991, while hepatobiliary scintigraphy has been a diagnostic option since the mid 1970s. The decreasing numbers of tests performed since the introduction of laparoscopy may, therefore, be due to the increased use of the CCKHIDA scan. The CCK-HIDA was the first specific test for chronic acalculous disease, and its increase coupled with decreased utilization of other preoperative evaluations may indicate increasing physician awareness of the disease and the appropriateness of the CCK-HIDA as a predictor of postoperative success after cholecystectomy. In a number of studies, 75-90% of patients with a clinical diagnosis of chronic acalculous cholecystitis have been reported to be rendered pain free by cholecystectomy. 4 – 9 Some series report a 70-75% cure rate in patients based on symptomatology only; whereas, other reviews reported 80-90% resolution using CCK-HIDA as preoperative predictor of success. 6 – 13 In this study, 22% of patients had symptoms postoperatively, including 2.7% having an additional operation within one year of cholecystectomy regardless of the era. Patient demographics indicate that this is an operation of white females, more so since the advent of laparoscopy. Although many believe there is a preponderance of females with biliary disease, an etiologic factor has not been identified that explains this overwhelming majority of patients with acalculous disease. The concurrent racial demographics during this study are not precisely known; however, there was a statistically significant increase in the number of white females with chronic acalculous cholecystitis compared with patients with cholelithiasis. This may reflect an unknown bias in this review and may deal with access to health care, insurance status, or referral patterns from more outlying rural areas more likely to consist of Caucasians. In both eras combined, 23% of patients had normal findings on pathologic review. Other investigators demonstrated no relation between decreased gallbladder ejection fraction and the findings of chronic cholecystitis on pathology. 4 Symptoms occurring with the CCK-stimulated hepatobiliary imaging studies also are not specific for cholecystitis. Patients with irritable bowel syndrome may also have reproduction of pain with CCK stimulation. 14 Others have suggested that chronic acalculous biliary disease may be associated with irritable bowel syndrome as a global dysmotility syndrome. 15 In one series, injection of CCK increased colonic motor activity in all 20 patients tested with irritable bowel syndrome. This was especially severe in 40% of patients who complained of abdominal pain after food. Half of these patients developed reproduction of symptoms with the CCK. 14 Cautious interpretation of patient reproduction of symptoms with CCK administration appears to be warranted. The change from open to laparoscopic cholecystectomy was accompanied by more white females undergoing operation for acalculous biliary disease. The number of preoperative diagnostic evaluations has decreased since the advent of laparoscopy. Over 90% of these patients now have an ultrasound, a CCK-stimulated HIDA, and one additional evaluation prior to cholecystectomy. The true incidence of persistent symptoms may be underestimated in this study because of incomplete long-term follow-up in only half of the patients in this series. | Study | biomedical | en | 0.999997 |
10527336 | The diagnosis of occult gastrointestinal bleeding of obscure origin is one of the most vexing problems confronting physicians. 1 Despite impressive technological advances during the last 30 years, the management of bleeding from the lower gastrointestinal tract remains a diagnostic and therapeutic challenge. Although a generally accepted treatment protocol for upper gastrointestinal tract hemorrhage has been established, there is no similar consensus for the management of hemorrhage originating distal to the Treitz ligament. 2 A 29-year-old white male was seen in the emergency room of the American-British Hospital of Mexico City, referred from a rural clinic in one of the neighbor states of Mexico City with a 4-day history of severe gastrointestinal bleeding characterized at first by melena and then hematochezia. Six units of packed red blood cells were transfused to the patient before his admission to the emergency room. Past history was unrewarding except for a laparoscopic fundoplication nine months before for severe gastroesophageal reflux disease. Clinical examination revealed generalized pallor and red blood on rectal digital exam. Blood pressure was 100/60 mm Hg with a pulse of 90 beats per minute. A complete blood count demonstrated a hemoglobin of 8.6 mg/1. An emergency esophagogastroduodenoscopy was negative for bleeding and showed a properly functioning fundoplication; a colonoscopy performed two hours later after bowel preparation showed fresh blood in the large bowel coming from the ileum. The next morning the patient was taken to the operating room, and, under general anesthesia, a 10 mm trocar was placed in the umbilicus. The abdominal cavity was inspected and immediately a 6 × 5 cm ileal cystic dark mass was identified in the pelvic area . Another 10 mm trocar was placed in the right flank and a 5 mm one in the left flank. The whole small bowel was inspected from the ligament of Treitz to the ileocecal valve and the tumor was located at 50 cm proximal to the latter. The umbilical incision was extended 3 cm downwards, the wound protected, the tumor exteriorized , and a 20 cm ileal resection was performed, followed by a termino-terminal stapler anastomosis. The postoperative course was uneventful: a liquid diet was tolerated the following day, a normal bowel movement occurred on postoperative day 2, and the patient was discharged on postoperative day 4 and remains asymptomatic 18 months later. The specimen was a segment of small intestine which measured 17 × 2.5 cm. On its outer surface, a neoplasm was identified pending from the antimesenteric border. It had a reddish-brown hue, bosselated surface with areas of recent hemorrhage and measured 6 × 5.5 × 4 cm. On sectioning, it had a firm consistency, and the surface was trabeculated, gray-pink and with focal hemorrhage at the periphery. The base of implantation was 3 cm wide, and part of the neoplasia protruded through the bowel wall, creating a dumbbell shape . This was associated with an extensive ulcer overlying the mucosa. Microscopically, the tumor was hypercellular and made up of bundles of fusiform cells with bland nuclei, homogeneous chromatin pattern, occasional small, inconspicuous and usually single nucleolus. The cytoplasm was eosinophilic and homogeneous with unapparent cell borders. There were 2 mitotic figures in a 150 hpf count, no necrotic foci, no pleomorphic cells nor “skeinoid” fibers, and only scattered hemorrhagic areas . The immunohistochemical profile showed focal positivity for ps-100, CD34 (QBENd/10); actin antigen (PCNA) was strongly positive in 4.1% of neoplastic cells. DNA analysis (CAS-200) showed a hypodiploid population (DNA index: 0.4, s-phase: 3.3). Low risk small bowel stromal tumor (GIST), based on size (6 × 5.5 cm), no necrosis, 2 mitotic figures in hpfs and PCNA index lower than 10%. Etiology of acute lower gastrointestinal bleeding is closely associated with the patient's age. In adolescents and young adults, Meckel's diverticulum, inflammatory bowel disease and polyps account for the majority of cases. In adult patients up to 60 years of age, diverticulosis, inflammatory bowel disease, and polyps are the most common cause, whereas in patients older than 60 years angiodysplasia, diverticulosis and cancer are the most common etiologies. Most acute lower gastrointestinal bleeding episodes arise from the colon; however, 15% to 20% originate in the small intestine. 2 , 3 It must be mentioned that the yield of small bowel series performed to evaluate patients with obscure gastrointestinal bleeding is 2% to 3% for small bowel tumors; use of enteroclysis, or double-contrast small bowel series, may increase the yield 6% to 10%. “Push enteroscopy” should be the next step in the evaluation of all cases of gastrointestinal bleeding of obscure origin because diagnostic yield has been reported to be that of 5% to 20% for small bowel masses. Intraoperative enteroscopy increases diagnostic yield by 20% to 30%. 1 Other tests such as CT scan and endoscopic ultrasound have occasionally showed good results. 3 In this case, the colonoscopy was of great importance because it gave us the information that the bleeding site was located within the limits of the jejunum and the ileum. Following the endoscopic procedures, it was decided that the patient should be explored in the operative room given the negligible morbidity of a diagnostic laparoscopy. The rationale was that arteriography has a relatively high complication rate (up to 9%) that includes arterial thrombosis, embolization, and renal failure and success rates vary widely (14% to 72%); that radionuclide scanning is time consuming, and several reports in the literature are not encouraging 3 ; that the bleeding site was located somewhere between the proximal jejunum and the ileum and given the patient's age the likelihood of an etiologic factor that could be readily identified by laparoscopy. Provisions were made to perform an intraoperative enteroscopy if the need was present. Primary benign and malignant as well as metastatic neoplasms of the small bowel are rare entities, comprising less than 5% of all gastrointestinal tumors and 0.35% of all malignancies. 1 Gastrointestinal stromal tumors (GISTs) have been the subject of considerable debate since their earliest descriptions, with controversy centering on the cell of origin, line of cellular differentiation and the prediction of clinical behavior. 4 – 6 It is hard to estimate the exact incidence of GIST from previous reports in the literature. 5 Both benign and malignant stromal tumors may occur at any age and either sex. 7 Whereas most of them are asymptomatic, the most common presenting symptom is gastrointestinal bleeding; other manifestations are obstruction, intussusception or, rarely, perforation. 1 , 5 , 7 Despite advances in technology, preoperative diagnosis of GIST - specially of those in the small bowel - is often difficult and usually requires laparotomy. 8 Most of these tumors are located in the stomach (47%), small bowel and rectum (11%), colon (7%), duodenum (5%) and esophagus (5%). 9 Both benign and malignant tumors can share a similar appearance, even when the latter tend to be bigger than the former. From a microscopic standpoint, these neoplasms have a wide variation regarding growth patterns. There is not, however, a single immunohistochemical profile that allows one to predict the neoplasm's biological behavior. Generally accepted malignancy criteria include a diameter of more than 5 cm, invasion to nearby structures, necrosis, hypercellularity, increased nucleus/cytoplasm ratio, mitotic rate greater than 1-5 per 10 hpf, and infiltration of the overlying mucosa. 5 Sometimes, the term “uncertain biological conduct” should be adopted. In these cases, evaluation of proliferative activity by immunohistochemical methods appears to be promising, though inconstant. 4 , 10 – 12 The greatest value of tests using proliferative markers is the possibility to re-classify with more precision tumors that were considered as “borderline” or of “unknown biological conduct” into high or low-risk groups. Most common sites of metastasis are the liver and the peritoneum. 13 , 14 Lymph nodes are affected in 7%-10% of cases, and rarely are extra-abdominal structures invaded: lung, subcutaneous tissue, brain, thyroid, bone, kidney, adrenals, heart, larynx, skin and spermatic cord. 5 As GISTs are usually radio-resistant and insensitive to chemotherapeutic agents, the only curative therapy is surgical excision. 8 It is likely that this tumor was present during the previous laparoscopic surgery, but a careful review of the video a posteriori did not reveal any abnormality during the abdominal inspection (although an examination of the small bowel was not done), and the patient did not have any evidence of anemia and/or any other gastrointestinal symptoms. Laparoscopic surgery for small bowel obstruction, perforated diverticulitis, and for benign and malignant tumors of the small and large intestine has produced excellent results in experienced hands, although in some cases may require conversion to laparotomy and/or an assisted procedure. 15 – 18 In the case herein presented, the tumor was localized immediately with the aid of a simple diagnostic laparoscopy, which allowed a successful assisted resection. New applications of minimally invasive surgery are continuously being described. It is recommended that in some selected, well-evaluated patients with lower gastrointestinal bleeding of unknown origin and stable vital signs, laparoscopy may provide a precise diagnosis and adequate treatment within the same procedure, avoiding the need for time-consuming studies with substantial morbidity and expense. | Review | biomedical | en | 0.999997 |
10527337 | Endometriosis associated with massive ascites is sufficiently rare with less than 20 cases being reported since 1954. l Most cases of hemorrhagic ascites are found in patients with underlying malignancies, such as hepatoma or ovarian carcinoma, in tuberculosis, or with a perforated duodenal ulcer. Endometriosis, however, is an exceptionally rare cause. 2 A 43-year-old Hispanic female Gravida 3 Para 3 was transferred from an outlying hospital with complaints of acute onset abdominal pain located primarily in the right lower quadrant. The patient described the pain as being sharp in intensity. It was not associated with any physical activity. Bowel movements were normal, and she denied nausea or vomiting. Her only previous complaint had been generalized abdominal bloating since 1990. She was admitted through the emergency room at the outlying hospital where she underwent extensive evaluation. Laboratory investigations revealed a normal initial complete blood count (CBC) with an increase in white blood cell count (WBC) to 15 900 thousand, containing 91% polymorphonuculear neutrophiles, 6% lymphocytes, 2% bands, and 1% monocytes, on hospital day two. The patient was transferred on this date and taken to surgery within two hours of admission. The preoperative white count was down to 11 800 with a similar differential. Urinalysis with culture and sensitivity was negative for infection. Chem 7 and liver function tests were normal. BHCG was negative. Chest X-ray revealed right lower lobe subsegmental atelectasis. Abdominal flat plate revealed nonspecific gas pattern. Pelvic ultrasound demonstrated a complex mass in the cul-de-sac with septations extending into the right lower quadrant of the abdomen as well as a significant amount of intraperitoneal fluid in the pelvis and upper abdomen. The patient was hospitalized for two days. Despite Demerol injections every three hours, her pain did not subside. She was placed empirically on Cefotan 1 gm every 12 hours 24 hours prior to transfer to Woman's Hospital. In general, the patient's clinical picture was worsening with no evidence of specific etiology for her abdominal pain. Consultations were obtained, and she was transferred to a tertiary care hospital for further care. Her medical history was significant in that she had been diagnosed with Stage IV endometriosis and extensive pelvic adhesions. She had undergone a laparoscopic procedure in 1990 for vaporization of endometriosis and a left salpingo-oophorectomy. On her admission to the hospital, she had a markedly distended, tender abdomen. Bowel sounds were hypoactive. Diffuse rebound tenderness and a fluid wave were present. She was fairly anxious. Pelvic examination was difficult to access uterus and adnexa secondary to distension. She was transferred to the operating room within two hours of admission. The working diagnosis at that time was a ruptured endometrioma vs ruptured ovarian cyst. Open laparoscopy revealed the large amount of brown, greenish fluid in the pelvis and abdomen. An exploratory laparotomy was performed. Multiple endometrial implants were present throughout the anterior abdominal wall, omentum, small and large bowel, as well as the pelvic organs. The total ascitic fluid measured approximately 2000 cc. The fluid, although not sent for cell block and cytology, had the appearance of a transudate rather than exudate. Cultures of the fluid were negative. Once the fluid was aspirated, the pelvis was evaluated more clearly and a ruptured endometrioma was noted on the right ovary. There was no evidence of bowel perforation. A total abdominal hysterectomy and right salpingo-oophorectomy were performed. Final pathology revealed proliferative endometrial pattern, myometrium with indistinct nodular areas of adenomyosis and leiomyoma right ovary, which measured 4×5×7 cm containing a 5 cm ruptured endometrioma without evidence of an adjacent functional cyst. There were no signs of inflammation in any of the pelvic tissues examined. The simultaneous occurrence of ascites and endometriosis is rare. The first case was reported in 1954 by Brews. 1 The combination of pleural effusions is even more rare with fewer than ten cases in the world literature. 3 The exact cause for ascites associated with endometriosis remains obscure. Bernstein et al in 1961 proposed a mechanism whereby the rupture of chocolate cysts release blood and endometrial cells into the peritoneal cavity. The formation of ascites and dense adhesions are the result of irritation on serosal surfaces caused by free blood in the peritoneal cavity. 4 Ascites has also been found in extensive endometriosis without rupture of chocolate cysts. Theoretically, transdiaphragmatic lymphatics allows spread of ascitic fluid into the pleural cavity, as seen with Meig's syndrome. 5 In reviewing cases since 1954, the patients were generally young with an average age of 27 years ( Table 1 ) . The majority of patients were nulliparous. The most common presentation was increasing abdominal girth, often accompanied by pain and dysmenorrhea. The average amount of ascitic fluid was 3,404 cc with the fluid varying between 150 to 7500 cc. The fluid was characteristically dark brown or bloody. Cytology was negative for malignancy. Pleural effusions were present in 33% of the reported cases. 1 – 6 The present case report demonstrated similar findings on presentation to the hospital. The treatment of choice for a ruptured endometrioma is yet to be established ( Table 2 ) . The most definitive therapy is surgical with hysterectomy and removal of both ovaries. This method, however, seems too radical for women wishing to preserve their fertility. As a consequence, medical therapy with hormonal management has been attempted. Hormonal therapy includes progestin, progestin and estrogen, depomedroxyprogesterone acetate, danazol, or GnRH agonists. 5 The physician must consider endometriosis in the differential diagnosis whenever both ascites and a pelvic mass are found in the same patient. Pleural effusion may be associated with endometriosis, but this finding is very rare. The definitive therapy appears to be surgical castration since no recurrence of ascites or progression of endometriosis has been reported in the management. 4 However, ascites has been suppressed with hormonal therapy, and this appears to be promising in the treatment of this complication, especially for women wishing to retain their fertility. 6 | Other | biomedical | en | 0.999995 |
10527338 | The role of laparoscopic herniorraphy (LH) remains controversial. The totally extraperitoneal (TEP) method is essentially a laparoscopic modification of Stoppa's repair. 1 Certain potential intraoperative complications have been reported with laparoscopic hernia repair. 2 We report a case of pneumomediastinum and subcutaneous emphysema of the neck, without pneumothorax, after TEP repair. A 52-year-old man, American Society of Anesthesiologists (ASA) level I, 75 kg, 173 cm, with left indirect groin hernia was admitted for elective inguinal repair using the TEP operation. After an uneventful intubation, TEP repair of the hernia utilizing three midline trocars technique was performed. At the beginning of the procedure, SO 2 was 98%, blood pressure 140/65 and FetCO 2 was 34 mm Hg. Operation time was 32 minutes, with a mean intra-abdominal pressure of 11-12 mm Hg. During the procedure, a small noncomplicated break of the peritoneum was detected and closed with staples. No hemodynamic changes were detected. Immediately after extubation, the patient had severe chest pain, O 2 saturation decreased to 84%, FetCO 2 increased to 44 mm Hg, and subcutaneous emphysema of the neck was detected. There was no emphysema of the abdomen or of the back. A chest film and thoracic computed tomographic (CT) scan confirmed the presence of pneumomediastinum without pneumothorax. Fiberoptic bronchoscopy did not reveal any tear in the upper airway. After two hours of mechanical ventilation, the emphysema resolved, and the patient was extubated without any problem. The patient was discharged asymptomatic 48 hours after surgery. Theoretical advantages of laparoscopic hernioplasty are less pain and a quicker return to normal activity. Complications of TEP laparoscopic hernioplasty are reported infrequently. Bowel, bladder and vessel injuries, bleeding in the preperitoneal space, severe testicular pain, hemoscrotum or nerve entrapment 1 noted with TEP hernioplasty have also have been described in open procedures. The detractors of LH mainly criticize the laparoscopic approach as usually being performed under general anesthesia with higher costs and an unknown recurrence rate. However, large series have been published showing a low recurrence rate (0% - 1%). 1 In fact, LH is now performed as a day-case procedure. 3 TEP repair is performed by dissecting the preperitoneal space and insufflating carbon dioxide to maintain an operative space without pneumoperitoneum. This method potentially avoids the risks of intra-abdominal organ lesions and peritoneal adhesions. Air may enter the mediastinum from the esophagus, trachea, bronchi, lung, neck, abdomen or retroperitoneal space, producing mediastinal emphysema or pneumomediastinum. Pneumomediastinum is a very rare complication of intra-abdominal laparoscopic procedures. 4 , 5 CO 2 usually passes through the hiatus secondary to a congenital anomaly, weak points, defects or a tear in the diaphragm. High-working CO 2 pressure or a prolonged insufflation time can also lead to pneumomediastinum. A recent article described a TEP procedure complicated by hypercarbia and the development of massive subcutaneous emphysema without hemodynamic instability. This was thought to be due to a high intra-abdominal insufflation pressure that ranged between 14 and 18 mm Hg. 2 Two cases of pneumothorax have also been previously described during TEP. 6 , 7 It is thought that pneumothorax as a complication of TEP is most likely related to high insufflation pressure and the length of the procedure, itself. Both cases occurred in healthy patients without hiatal or diaphragmatic hernia, and the condition was solved without the placement of a chest tube. Pneumomediastinum was present in both cases. A retroperitoneal route could be postulated on extraperitoneal procedures, but no retroperitoneal air was present on CT, and no back emphysema was detected. In our case, a hiatal route is the most reasonable explanation for the occurance of pneumomediatinum as insufflation time was short, working pressure was low, and bronchoscopy ruled out an airway disruption. We conclude that pneumomediastinum can occur after extraperitoneal laparoscopic hernia procedures due to small tears in the peritoneum that occur even with low-working pressures. Only early diagnosis and treatment can avoid serious complications. | Clinical case | biomedical | en | 0.999998 |
10527339 | The world has never been changing faster than it is today. At the end of the 20th century, we are experiencing one of the greatest technological and social revolutions ever: the beginning of the information age. 1 Especially revolutionary is easy access to information provided by the World Wide Web. Available resources include commentaries and reviews, bibliographical and statistical databases, discussion groups and newstickers, multimedia textbooks and journals, clinical decision aids and patient simulations, case studies, job boards and information on residency, educational software and information on upcoming meetings, as well as many other sources of information. Indeed, in the past few years nothing has affected medical education and practice more than the Internet. The fast availability of current medical literature and the availability of tools for easy access to information, as well as for the easy production of information, have confronted research physicians, scholars, and students with new kinds of problems, many of which concern us personally. Some of these problems will be discussed in this paper. 2 We must remember that a significant part (probably more than 95%) of the information present in Cyberspace is published there directly. This means that this information bypassed traditional publishers and established review systems. Literally, anyone with control of a Web site can claim to be an expert. At the same time, Web sites often provide information about diseases that may be treated by products and services sold by businesses that maintain or support the site. Consequently, the information provided may be biased to promote the commercial interests of the site sponsor. 3 If the author of information appearing on a Web site is known, we can research his or her expertise, using, for example, MEDLINE. Unfortunately, numerous Web sites do not carry an author's name or the authorship is not clear, so that such “tracking down” becomes impossible. Even more complicated is dealing with image and sound files. These electronic documents lack certain components that are used to construct Web site citation. Image and sound files do not usually contain information about authorship. Another crucial bibliographic element is publication date, which is difficult to determine. We must distinguish between dates of publication and “last update” and always remember that the edition (version) number is rarely published. There are numerous style guidelines that deal with electronic sources of information; they can be found both in print media and on the Internet. Since there is no standard work widely accepted by Web surfers and librarians, many scholars have summarized existing recommendations, adapted them or extended them for a particular academic discipline, or have presented their own approach. The most popular style guide for citing electronic sources is that published by Xia Li and Nancy B. Crane, reference librarians at the University of Vermont-Burlington. Other widely accepted styles are those of the APA (American Psychological Association), the MLA (Modern Languages Association of America), the model of Melvin E. Page, a history scholar and experienced Internet user from East Tennessee State University, and various adaptations of Turabian styles. Despite the differences, each style guide attempts to do the same thing: indicate to the reader how to reach the requested original information source. 4 All of the systems and models of citation of Internet-based sources, however, deal with the style of citation and less with the basic shortcoming of the WWW sources: their extremely short half-life. This leads us to pose the unorthodox questions, Why do we cite? Do we really have to do it? Scholars cite their sources of information for three main reasons: 1) to insure that the information conveyed is accurate, 2) to guarantee their readers access to the full context in which the material was cited, and 3) to credit authors) (“intellectual honesty”). In short, the essence of citation is verification of information. Clearly, neither intellectual honesty nor accuracy can be verified if the source consulted is no longer available. The reader cannot analyze the researcher's use and interpretation of the evidence. Frequently, only months or even weeks (sic!) after publishing, an electronic document gets deleted from a university server. Internet addresses (so-called “URL” - Uniform Resource Locator) are often changed by Webmasters or system administrators for technical purposes, and Web sites may disappear without any trace from cyberspace, due, for example to lack of sponsors. Repeatedly, electronic messages are moved after a certain period of time and stored in the administrator's archives, which are not generally accessible to the public. This is true especially of list-servers' messages or newsgroup postings. Unfortunately, this phenomena is not a sporadic one. When the author of this paper checked ARS MEDICI, a collection of 20,000 medical Internet addresses published on a CD-ROM in 1997, it turned out that over 50% of all addresses were dead links. 5 Can we imagine any scientific paper citing references of which less than 18 months later only 50% are available? A bibliographical disaster! Another example: What if someone (computer system manager, Web site author or hacker) modifies a document AFTER a surfer has cited a particular electronic document? For example, the author of a Web site on endoscopy presents some facts on the history of that procedure under URL: www.example-endoscopy.com/history.html . There, a surfer may learn that the first surgeon who performed laparoscopic cholecystectomy (LC) was Phillipe Mouret of Lyon, France, in 1987. Some time later, however, the author of the site discovers that it was E. Mühe of Böblingen, Germany, who had carried out the first LC in 1985, and not the aforementioned French surgeon. Consequently, the author alters the contents of the Web site because of this “new” information but not its address (URL) -- a common routine in Cyberspace known as “updating.” Now, it is possible to have two groups of researchers pointing to the above-mentioned Web site: one group, who picked up the historical information before the site was updated (“Phillipe Mouret was the first”), and the other group, who reached the Web site after it was updated (“Erich Mühe was the first”). As a result, we are confronted with an unacceptable situation -- both groups would quote the same reference (Internet address), but both would refer to totally different facts. The situation might conceivably become more problematic (or even dramatic) if this phenomena occurs on Web sites dealing with clinical issues, for example, application of drugs, interpretation of labor findings, or recommendation of certain operation techniques. The law dealing with information obtained from or through the Web is not well defined. Potential legal issues include responsibility for diagnostic and therapeutic procedures based on Internet information. Equally troublesome is the question of interaction between physicians and patients over the Internet, when, for example, it may occur that a physician practices medicine without being licensed in the state or country in which the patient resides. Clearly, there is no foolproof mechanism to prevent erroneous information from being placed in print media, but with printed material there are effective methods to store the information. Indeed, in the case of print media, we can reach the source directly at any time through any number of means, such as interlibrary loans. Due to well developed library systems, it is not a problem to reach, for example, the works of Theodor Billroth , William S. Halsted , or Rudolf Virchow , written more than 100 years ago. In the case of electronic sources, we do not have such a system. Various solutions have been presented both in print media and on the Web to manage these problems. Many expert users propose downloading (or printing) electronic documents and posting the messages upon request. This is only a partial solution: if a reader cannot retrieve the source by himself or herself, scholarship becomes more an act of faith rather than an act of research. A citation, from a scientific point of view, becomes more or less meaningless. Neither downloading nor printing sources at the moment of their quotation makes the information available for other surfers once it is deleted. It is no wonder that archivists and librarians all over the world have launched various projects to collect electronic sources. 6 It is nearly impossible to imagine a library collecting all Web information along with countless updates even in a single language. Theoretically, a variety of ways of solving the problem of availability to at least portions of documents are feasible. A thinkable solution would be the foundation of a central server, let us call it “Online-Library of Medicine.” It would be controlled by an authority -- institutional or not. An author would submit his or her electronic document; “Online-Library of Medicine” would collect, analyze and register the publication. “Online-Library of Medicine” also would approve the electronic document and make it available on its own server. And only such “approved by Online-Library of Medicine” sources -- both on the author's or the Library's server -- would carry weight as quotable sources for the scientific community. Most importantly, it would be accessible to anyone at anytime. Certainly, approved documents would be available also as printed matter to those having no Internet access -- for example, through a local librarian or interlibrary loan system. In order to provide reliable and repeatable data, anonymity must be replaced by clearly stated authorship and date of first publication, and the document itself must be stored on a server under intellectual control. Presently, there are no good answers to the problems regarding quality control, short lifetime, and lack of accessibility to Internet sources. We can only follow general rules and make suggestions by analogy to ink-on-paper style. The further cooperation of archivists, librarians, expert users, motivated providers, information system developers, computing professionals, and, finally, health specialists is necessary in order to provide the medical community with Internet data capable of being citing. It does not matter what solution will be developed in the future to solve the key issue of the availability of a previously consulted information source, “Online-Library of Medicine” or any other solution. But one thing is certain, we urgently need a system making Internet resources available for all surfers, even years after the resources were first published online. Until that time, we must keep in our minds the motto “caveat lector” (let the reader beware), or, rather, in the spirit of our time: click c@refully before you cite. Due to limited space, it was not possible to include in this paper detailed models of all of the above-mentioned styles of citing (APA, etc.) They can be found on the author's Web site under www.endo-highlights.com/internet/citing.html . For their actuality, but not contents, the author takes full responsibility. A Short Checklist for Reliability of Internet Sources is provided and designed to make the surfer more aware of the issue of the quality of Internet-based Sources ( Table 1 ) . | Other | other | en | 0.999999 |
10532961 | Rhodopsin is the visual pigment of the rod photoreceptor and catalyzes the activation of the G-protein, transducin. Seven transmembrane segments of opsin form a pocket to bind 11-cis-retinal (11 c Ret), 1 forming a chromophore with the lysine (K296) as a protonated Schiff base (PSB + -H). The chromophore isomerizes to all-trans-retinal within 200 fs . Energy uptake by the pigment is efficient and related to the steric strain of isomerization and the charge separation of the cationic PSB + -H from its anionic counterion (E113 − ) . Rhodopsin then undergoes a series of thermal transitions over the time scale of picoseconds through milliseconds to achieve the metarhodopsin-II (Meta-II) spectral conformation. At least two critical and sequential proton exchange mechanisms occur during Meta-II formation: the net transfer of the Schiff base proton to the counterion at E113 − and the uptake of two protons into the cytoplasmic membrane surface . These charge motions are reflected in transitions between at least two respective Meta-II states (Meta-II a and Meta-II b ) that share the same absorption (λ max 380 nm). The transition from the Meta-I to the Meta-II state is the only endothermic state change that occurs during the thermal dark reactions. This indicates that the spontaneous transition into these states is associated with a large positive entropy. A significant molecular volume increase occurs during the lifetime of the Meta-II states . The causes of volume expansion are likely to involve changes in sidechain interactions within the membrane, movement of α helices, and the configuration of the cytoplasmic loops that are temporally correlated with formation of the R* state, which allows transducin docking . Several critical state transitions in rhodopsin activation are thus spectrally silent. Moreover, the underlying molecular biophysics and forces driving these conformational changes remain unsolved. Proton transfer from the Schiff base to E113 − is sufficient to activate a chain of molecular events which result in R* . Other tools such as Fourier transform infrared spectroscopy or electron spin resonance can sample conformation changes outside the chromophore environment. But, like time-resolved absorption studies, these are currently limited by the need for hundreds of micrograms or milligrams of detergent-extracted and purified rhodopsin, or cysteine mutagenic engineering to allow site-specific attachment of spin probes. Compared with Fourier transform infrared spectroscopy, only the more sensitive electron spin resonance technology can resolve environmental transitions on a millisecond time scale. These tools have nonetheless contributed greatly to our current understanding of the rhodopsin activation process , but await the development of cellular expression systems where the harvest of milligram-order quantities of mutated visual pigments is less of an experimental limitation . The early receptor potential (ERP) is a charge redistribution in rhodopsin associated with protein conformational changes . This signal can be measured at the surface of the living eye, across the retina, or across the membrane of single isolated photoreceptor cells. The ERP depends on rhodopsin being uniformly oriented in the plasma membrane and occurs in two distinct phases . The depolarizing R 1 component correlates with flash presentation and likely reflects the molecular process of charge separation during isomerization , but its underlying mechanism is not yet established. The millisecond-order R 2 phase is hyperpolarizing and correlates with conformational changes leading to the biochemically active R* or Meta-II intermediates . Absorption of light by different “spectral” states of rhodopsin generates unique ERP signals . This suggests that the “electrical” states of rhodopsin reflect unique charge distributions during conformational changes. Thus, rhodopsin activation results in both spectral and electrical state transitions. A clear correlation has been established between spectral and electrical states . However, the measurements of these states reflect different molecular properties. Absorption state transitions reflect the different environments of the chromophore. Since the chromophore lies predominantly in the membrane plane, these transitions reflect principally in-plane charge redistributions. Electrical transitions, which are measured across the membrane, reflect molecular events with vector contributions largely orthogonal to the dipole absorption state of the chromophore environment. Because of this, electrical measurements assess a vector of the activation process that is distinct. An additional advantage is that electrical measurements can simultaneously sample the entire coordinated ensemble of charge motions, dipole reorientations, or interfacial charge transfer reactions across the full thickness of the membrane plane and its local boundary surface layers. With modern cellular electrophysiological tools, charge motions can currently be examined with submillisecond time resolution. Supplementary techniques are likely to extend recording to the nanosecond time domain. The early receptor current (ERC) of rhodopsin activation is the direct measure of charge flow that underlies the ERP. This nonlinear capacitative current shows saturation, dependent upon the amount of rhodopsin molecules available for activation . Protein conformation-dependent currents are quite common , probably the best example being the gating currents of ionic channels that have features in common with the ERC such as signal waveform and bandwidth . Studies of channel gating currents has clarified the role of the molecular structure during voltage-dependent gating. For example, the localization of gating charges to the S4 helices, and the motions of these elements in the electrostatic field, was advanced through gating current studies of expressed ionic channels. Similarly, the ERC might be used to study the forces that govern rhodopsin state transitions while it resides in a physiologically intact membranous environment, or to analyze electrical state transitions that may be spectrally silent to UV-visible or infrared absorption, or occur in environments not accessible to spin probe introduction. The ERC of rhodopsin activation in intact photoreceptors has been elegantly studied using gigaohm-seal, whole-cell patch clamping techniques . Therefore, techniques were developed in which the ERC could be recorded in a unicellular expression system containing high levels of normal rhodopsin . This method allows assay of rhodopsin activation with seven to eight orders of magnitude less material than other time-resolved techniques. High fidelity ERC currents are routinely recorded from single fused giant cells containing on the order of a picogram of regenerated rhodopsin (1.5 × 10 7 molecules). In the current work, the ERC approach is used to investigate activation properties of expressed normal or wild-type (WT) human rhodopsin for comparison to known properties of the native pigment previously studied in situ. Human opsin is expressed and regenerated in a membrane environment of transformed HEK293S kidney cells at densities comparable with that in intact photoreceptors . Properties of the WT human visual pigment studied with the ERC are consistent with charge motions originating from activation of the ground state of rhodopsin, within the normal range of photosensitivity, and according to the principle of univariance. ERC signals can be regenerated not only with 11 c Ret but also with 9-cis-retinal (9 c Ret). This is a first step toward analogue pigment investigations with the time-resolved ERC tool. Finally, ERC signals were recorded from two mutant human opsin pigments (D83N, E134Q) that have R 2 relaxation properties distinct from WT. Initial steps are taken toward the development of a quantitative and analytical approach to investigate rhodopsin structure–function relationships using this electrophysiological method. This is especially relevant because the millisecond-order conformational events during rhodopsin activation are now thought to be electrostatically mediated . This study is the first to apply the ERC method to characterize rapid electrical processes during activation in expressed mutant and analogue visual pigments in comparison with the normal process. The ERC approach has the sensitivity and temporal resolution to significantly advance knowledge of the underlying molecular biophysical chemistry of rhodopsin activation. Human opsin-expressing HEK293S cell lines were used for ERC recordings . Stable mutant opsin HEK293S cell lines were developed using cytomegalovirus expression vectors that were transfected using calcium phosphate and selected in G418 (500 μg/ml). Development and characterization of human opsin cell lines is described elsewhere . The WT human cell line used in these experiments was WT-12 (∼3 × 10 6 opsins/cell), the D83N mutant cell line was D83N-11 (∼8 × 10 5 opsins/cell), and the E134Q cell line was E134Q-7 (∼2 × 10 5 opsins/cell) . All cell lines were grown at 37°C and 5% CO 2 in DMEM/F12 containing 10% calf serum, antibiotics, and 2.5 mM l -glutamine on poly- l -lysine–coated coverslips. Cells on coverslips were chemically fused to form giant cells by a 1–2 min exposure to 50% (wt/vol) polyethylene glycol (1,500 g/mol) in 75 mM HEPES, pH 8.0 (Boehringer-Mannheim Biochemicals) . Giant cells with a single plasma membrane were evident within hours after fusion. ERCs were recorded between 1 and 3 d after fusion. Fused HEK293 cells amplify the amount of expressed integral plasma membrane proteins and allowed high fidelity measurements of sodium channel gating currents and rhodopsin ERCs . Cells on coverslips were washed in regeneration buffer and placed in a light-tight container in the darkroom at room temperature (22–25°C). Regeneration buffer was (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl 2 , 1.0 MgCl 2 , 10 glucose, 10 HEPES-NaOH, pH 7.2, and 2% (wt/vol) (∼290 μM) fatty acid–free bovine serum albumin (FAF-BSA; Sigma Chemical Co.). Concentrated (mM) 11 c Ret or 9 c Ret stocks (in ethanol) were added in small volumes to this solution to a final concentration of 50 μM in preliminary experiments and 25 μM in the final protocol with 0.025% (vol/vol) α- d -tocopherol (vitamin E) added as an antioxidant. Vitamin E is found in high concentrations in photoreceptor cells and may serve as an antioxidant . Vitamin E appeared to improve the longevity and quality of the recordings, likely because of its antioxidation effects on membranes and retinals. Primary regeneration was conducted for at least 30 min and coverslip fragments were then washed in bath recording buffer (without 11 c Ret or 9 c Ret) and transferred to the recording chamber, which contained E-1 buffer (see below). FAF-BSA molecules are a pool of chromophore receptors that efficiently solubilize hydrophobic retinals into aqueous solvents and deliver 11 c Ret loads rapidly and stoichiometrically to regenerate rhodopsin from opsin . Optically clear solutions and effective solubilization result when ethanolic stocks of chromophore are introduced into 2% FAF-BSA buffers, whereas without FAF-BSA a scattering dispersion results that is indicative of chromophore partition into aggregates or micelles. Delivery of chromophore to cells is facilitated because a solubilized chromophore can partition more efficaciously into cellular membranes while chromophores bound to FAF-BSA may also be absorbed by pinocytosis. 11 c Ret was a gift of the National Eye Institute and Dr. Rosalie Crouch (Medical University of South Carolina, Charleston, SC) and 9 c Ret was obtained from Sigma Chemical Co. Rhodopsin regenerated in fused giant cells was activated by an intense flash microbeam apparatus described in detail elsewhere . In brief, light from a xenon flash tube is collimated, filtered, and condensed into a 1-mm-core fused silica fiber optics for transmission to the epifluorescent port of the microscope (Diaphot; Nikon Inc.). The objective lens is used to condense the fiber output into a microbeam spot parafocal with the specimen plane where the giant cell is situated. The spot-size diameter [full-width-half-maximum (FWHM)] in these experiments is 228 μm, which is about three times the size of the largest giant cell used. In routine flash photolysis, three-cavity bandpass filter elements (350, 430, 500, and 570 nm) were used that had 70-nm bandpass (FWHM) centered on peak transmission wavelength. To acquire action spectra data, 30 nm FWHM bandpass filters (centered at 400, 440, 480, 500, 520, 580, and 620 nm, and a 540-nm filter with a 10 nm FWHM bandwidth) were used. The throughputs of all filters, except those at 350 (70) and 400 (30) nm, do not overlap with the absorption spectra of free chromophore (peak ≈ 374 nm) such that isomerization (cis → trans or trans → cis) of any free chromophore is not expected during flashes used to elicit ERCs. Unless otherwise mentioned, flashes were delivered at the maximum capacity of the instrument. Intensities were 10 8 –10 9 photons/μm 2 across the near UV/visible band. Flash microbeam intensities were measured using a calibrated photodiode placed over the specimen plane of the microscope. To regulate flash intensity output , the voltage on the flash tube energy storage capacitor was adjusted. Flash duration was only ∼14 μs, insuring that the Meta-I ⇔ Meta-II equilibrium (milliseconds) generated at room temperature in these experiments was not perturbed by photoregeneration to other states . Flash duration did overlap with lifetimes of bathorhodopsin, blue-shifted intermediate, and lumirhodopsin such that photoregeneration from these states is possible. Since we were largely concerned with the millisecond-order R 2 phase of the ERC, any photoregeneration from early bleaching intermediates should not perturb the charge motions occurring during R 2 , which correlate with the time scale of the Meta-I ⇔ Meta-II transition. Shielding and fiber optic transmission prevent contamination of the patch-clamp electronics with flash-associated noise. Photosensitivity (P t ) is the product of quantal efficiency (γ) and the wavelength-dependent absorbance cross section (α λ ). The absorbance cross section of wild-type human rhodopsin is 1.53 × 10 −8 μm 2 (calculated from an extinction coefficient of 40,000 M −1 cm −1 at 493 nm and γ is 0.67, leading to a P t of 10 −8 μm 2 for normal human rhodopsin at peak extinction (493 nm). P t can be used to estimate the fraction of rhodopsin molecules absorbing at least one photon per flash using the zero-order term of the Poisson equation [1 − P o = 1 − exp(−P t · i )], where i is the flash intensity (photons/μm 2 ) and P o is the fraction that absorbs no photons [Poisson Eq.: P n = (P t * i ) n *exp(−P t * i )/ n !, where n is the number of absorptions per chromophore]. In this calculation, one adjusts α λ by the ratio of absorbance at the wavelength of interest to that at peak extinction. γ is assumed to be constant and independent of wavelength. For the 70-nm bandpass filters used in these experiments (centered at 350, 430, 500, and 570 nm), the fraction of molecules absorbing at least one photon were estimated to be 0.159, 0.716, 0.963, and 0.273, respectively. For the 30- and 10-nm bandpass filters used in these experiments (centered at 400, 440, 480, 500, 520, 540, 580, and 620 nm), the fraction of rhodopsin molecules absorbing at least one photon were estimated at 0.226, 0.626, 0.831, 0.80, 0.733, 0.44, 0.122, and 0.013, respectively. These calculations assume no orientational factors, no self-screening effects, and transparent cellular media. Thus, microbeam flash intensities were not expected to be experimentally limiting for flash photolytic stimulation of expressed rhodopsin pigments, except perhaps for the 620-nm stimulus. The maximum extent of rhodopsin bleaching (i.e., formation of Meta-II) after a single flash is 50% because of second (or even-numbered) photon reabsorptions during the lifetimes of early states that have high quantal efficiency to photochemically regenerate the ground state (e.g., bathorhodopsin, lumirhodopsin). Flashes at 400, 580, and 620 nm were likely to elicit only single photon absorptions (>90%). Flashes at other wavelengths (440, 480, 500, 520, and 540 nm) were more likely to include even-numbered absorptions (relative fraction of total for even numbered absorptions 0.31, 0.415, 0.405, 0.367, and 0.22, respectively). The absolute flash intensities (10 8 photons/μm 2 ) at the various center wavelengths (parentheses) used in action spectra experiments were as follows: 1.20 (400 nm), 2.29 (440 nm), 2.27 (480 nm), 1.96 (500 nm), 2.02 (520 nm), 1.49 (540 nm), 2.49 (580 nm), and 1.68 (620 nm). The relative ratios of absolute flash intensities relative to that at 500 nm were 0.61, 1.16, 1.15, 1.0, 1.03, 0.76, 1.27, and 0.85, respectively. To scale charge motions for action spectra , the reciprocal of these scale factors were used to multiplicatively scale the integrated charge motions. Cells on coverslips were imaged using infrared light (high pass cutoff 830 nm) at 80–160× by an inverted microscope (Diaphot; Nikon Inc.) equipped with Nomarski differential interference contrast, a CCD camera, and a TV monitor. The microscope was housed in a Faraday cage in a dark room. Microelectrodes were fashioned from borosilicate glass using two stage pulls and coated with Black Sylgard (Dow Corning Corp.). Electrodes were routinely filled with one of two intracellular solutions (with or without 10 mM HEPES-CsOH) containing (mM) 70 tetramethylammonium (TMA)-OH, 70 Mes-H, 70 TMA-F, 10 EGTA-CsOH, 10 HEPES-CsOH, pH 6.5; these solutions yielded ERCs with no qualitative changes and are called I-1. The internal pH was chosen to be 6.5 to forward bias the Meta-I ⇔ Meta-II equilibrium strongly in favor of Meta-II . In the E134Q experiments, intracellular buffers of otherwise identical composition to I-1 were formulated (I-2, I-3, I-4, I-5) to have intracellular pHs of 6.0, 7.0, 7.5, and 8.0, respectively. Bath solution contained (mM): 140 TMA-OH, 140 Mes-H, 2.0 CaCl 2 , 2.0 MgCl 2 , 5.0 HEPES-NaOH, pH 7.0 (E-1). pH was titrated by addition of HCl. Gigaohm seals formed readily with these solutions, electrode/patch/seal capacitance was compensated electronically, and further suction was used to enter whole-cell recording. Inward and outward rectifying conductances are present in HEK293 cells but were suppressed with permeant ion replacement solutions used in the pipette and bath. Without serum used in the growth medium, fused giant cells in E-1 recording buffer rounded up into approximately spherical shapes that remained well attached to the treated coverslips at their base. The patch-clamp instrument was an Axopatch 1C with a CV-4 resistive feedback headstage and the later was used with a gain of 1 (0.5 Gigaohm feedback resistor; Axon Instruments). Since the ERC is a capacitative current, whole-cell capacitance (C mem ) and series resistance were not compensated, because this has the potential to alter the waveform. Membrane holding potential was clamped at 0 mV unless otherwise noted in the legends. Whole-cell capacity current was measured by a +20 mV/4 ms test pulse from a holding potential of −80 mV and C mem was computed by integrating the capacitative current waveform to obtain charge (Q) (Q/V = C mem ) after substraction of ohmic current. Cell surface area was computed from the measured C mem (1 μF/cm 2 ). Ramp voltage clamps were delivered to test for a high resistance membrane and low level leakage. Cells with large leakage were discarded. Whole-cell currents were recorded at 5 kHz bandwidth using an eight-pole Bessel filter. Flashes were controlled and ERC and flash stimulation data acquired using pCLAMP 5.51 (CLAMPEX) and digitized (200 μs/point) by a Labmaster (100 kHz) interface board (Scientific Solutions Inc.). This acquisition rate was selected to provide the best possible representation of the R 1 signal, which is still undersampled, while critically allowing the full time course of the R 2 component to be acquired out to 100 ms. All ERC data was processed and analyzed using the Origin4.1 package (MicroCal Software, Inc.). Nonlinear least squares fitting was conducted using a Levinberg-Marquardt algorithm. Polyethylene-glycol–fused giant HEK293S cells are used to amplify the amount of regenerable plasma membrane opsin (WT or mutant) in single large cells to improve the signal-to-noise ratio (SNR) during ERC data acquisition. These are prepared from single cells that are stably transformed to constitutively express WT or mutant human opsins in the range of 1–10 × 10 6 molecules . Giant cells share with unfused cells a uniform distribution of immunocytochemically reactive opsin in their plasma membranes (not shown). The increased plasma membrane surface area of fused cells contains larger numbers of opsin molecules in linear proportion to the number of cells that participated in the fusion events . R 2 charge motion is linearly dependent on the number of light-activated rhodopsin molecules in the plasma membranes . Therefore, the fused cell system was a rational choice for recording larger ERC signals at greater SNR. The largest fused cells had total WT human ERC R 2 charge (Q) about an order of magnitude greater (2.0 vs. 0.29 pC) than amphibian rod photoreceptors . Fused cells increased total ERC charge, improved SNR, and allowed spontaneous regeneration of pigment after bleaching by simple dark adaptation without further addition of chromophore beyond the primary regeneration. Fig. 1 A shows a giant cell ERC obtained on the first flash series (500 nm) after a 30-min regeneration. Flash stimuli were given at 500 nm to extinguish the ERC signal into background whole-cell noise. Both the submillisecond negative R 1 current and the millisecond-order larger, and positive R 2 current were routinely observed and essentially identical to those recorded in photoreceptors . While not detectable in single unfused cells, in giant cells the R 1 phase of the ERC was recordable, apparently due to increased total rhodopsin and improved SNR. After subtraction of baseline current, integration of each ERC lead to the charge motion (Q i ) in femtocoulombs attributable to each phase. Successive flashes progressively extinguished both phases of the ERC until no responses were observed above background noise. This was consistent with bleaching of plasma membrane rhodopsin due to photolysis. The observation that giant cells spontaneously recovered ERC signals after a complete bleach by simple dark adaptation for 10–15 min without added chromophore was quite surprising. Unless otherwise stated, cells were not exposed to additional 11 c Ret once the coverslip was removed from regeneration buffer and placed in E-1 buffer in the recording chamber. Subsequent flash photolysis after post-bleach dark adaptation lead to robust ERC signals that were again extinguished by additional flashes at 500 nm . Another period of dark adaptation promoted spontaneous recovery of the ERC that was again extinguished by successive 570-nm flashes . The R 2 but not R 1 signals were recorded after spontaneous regeneration of visual pigment that occurs during 10-min dark adaptation (see discussion ). Fig. 1 D shows the kinetics of the spontaneous recovery of total ERC charge upon dark adaptation for two similarly sized fused cells (67.5 and 65.6 μm diameter). Cells were bleached and dark adaptation was allowed to occur for variable time periods before additional 500-nm flashes were given in rapid succession to extinguish the ERC signal. The total charge (Q ∞ ) present at each time point of dark adaptation was obtained by summing all Q i values to yield Q ∞ at that time point. Q ∞ values were normalized to the maximum charge determined for each cell so that regeneration data could be compared. The data were fit by a single exponential accumulation curve. The initial regeneration rate is fast and the process begins to approach steady state by 10 min of dark adaptation. The half time of ERC regeneration was 3.2 ± 1.1 min. If primary regeneration in 2% BSA fully equilibrates WT-HEK293S cells with 11 c Ret at a concentration (25 μM) in considerable excess over the expected molarity of opsin in fused giant cells (≈2.4 μM), then spontaneous regeneration can be described as a process with pseudo–first order kinetics from a single compartment containing chromophore. We routinely dark adapted cells for either 10 or 15 min between successive extinctions to allow visual pigment and ERC signal recovery to the stationary plateau or, at a minimum, to permit a criterion level of regeneration for any comparison of charge motions under different conditions tested on a single cell. The source of chromophore that promoted spontaneous ERC recovery was investigated. Single fused giant cells regenerated with 11 c Ret were subjected to primary extinction by successive flashes at 500 nm in normal bath solution (E-1 without chromophore). Dark adaptation promoted spontaneous pigment regeneration and allowed for a secondary extinction of the ERC. Immediately after the second extinction and throughout the next dark adaptation, the chamber volume was replaced with E-1 containing 10 mM hydroxylamine (NH 2 OH, pH 7.0) and an additional 5 min of dark adaptation preceded the next series of successive 500-nm flashes. ERCs were elicited and had Q i values comparable with those seen during the primary and secondary extinctions without NH 2 OH. Q i extinctions before and after NH 2 OH are shown . When NH 2 OH was present in the bath many more flashes (in this cell 23 flashes) were required to completely extinguish the ERC R 2 charge in comparison to three to five flashes during the primary and secondary extinctions. This suggested that 10 mM NH 2 OH decreased photosensitivity and this effect will be examined fully in a subsequent study. Two dark adaptations (10 min each) in 10 mM NH 2 OH were followed by some but not full ERC signal recovery, which was then subsequently extinguished by additional flashes. ERC signals continued to be recordable on the time scale of tens of minutes in the presence of constant 10 mM extracellular NH 2 OH, a concentration far greater than the initial loading of chromophore (25 μM). Fig. 2 B shows the exhaustion course of Q i vs. flash number for another giant cell before and after introduction of 10 mM NH 2 OH into the bath solution. In NH 2 OH total R 2 charge decreased with successive extinctions and dark regenerations until no significant signals remained. Bath solution was then replaced with fresh E-1 containing 25 μM 11 c Ret in 2% FAF-BSA (without NH 2 OH). Strong ERC R 2 signals were regenerated comparable in total charge with that seen during the primary extinction. These experiments demonstrate strong evidence that the source of 11 c Ret during spontaneous dark regeneration is internal to the cell that is recorded. Additional observations support an intracellular origin for 11 c Ret during dark adaptation. First, the cell repeats dark-adaptive ERC regeneration until the signals expire, and then they do not again regenerate unless 11 c Ret is reapplied to the cell. This rundown is evident in the extinctions in NH 2 OH in Fig. 2 and suggests that cells exhaust their store of 11 c Ret. Second, in some cells the plasma membrane appeared wrinkled or the cytoplasm appeared optically smooth after expiration of ERC signals. This suggested a change in the structure of internal membranes associated with regeneration ability. Finally, the greatest number of “visual cycles” in this study was eight and was encountered in the largest fused giant cells (≈80 μm diameter) and, although not yet systematically investigated, the number of regenerations appeared to scale in proportion to cell size. Given the hydrophobic nature of 11 c Ret, it is likely that it is stored by partitioning into internal cellular membranes, and then repartitions back to the plasma membrane during dark adaptation to regenerate visual pigment (see discussion ). A previous study of ERC spectral sensitivity used 70-nm bandpass filters and demonstrated a broad action spectrum consistent with the ground state of human rhodopsin pigment . However, the possibility remained that other intermediates such as Meta-III 465 , pseudophotoproducts (Meta 470 ) or even isorhodopsin could contribute to the action spectrum. To investigate this issue further, the spectral sensitivity of ERC R 2 charge motion was measured with stimuli generated with relatively narrow 30-nm band-pass (FWHM) interference filters. In these experiments, the ERC R 2 charge from only the first flash after each 10-min recovery period was obtained between different filter settings. Rather than extinguish the signal, we did a criterion dark adaptation for 10 min between each single flash at a given wavelength. Fig. 3 shows first-flash ERC signals at several different wavelengths from a single giant cell. All ERC signals in the figure were multiplicatively scaled by the ratio of absolute flash intensities measured relative to 500 nm so that the spectral sensitivity can be more readily appreciated by observation. The ERC currents vary in amplitude depending on the wavelength of stimulation. To generate the action spectrum, the ERC charge (Q i ) was obtained at each wavelength and the charges were corrected for differences in photon density delivered with different filters so that action spectra at equal photon exposure would be generated. To allow comparison of action spectra from different fused cells containing different amounts of rhodopsin and ERC signal size, spectral sensitivity data were normalized to the maximum integrated R 2 charge at whatever wavelength it occurred. For all three cells, the largest R 2 charge occurred with 500-nm stimulation. In Fig. 3 , the mean ERC spectral sensitivity data (±SEM, n = 3) are plotted versus wavelength of peak stimulation. By stimulating with only a single flash at each wavelength and allowing sufficient time for regeneration, less stress is placed on the retinal load in the cell and the relative percent of bleaching is considerably less than when the ERC was extinguished by successive flashes at a single wavelength. This should allow greater sensitivity to detect differences because the fraction of pigment that must regenerate between each single flash bleach is smaller and data can be obtained from a uniformly regenerated population of pigment molecules with low variability. Instead of fitting spectral sensitivity data with a Lorenztian/Gaussian peak function (e.g., Voigt) , the absorbance spectrum of WT human rhodopsin was normalized with respect to the maximum visible band at 493 nm (α band) and overlaid with the ERC action spectrum. WT rhodopsin was immunoaffinity purified from the same WT-expressing cell line used in these ERC experiments . The absorbance spectrum of WT human rhodopsin (α band peak at 493 nm) provides an excellent fit to the ERC action spectrum obtained from cells expressing WT human opsin that was regenerated with 11 c Ret. The ERC action spectrum peaked around 493 nm, consistent with the major absorption band of ground state human rhodopsin regenerated with 11 c Ret. Moreover, the bandwidth of the α peak of the human rhodopsin absorbance spectrum also fits the ERC data well. It is important to mention that all of the action spectra data were obtained from cells after they had undergone a primary bleach; that is, under conditions where long-lived bleaching intermediates or photoregenerated pigments might have been present. Normal human rhodopsin has an absorbance spectrum that is slightly blue shifted (≈493 nm) with respect to the bovine pigment (498 nm) , and this is maintained on expressing human rhodopsin in HEK293S cells. Assuming that absorption maxima of spectral states of human rhodopsin would scale relative to the bovine pigment (isorhodopsin 487 , Meta-III 465 , and Meta 470 ), one would expect peaks at 482, 460, and 465 nm, respectively, in the human pigment. Therefore, if there are spectral states of bleaching intermediates or isorhodopsin present during ERC data acquisition, then they must represent a very small unresolved component of the charge motion. From this we conclude that the pigment underlying the expression ERC is WT human rhodopsin, and that during dark adaptation 11 c Ret from within the cell reacts with bleached opsin to form a PSB + -H and a normal ground state pigment. Successive flashes at a single wavelength and intensity promoted progressive loss of ERC R 2 charge until no further signal was obtained above background current noise. Since the interstimulus intervals between successive flashes were only ∼10 s, pigment regeneration was minimal between stimuli, and regeneration should not contribute to the extinction progression. The spectral sensitivity of the ERC governs not only the efficacy of successive bleaches, but also single bleaches. Fig. 4 A shows the Q i extinction of R 2 in response to successive flashes at 570 and then 500 nm in a single giant cell. Cumulative flash intensity delivered is used as the dependent variable. After ERC charge was effectively extinguished into noise by 570-nm flashes, 500-nm flashes of greater effective intensity were immediately delivered. The additional extinguishable ERC charge found with 500-nm flashes indicated residual ground state rhodopsin in the cell after the 570-nm flashes. This resulted because 570-nm flashes are not as effective at eliciting ERC currents as are flashes at 500 nm given the relative absorbance of WT human rhodopsin at 570 vs. 493 nm (peak absorbance) (OD 570 /OD 493 = 0.436) . Similar findings occur when bleaching at other wavelengths is followed by flash photolysis at 500 nm. The relative probability of activating rhodopsin, taken as the ratio of absorbance cross sections, is only ∼12% at 570 nm relative to 500 nm (α 570 /α 493 = 0.115), assuming equal photon density at the two wavelengths. At the maximal flash strengths used, the fraction of rhodopsin molecules absorbing at least one photon at 500 vs. 570 nm was estimated to be ∼0.96 and 0.27, respectively. Thus, the flash system does not deliver sufficient photons at 570 nm to compensate for the lower probability of activation. This illustrates that detection of rhodopsin charge motions depends on the unitary charge motion, which should be the same at any wavelength (see univariance below), and the number of activated rhodopsin molecules that mobilize charge and sum into an ensemble ERC current. Even at peak wavelength (≈500 nm), a given flash intensity may not be sufficient to generate ERC currents above noise because the ensemble ERC current lies within the noise band. Single exponential exhaustion curves are fit for both data sets at the two respective wavelengths. ERC extinction data for 570-, 500-, or 430-nm flashes from many experiments were always fit by single exponential curves. This is consistent with the ERC charge motion being proportional to the amount of rhodopsin that remains unactivated before each flash is given. Extinction data were fit by the following model using a nonlinear least squares method: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}Q_{{\mathrm{i}}}=Q_{{\mathrm{{\infty}}}}{\cdot}{\mathrm{exp}} \left \left(-I{\cdot}P_{{\mathrm{t}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where Q i is the charge motion resulting from a single flash, Q ∞ is the total charge before any flashes are given, I is the cumulative flash intensity, and P t is the photosensitivity. The bleaching process follows an exponential extinction that was further tested by a natural logarithmic transform of Q i values and linear fitting . was used to determine P t for several giant cells subjected to flash photolytic exhaustion under conditions of different flash stimulation wavelength. The charge extinction data for the initial (primary) and three subsequent (secondary) bleaches at 500 nm and single bleaches at 430 and 570 nm in a single large giant cell (≈80 μm) are shown in Fig. 4 C. The charge extinction data sets for each bleach were normalized to the maximum charge on the first flash, and then was fit to each data set and overlaid. The fit by the single exponential model implies that each flash promoted activation and extinction (bleaching) of a fraction of remaining ground state pigment. In some flash stimulation series, there was a residual content of charge that was slow to extinguish (10–20%). P t for the primary bleach at 500 nm was estimated to be ∼3.3 × 10 −9 μm 2 , but this is less reliable as there were only three points to fit. The P t values obtained from fitted curves at 500 nm for the three secondary bleaches were 3.28 × 10 −9 , 3.04 × 10 −9 , and 2.24 × 10 −9 μm 2 . P t was stable over successive secondary bleaches and similar to that of the primary bleach. This is evidence in addition to the action spectrum that the ground state of rhodopsin is regenerated with 11 c Ret during dark adaptation between successive flash cycles and there was no accumulation of other intermediates with significantly different photosensitivities. The mean (±SEM) for P t values from several cells are shown in Fig. 4 D at 500(70), 430(70), 570(70), and 500(30) nm, where the filter bandwidth is indicated parenthetically. Both parametric and nonparametric (Kruskal-Wallis) analysis of variance tests were used to evaluate whether the means of the five conditions were different. No statistically significant difference was found between P t estimates of human rhodopsin during the initial and secondary extinctions after spontaneous regeneration. However, the trend toward a lower P t at 430 nm is consistent with the ratio of absorbance of rhodopsin at 430 vs. 500 nm (α 430 /α 493 = 0.423) . The P t of WT human rhodopsin is estimated to be ∼1.0 × 10 −8 μm 2 (see materials and methods ). The mean value of P t [500(70) nm] determined from 11 extinctions in 6 cells, all after spontaneous regeneration was 2.6 ± 0.4 × 10 −9 μm 2 and the maximum value measured was 5.0 × 10 −9 μm 2 . P t values measured using the extinction of R 2 charge are consistent with but lower than that expected of a rhodopsin chromophore. This is likely due to the suppressive effect on P t of photoregeneration resulting from multiple photon absorptions per rhodopsin molecule at the flash intensities used. For example, with each 500(70)-nm flash, the estimates on even numbered absorptions are ∼50%, which would underestimate P t by a similar amount (see discussion ). In recent experiments, stimulus intensity (at 500 nm) was reduced (by 85%) to decrease the probability of multiple hits per molecule, allowing P t estimation to a mean value of 8.5 × 10 −9 μm 2 , which approximates that expected from the extinction coefficient (Brueggemann and Sullivan, manuscript in preparation). As first demonstrated by Makino et al. 1991 , the ERC of photoreceptors has invariant kinetics regardless of the wavelength or intensity of the flash, although the amplitude of the signal scales in linear proportion to the amount of rhodopsin activated, provided that the flash strength is below saturation. Any rhodopsin molecule absorbing a single or odd number of photons will have a finite probability (constant γ = 0.67) of successful activation and will contribute to the kinetics of ERC charge flow. At fixed wavelength, variation in stimulus intensity is expected to affect the probability of absorption if each activated rhodopsin molecule makes an independent and additive (linear) contribution to the ERC, and photoreversal to other states by second photon absorptions is minimal. Fig. 5 shows ERCs resulting from 500-nm stimulation at two different flash strengths. When these responses are normalized and overlaid for comparison, the kinetics of the R 2 relaxation at different intensities are not distinguishable. The ERC response versus flash intensity was measured. The Q i response of the first flash in an extinction series at a constant intensity is plotted versus intensity. A linear fit of the charge motion versus absolute intensity was found. This indicated that the flash intensities used were below saturation for the cellular expression system. This result is consistent with known properties of the ERC/ERP. Each activated rhodopsin molecule undergoes conformational changes to contribute a quantum of charge motion to the overall R 2 signal . According to the univariance principle, the energy of the photon (wavelength) should not affect the activation kinetics of independent rhodopsin molecules. Photon energy only affects the probability of absorption because the molecular cross section is a function of wavelength. Fig. 6 shows ERCs acquired from a giant cell at 430, 500, and 570 nm. ERC waveforms were normalized to the smoothed peak of the R 2 current for comparison. A double exponential curve was generated to fit the relaxation kinetics of the R 2 signal from the 500-nm stimulus. This template was then overlaid with the R 2 signals from the 430- and 570-nm responses. The 500-nm template fits the R 2 relaxations of the 430- and 570-nm responses rather well, even though the SNR was lower with 570-nm stimuli because the absolute response was smaller. Although the amplitudes of the ERCs and total charge motion of the R 2 signal vary with wavelength, the kinetics of R 2 relaxation are similar. Similarly, the large 500-nm R 2 response in Fig. 3 was fit with a double exponential function, and this template was overlaid with the large 440-, 480-, and 520-nm ERC responses and provided a good fit. The 400- and 540-nm ERC responses were of lower SNR and were not fit well by the template. ERC data in Fig. 3 was collected with 30 nm FWHM stimuli. In the rhodopsin expression system, photon energy does not affect the kinetics of the state transitions in rhodopsin, which is consistent with the univariance principle. Previous studies have shown that the ground state of rhodopsin can be photoregenerated from Meta-II 380 by near UV flashes delivered concurrent with its lifetime . Photolysis of Meta-II generated inverted ERP signals . To investigate whether photoregeneration could be assayed with ERC measurements, 500-nm flashes were used to extinguish the ERC into background cell noise, whereupon 350-nm flashes (70 nm FWHM) overlapping the Meta-II absorption (350 nm, 8.02 × 10 7 photons/μm 2 ) were delivered immediately and in rapid succession. The number of near UV flashes (1–5, 5, 10, and 20) delivered was varied to affect an increasing dose of photoregeneration stimulus to the Meta-II remaining in the cell. Immediately after the near UV flashes were delivered, additional flashes at 500 nm were used to measure the level of regeneration as the ERC R 2 charge. Under the conditions of these experiments, the expected lifetime of Meta-II at room temperature in a membrane environment is on the order of several minutes . Fig. 7 A shows ERC responses from an experiment where 5, 10, or 20 350-nm flashes were given to promote photoregeneration of ground state rhodopsin from Meta-II. The top trace in each case is the ERC response to the first 500-nm flash in the series used to initially extinguish the signal. The middle shows the response to the last 500-nm flash indicating the level of extinction into the whole-cell current noise. The bottom shows the first flash at 500 nm after 5 (left), 10 (middle), or 20 (right) UV flashes. As the number of UV flashes is increased, the size of the recovered 500-nm ERC signal becomes similar to that found before the UV flashes. The ERC R 2 current waveform in response to 500-nm flashes after the UV flashes is comparable in size and kinetics to the 500-nm signal that preceded bleaching whether 5, 10, or 20 UV stimuli were delivered. This data suggested that the ground state of the pigment (11 c Ret) is regenerated during the photoconversion process. In this cell, the amount of ERC R 2 charge increased over the time period of successive extinctions. The underlying mechanism is not yet clear (see discussion ). Fig. 7 B shows the percent of ERC charge recovered after 5 (65%), 10 (74%), or 20 (87%) UV flashes, measured against the UV flash dose. The period of time needed to deliver 5, 10, or 20 UV flashes was 27, 67, or 182 s, respectively, in this experiment. Since the kinetics of spontaneous regeneration has a time constant of ∼3 min , ERC responses after 10 or 20 UV flashes could be significantly affected by chemical regeneration with chromophore rather than photoregeneration. Therefore, the charge recovery in response to one to five UV flashes was examined . By the third UV flash, the process of regeneration had stabilized (∼55%) at the UV intensity used and no significant additional charge recovery was found when two additional UV flashes were given. The amount of charge regenerated by three to five UV flashes was similar to that which regenerated after five flashes of the same intensity in the experiment described above. Only 11 s were required to deliver the three UV flashes, making it highly unlikely that any significant chemical regeneration had contributed to the 55% charge recovery. Thus, UV stimuli promoted a large fractional recovery of R 2 charge in a time frame that is much more rapid than the kinetics of spontaneous ERC regeneration by dark adaptation. These results strongly suggest that the ERC can be photoregenerated by additional photons overlapping the Meta-II bandwidth. The quantal efficiency appears favorable to studying the mechanism in greater detail. In most cells receiving UV stimuli, no apparent ERC charge motion occurred above the noise level of the cell. However, in a few large giant cells, apparent UV flash–induced ERC signals were identified even without any signal smoothing to suppress noise. An example is shown in Fig. 7 C, where a response to a UV (350-nm) flash is generated immediately after extinction of ERC signals with 500-nm stimuli. The UV-induced ERC signal is small and has a negative (inverted) R 2 -like response. An unconstrained third order polynomial fit the UV R 2 signal and also demonstrated the inverted R 2 signals. An ERC generated with a 500-nm flash after dark adaptation in the same cell is shown to demonstrate the magnitude of the normal R 2 charge motion. The time to peak of the positive 500- and negative 350-nm–induced R 2 signals was 5.9 and 12.2 ms, respectively. The ratio of the inverted to noninverted R 2 charge was 0.175 (0.099 in another smaller cell). Cells were regenerated with 9 c Ret to test the feasibility of ERC investigation of rhodopsin activation in analogue visual pigments. Analogue visual pigments are usually formed from WT opsin and a synthetic retinal known to have unique properties (e.g., to block Meta-II formation), but could also be formed from synthetic retinals and site-specific opsin mutants. The naturally occurring 9 c Ret analogue forms isorhodopsin, a stable ground state pigment that is generated in a photostationary state with rhodopsin and bathorhodopsin . Once isorhodopsin is photoactivated to bathorhodopsin, the same sequence of bleaching intermediates occur as compared with normal rhodopsin activation. ERCs were recorded in three of four fused cells regenerated in 9 c Ret and signals were uniformly small. This may in part be related to the cell sizes used [C mem 85.7, 11.6 (probably a single cell), and 48.6 pF]. Fig. 8 shows primary (left) and secondary (middle) extinctions of ERCs with 500-nm flashes for a fused cell regenerated with 9 c Ret. ERC signals in fused WT-HEK293 cells regenerated with 9 c Ret were smaller than those regenerated with 11 c Ret in the population of cells studied. However, the ERC R 2 waveform was similar. P t was determined by flash series extinction at 500 nm (70 nm) for this cell and found to be 1.03 × 10 −9 μm 2 , which is similar to the P t we calculate for isorhodopsin at peak extinction at 483 nm (5.55 × 10 −9 μm 2 ). This value is arrived at by first calculating the molecular cross section (α λ ) from the extinction coefficient for isorhodopsin at 483 nm (44,000 M −1 · cm −1 ) (α λ = 3.82 × 10 −21 * ∈ λ ) (α λ = 1.68 × 10 −8 μm 2 ) and multiplying α λ by the quantal efficiency of isorhodopsin of 0.33 . At the flash intensities used in these experiments (4.08 × 10 8 photons/μm 2 ), ∼90% of isorhodopsin pigment molecules should absorb at least one photon, with ∼55% of these being odd-numbered isomerizations that would proceed forward to bleaching and ∼45% being even-numbered isomerizations that would result in photoconversion to ground state species. Thus, P t could be suppressed by photoregeneration at the flash intensities used. One way to exploit the sensitivity of the ERC technique is to investigate charge motions in mutant rhodopsins. We generated stable, high-level producing (∼10 6 opsins/single cell), HEK293S cell lines of several human rod opsin mutants altered at single amino acids that could support proton exchange processes in the membrane region . Here we demonstrate the nature of the ERC signals obtained from D83N and E134Q opsin pigments regenerated with 11 c Ret. Our previous work suggested that the kinetic relaxation of the R 2 phase of WT human rhodopsin in WT-HEK293S giant cells was kinetically complex . In that study, we found that double exponentials were typically required to reliably fit large R 2 relaxations >100 ms after the flash. In this study, we further our kinetic analysis of WT R 2 relaxation and compare R 2 relaxations from the two mutant pigments to WT. Fig. 9 shows ERC signals in response to the first 500-nm flash after primary regeneration and secondary recovery in fused giant cells containing WT, D83N, or E134Q human rhodopsins. The WT pigment generated strong R 1 signals during the primary bleach. R 1 signals are rarely seen during the secondary or subsequent extinctions indicating that, if present, the size is below the limits of detection at the flash intensities used. Large (>40 pA) WT ERC signals typically require two exponentials to fit the time course of the R 2 relaxation over the first 100 ms. Residuals are shown beneath the ERC waveforms. R 1 signals were not observed in D83N rhodopsin during primary extinction in cells with R 2 charges of the same order as seen in fused WT cells that had R 1 signals. Like WT, the D83N R 2 relaxation typically requires two exponentials to reliably fit its relaxation. However, D83N signals appear to lack the “stretched” exponential appearance seen in many large WT signals during the 100 ms after the flash. The E134Q ERC signal was distinctly different from the WT signal. R 1 signals were not observed during primary extinctions. Moreover, the outward R 2 signals in E134Q rhodopsin-expressing cells were markedly simplified in comparison with WT or D83N ERCs. The relaxation was brief and required only a single exponential to fit its decay. Like WT ERCs, D83N and E134Q ERC signals extinguished with successive flashes and had spectral sensitivity consistent with pigments absorbing ∼500 nm . To begin to characterize R 2 relaxation, single or double exponential functions were fit to a large number of WT, D83N, and E134Q R 2 signals from many cells of similar size range. Time constants associated with R 2 relaxation, but not R 1 , are essentially independent of C mem . Time constants were extracted from the first and second exponential terms (τ a , τ b ). Since the identification of the respective components is dependent upon their weighting, it is possible that the τ b constant could be assigned to the τ a data set if the weighting of the τ a component is small or unreliable (e.g., lower SNR). Therefore, all the time constants obtained for R 2 relaxation were placed into a total ensemble, and histograms were generated from these populations for the WT, D83N, and E134Q datasets . The WT pigment demonstrated a broad skewed distribution suggestive of density around three time constant ranges, whereas the E134Q distribution was simple and symmetrical and the D83N distribution was intermediate. To quantitatively characterize the ensemble of time constants, Gaussian distribution functions were fit to each histogram. The WT histogram was reliably fit by a sum of three gaussian distributions, centered at 4.1, 12.5, and 26.4 ms ( Table ). There were also residuals with time constants longer than the three fitted distributions. This analysis demonstrates the kinetic complexity of the WT R 2 relaxation and supported the conclusion of a minimum of three charge states with distinct lifetimes (see discussion ). The D83N histogram was reliably fit to the sum of two Gaussian functions centered at 3.7 and 10.7 ms, still leaving some residuals. These time constants are comparable with the two fastest time constants measured in the first 100 ms of WT R 2 relaxations, whereas the third τ found in WT appears to be missing ( Table ). The E134Q histogram was distinctly different from both WT and D83N, requiring only a single Gaussian function with a peak centered at 4.4 ms. This single peak overlaps with the fastest time constant seen in the WT and D83N pigments ( Table ). This work establishes an initial approach to parameterize the R 2 relaxation. The intent is to use this approach as a means to quantify differences between WT and mutant ERC kinetics during the biochemically important time period of the R 2 signal. In earlier work, we established that, after regeneration with 11 c Ret, ERC signals could be recorded from both single and fused HEK293S cells expressing high levels of WT human opsin to their plasma membranes . The ERC originated from plasma membrane opsin that was regenerated with chromophore and the amount of charge motion was proportional to the size of the fused giant cell, consistent with the quantity of opsin expressed in the plasma membrane. The fused cell technique offered the advantage of a larger SNR, permitting more complex experiments and observations of greater complexity in the ERC signal (e.g., the R 1 phase) when compared with the single cell system. In this early work, an action spectrum for the R 2 signal was consistent with a normal rhodopsin pigment, but the bandwidth of the filters used (70 nm) made it difficult to conclusively rule out the contribution to the ERC response of other spectral pigment states with absorption ∼500 nm. Critically, it was demonstrated that the application of time-resolved ERC recording to expressed rhodopsin improved measurement sensitivity between 10 7 - and 10 8 -fold when compared with other contemporary methods used to study expressed visual pigments. In the current experiments, we applied the expression ERC tool to investigate physical properties of WT rhodopsin expressed in HEK293S cells. ERC measurements are consistent with normal properties of WT human rhodopsin such as the absorbance spectrum, photosensitivity, univariance, and photoreversibility from Meta-II. The utility of the expression ERC tool was expanded on in the experiments reported here by demonstrating successful measurements of ERCs in cells regenerated with the analogue chromophore 9 c Ret. Moreover, ERCs of two mutant pigments D83N and E134Q demonstrate qualitative and quantitative differences with respect to WT ERCs. These results strongly suggest that the expression ERC approach could be productively expanded to investigate a broad range of rhodopsin activation properties of analogue visual pigments and mutant visual pigments, thus embracing a structure–function approach applied to both the chromophore and remote environments of rhodopsin. When fused giant cells are regenerated (primary) with 11 c Ret in the dark, ERC signals are obtained on high intensity flash stimulation (10 8 photons/μm 2 ) with essentially identical waveform to the ERCs of vertebrate amphibian photoreceptors . The total charge motion in fused giant cells typically exceeds that found in amphibian rod photoreceptors. Both the R 1 and R 2 signals of the WT ERC were observed. The R 1 signal is not well resolved kinetically and is not likely to be since R 1 is thought to report very early charge separation events in the chromophore environment that are orders of magnitude outside the bandwidth of whole-cell patch clamp recording. Thus, only the amplitude of R 1 charge could be measured, but not its rise time. The R 2 signal is completely resolved in these experiments given that the rise of R 2 to its peak is observable in most cells, as is the R 1 to R 2 transition during primary bleaching. The ERC time scale that was well resolved in these experiments extends out from ∼400 μs after the flash. This is sufficient to cover the entire time course of the Meta-I to Meta-II equilibrium at room temperature. At higher bandwidth and lower temperatures, the formation of Meta-I from lumirhodopsin could probably be examined. The temporal limits of investigation of rhodopsin electrical state transitions with whole-cell recording will be the rise time of the flash stimulus, the speed and sensitivity of the amplifier and digitization hardware, cellular capacitance and access resistance, and the decreased SNR expected due to greater noise at wider bandwidths. To regenerate rhodopsin from opsin apoprotein constitutively expressed in these cells, a FAF-BSA technique previously used to regenerate rhodopsin from opsin in intact rod outer segments was adapted to the HEK293S fused cell expression system. The regeneration kinetics (≈3 min) are similar to rates in intact photoreceptors . The combination of fusing single cells to form giant cells and the use of FAF-BSA regeneration permitted much larger ERC signals and the spontaneous recovery of the ERC signal during post-bleach dark adaptation. Experiments reported here indicated that the source of 11 c Ret during spontaneous dark regeneration is internal to the cell that is recorded. Several bleach and recovery cycles were required to completely exhaust the source of chromophore in the presence of extracellular NH 2 OH. If regenerating 11 c Ret chromophore originated from the outer surface of the plasma membrane or external to the cell (e.g., released by other cells on the coverslip or from recording chamber surfaces), then NH 2 OH should rapidly prevent ERC recovery by converting retinaldehydes to oximes, which do not form visual pigments. The only other potential source of chromophore is internal to the cell under recording, shielded from reaction with extracellular 10 mM NH 2 OH. Once NH 2 OH was applied to the bath, plasma membrane visual pigment was partially bleached and spontaneously regenerated over several cycles before the source of intracellular chromophore became exhausted and ERCs did not recover. Prompt recovery followed washout of NH 2 OH and perfusion of 25 μM 11 c Ret plus FAF-BSA in recording buffer. These experiments suggest that 11 c Ret can enter the retinal binding pocket when presented from either side of the membrane. The data also provides indirect evidence that NH 2 OH is impeded from permeating the plasma membrane of the HEK293 cells in E-1/I-1 solutions because if it had, then the spontaneous regeneration should have been promptly quenched. That ERCs are recordable when NH 2 OH has access to the extracellular surface of membrane-oriented rhodopsin indicates that visual pigment regenerated in the dark is not reacting with this agent, consistent with the resistance of the ground state of rod rhodopsin to NH 2 OH. NH 2 OH does not affect the rate of formation of Meta-II . Persistence of ERC R 2 signals in NH 2 OH also suggests that buildup of thermal bleaching intermediates such as Meta-II, Meta-III, or M 470 cannot be contributing to the charge motions of the ERC signals during secondary extinctions because the all-trans-retinal in the ligand pocket of these rhodopsin conformations would react rapidly with NH 2 OH to form opsin and the oxime adduct and extinguish the signal entirely. One of the major advantages of the FAF-BSA/α- d -tocopherol regeneration technique in fused giant cells is the spontaneous recovery of visual pigment and ERC signals without the need for reinstallation of 11 c Ret into the recording chamber. A similar process has been reported for retina isolated away from pigment epithelium and apparently reflects limited photoreceptor stores of 11 c Ret . Makino et al. 1991 also found spontaneous regeneration of ERC signals and used extracellular NH 2 OH (10 mM) to quench the process, indicating that a major fraction of chromophore originated from outside the cell (e.g., partitioned onto chamber surfaces) during active delivery of retinals to the recording chamber. In the experiments reported here, no 11 c Ret or 9 c Ret was unintentionally added to the chamber in our experiments other than perhaps a small amount on the small coverslip fragment holding the giant cells. The FAF-BSA/vitamin E technique appears to be an efficient way to load chromophore into HEK293 cells that express visual pigments. Similar success was found by McDowell 1993 for intact rod photoreceptors using FAF-BSA and for Jones et al. 1989 , where interphotoreceptor retinoid-binding protein was used. The experiments reported by McDowell 1993 were conducted with a 10-fold molar excess of 11 c Ret (25 μM) over opsin and demonstrated pseudo–first-order kinetics. Rotmans et al. 1974 demonstrated that the regeneration rate of opsin in bleached outer segment fragments (τ 1/2 ≈ 10 min) was similar to the decay of Meta-II at 25°C (≈7 min half-life) and proposed that 11 c Ret enters the ligand-binding pocket only after all-trans-retinal has vacated the environment. FAF-BSA can be viewed as a nonspecific retinoid binding protein . The likely mechanism of its action is that FAF-BSA effectively solubilizes chromophore in an aqueous environment and permits efficient transfer into lipid membranes. Once partitioned into cellular membranes, retinoid transfer into bleached pigments can be rapid . Curiously, the rate limiting enzyme in the visual cycle of rodent rod photoreceptors, all-trans-retinol dehydrogenase/reductase, is also expressed in kidney . This raises the interesting hypothesis that HEK293S cells may have some capacity for retinoid metabolism. HEK293S cells are a suspension-adapted clone of transformed human embryonic kidney cells. As such, they are likely to express both general human cell housekeeping genes as well as some genes specific for the kidney. This cellular system has been a popular expression environment for heterologous genes including human and bovine opsins over the last decade . Spontaneous regeneration of visual pigment after 11 c Ret loading in FAF-BSA/vitamin E provides parsimonious support of ERC experiments. The regeneration of ERCs in WT-HEK293S cells from an internal source of 11 c Ret provides a clear advantage for complex experiments where charge motions must be compared against different conditions (e.g., action spectra determination). Two properties of ERCs measured in this environment are under further active investigation (Brueggemann and Sullivan, manuscript in preparation). First is the loss of the inward R 1 signal after post-bleach spontaneous regeneration. The time to peak of the R 2 signal is slowed approximately twofold when the R 1 signal is present , possibly suggesting that the R 1 signal represents additional state transformations. In cells that were primarily loaded with 11 c Ret and studied under whole-cell recording for periods of time >30 min, a slow accumulation of R 2 charge (approximately twofold) occurred . The mechanism of this process is not yet clear. Strong evidence is demonstrated that ERC signals result from activation of the ground state of human rhodopsin in the plasma membrane of fused giant cells and that no other additional spectral states are contributing to these measurements. Evidence supports the conclusion that the identity of the chromophore regenerating visual pigment is 11 c Ret, which forms a PSB + -H with K296 of the opsin apoprotein. The action spectrum of the ERC of WT human rhodopsin, when stimulated through 30-nm bandpass filters, was well fit by a scaled normalized template of the absorbance spectrum of WT human rhodopsin purified from the same cell line used for ERC studies. The action spectrum was obtained from cells that had already had their ERCs extinguished and had undergone spontaneous recovery. These experiments allow the conclusion that the ground state of rhodopsin is the one that regenerates upon dark adaptation after primary ERC extinction. Thus, during dark adaptation, the Meta-II state must decay with hydrolysis of the Schiff base to form opsin, which then can subsequently react with 11 c Ret to form a protonated Schiff base at K296. From our previous work on spectral sensitivity, which was conducted with 70-nm bandpass filters, the action spectra was much broader than rhodopsin absorbance. Data from the experiments reported here shows that this outcome was the result of stimulus bandwidth, and not the presence of additional spectral states that were contributing to charge motion. Specifically, there were three pigments that could have contributed to “recovery” of the ERC when later stimulated with 500-nm stimuli: Meta-III 465 and Meta 470 , both resulting from thermal Meta-II 380 decay , and isorhodopsin resulting from photoregeneration from early intermediates. The formation and lifetime of Meta-III and Meta 470 are consistent with the kinetics found for ERC recovery (≈3.0 min). However, if these states contributed substantially to the ERC, the action spectrum would not have been fit by the absorbance of rhodopsin and the signals should have been promptly extinguished by 10 mM NH 2 OH . Given the difference in peak absorbance of bovine and human rhodopsin, these three pigments in isolation would have produced action spectra with peaks around 460, 475, and 483 nm. The human rhodopsin absorbance spectrum, peaking at 493 nm, provided a good fit to the action spectra peak and there was no evidence of a significant blue shift of the ERC data. In addition, the bandwidth of the ERC action spectrum was consistent with rhodopsin absorbance. Thus, we conclude that the pigment underlying the ERC after primary regeneration and secondary recovery is rod rhodopsin, which was not sensitive to NH 2 OH in the dark. The rapid recovery of ERC signal over 10-min periods suggests that long-lived photointermediates such as Meta-III or Meta 470 are not extensively populated in this expression system. If these or other pigment states are present, their contribution to the ERC must be minimal. This conclusion is substantiated by the similar P t values for ERC extinction after primary regeneration and secondary recovery. If additional states were involved it is likely that the P t parameter would have changed significantly. The ERC signal can be extinguished with successive flashes in a fashion similar to rhodopsin bleaching in photoreceptors and with similar photosensitivity. The mean and maximum P t values (2.6 × 10 −9 and 5.0 × 10 −9 μm 2 , respectively) determined from exponential extinction decays are consistent with a rhodopsin photopigment but are lower than expected (1.0 × 10 −8 μm 2 ), taking the product of the molecular cross section of human rhodopsin at 493 nm (α 493 = 1.528 × 10 −8 μm 2 , see materials and methods ) and the known quantal efficiency (γ = 0.67) . The mechanism for the approximately twofold suppression of P t is considered. One would not expect any significant pigment orientational effect in the fused cell system because rhodopsin is expressed in a membrane with an essentially spherical geometry at the time of recordings and any orientation should increase P t . The P t values obtained from ERC extinctions of R 2 in amphibian rods (7.6 ± 2.2 × 10 −9 μm 2 ) originate predominantly from an A 2 pigment with a lower peak extinction coefficient than human rhodopsin (≈30,000 vs. 40,000 M −1 · cm −1 ) . Since cells were regenerated with 11 c Ret, an A 1 pigment is expected and found upon purification of rhodopsin from the same cell line. Therefore, the suppressed values of P t must have a different origin. Our findings may reflect, in part, the variability of extracting the P t parameter from extinction experiments . The accumulation of bleaching intermediates is unlikely because the extinction coefficients of Meta-III or Meta 470 are similar to that for rhodopsin and would not be expected to suppress P t . Moreover, the accumulation of these intermediates was essentially excluded by ERC persistence in NH 2 OH (and further P t suppression) and the fitting of the ERC action spectrum by the absorbance spectrum of WT rhodopsin. Photoregeneration from early intermediate states with high quantal efficiency for photoconversion (e.g., bathorhodopsin, lumirhodopsin) can definitively lower P t estimates for the absorbing pigment . Makino et al. 1991 found no photoregeneration in ERC experiments on rod and cone photoreceptors when P t was measured using flash strengths that were typically about an order of magnitude less intense than used here but with durations ∼20-fold longer (300 μs) that probably overlapped with the Meta-I lifetime and the Meta-I ⇔ Meta-II equilibrium. Flashes of this duration were, however, unlikely to overlap significantly with the lifetimes of earlier intermediates that have higher quantal efficiency of photoconversion . At room temperature in these experiments, the only bleaching intermediates that could overlap with the 14-μs flash duration are bathorhodopsin, the blue-shifted intermediate, and lumirhodopsin. Lumirhodopsin is completely formed by 1 μs at room temperature , and is likely to have been the most populated state during the flashes presented. The flash duration in these experiments would not overlap the rate of Meta-I formation at room temperature (100 μs) . The stimuli used in these experiments should have no influence on the Meta-I ⇔ Meta-II equilibrium, which correlates in part with the R 2 time course . In extracting P t from R 2 extinction, we are assessing early photochemical processes of absorption through examination of charge motions of later conformational states that are thermal and not photochemical in origin. If we use the mean P t obtained at 500 nm (2.6 × 10 −9 μm 2 ), we can calculate the quantal efficiency assuming realistically that the molecular cross section is constant (1.23 × 10 −8 μm 2 ). This results in an estimate for γ of 0.21 that is only ∼32% of the known value of 0.67 determined from small bleaches. How could γ be decreased to explain the suppression of P t ? In fact, Williams 1964 , Williams 1965 , Williams 1966 , Williams 1974 have shown that γ is a function of light intensity. When the flash intensity is infinitely strong, γ should approach zero. γ is expected to decrease in proportion to the intensity of the flash stimulation because photoconversion becomes increasingly likely (even-numbered absorptions) from intermediates with lifetimes of the same order as the flash duration. Hagins 1955 and Williams 1964 , Williams 1965 , Williams 1974 have shown that the maximum fraction of rhodopsin bleached in a single brief flash can be only 50% because of this process. At the flash intensities used in these experiments (e.g., 500 and 70 nm FWHM, 4.08 × 10 8 photons/μm 2 ), we estimate the fraction of rhodopsin molecules absorbing at least one photon to be ∼0.963 using the crude estimates obtained from the Poisson distribution (see materials and methods ). However, only odd-numbered absorptions go on to bleaching, whereas even numbered absorptions photoconvert. The fraction of even-numbered photoregenerating absorptions is ∼50% at the flash intensity used. Therefore a maximum of 50% of rhodopsin molecules bleach with each flash, although there are sufficient photons in a single flash to bleach all molecules present. The amount of rhodopsin bleaching with each flash, then, is a constant fraction of unbleached molecules remaining. This explains why the extinction followed a single exponential decay function. It also explains why P t is suppressed by at least 50% in our measurements. The R 2 signal reports only those rhodopsin molecules that absorb an odd number of photons and move forward through later intermediates to bleaching. It is important to mention that the ERC R 2 kinetics should not be influenced at all by photoregeneration because the flash duration is about two orders of magnitude more brief than the electrical state transitions measured. The shape of the R 2 waveform does not change with sequential flashes even though the amplitude decreases and no evidence for photoregeneration was found when examining the extinction of the R 2 signal or the homogeneous kinetics of R 2 relaxation with different flash intensities. Thus, our findings are consistent with a significant suppression of P t of WT rhodopsin, likely because of the high probability of early state photoconversion using the stimuli employed in these experiments. The intermediate likely to be significantly populated during the highest photon fluxes in these experiments is lumirhodopsin since it is completely formed by 1 μs and is the only state present during the bulk of photon irradiance (rise time ≈ 5 μs). High intensities were used to obtain good estimates of the cross section (α) in the spectral measurements and were maintained throughout because of the high SNR obtained in ERC recordings. In fact, P t approximates the value expected when stimulus intensities (500 nm) are nearly a log-fold lower (see results ). In future experiments, the effects of a range of flash intensities on P t and quantal efficiency will be investigated. In addition, we will also investigate the mechanism for the marked suppressive effect (≈1 log) of NH 2 OH on P t , which appears to be a novel finding. We speculate that NH 2 OH has the capacity to bind in retinal binding pocket and perhaps alter the local chromophore environment in the ground state in such a manner as to result in altered photochemical properties. Experiments were conducted to test whether the ERC can be used to study rhodopsin photoregeneration from the Meta-II state. These experiments were motivated by earlier studies that reported reversed ERP signals from the Meta-II state upon near UV stimulation . As discussed above, photoregeneration from early intermediates is likely to be responsible for the suppressive effect on estimation of P t using the ERC method. To further explore photoregeneration as a molecular conformational process, we tested whether the ground state of rhodopsin could be achieved by photolytic activation of Meta-II, which has a lifetime accessible to xenon flash stimulation and cellular ERC measurement. Near UV flashes overlapping with Meta-II 380 absorption and lifetime were presented immediately after extinction of 500 nm ERC responses and promoted rapid recovery of the ERC signal in a manner inconsistent with the normal chemical regeneration by 11 c Ret. Meta-II is known to have an extinction coefficient slightly larger than rhodopsin, and photocoversion of Meta-II to rhodopsin has been reported to occur with a quantal efficiency of ∼0.2 . Using estimates of the extinction coefficient for Meta-II between 33,000 and 49,200 M −1 cm −1 and the Poisson equation, the fraction of Meta-II molecules absorbing at least one photon would be 25–35%/flash with 80–90% of these being single photon absorptions at the 350-nm photon densities delivered (8.02 × 10 7 photons/μm 2 ). The inverted ERC signals that were only seen in large cells are consistent with inverted ERP signals that have been recorded from the retina . These results are consistent with a net reversal of charge flow during photoregeneration from Meta-II. The reverse state path is likely to be different compared with the forward activation pathway given that the amount of charge is much smaller (10–20%) and appears to have a considerably slower time course with respect to the peak times of the positive R 2 signals that associate with Meta- II formation. Complete reversibility of the transitions would be expected to result in charge motions of the same magnitude but different polarity. Arnis and Hofmann 1995 recently showed that photoregeneration of rhodopsin from the active Meta-II state proceeds by isomerization of the chromophore and Schiff base reprotonation, but the ground state was achieved on a much slower time scale and was due to thermal and not photochemical conformational changes. Such slow events would not be seen in these current recordings, but might be reflected in ERP (voltage) recordings where the charge flow can be integrated on the membrane capacitance. Therefore, the small inverted ERC signals that we see with UV flashes could reflect dipolar rearrangements in the chromophore pocket or the reprotonation of the Schiff base, perhaps from the E113 protonated counterion, which is located more toward the extracellular surface of rhodopsin . Or it could reflect release of protons from the cytoplasmic surface . Future experiments will focus on the rate of thermal disappearance of the Meta-II state from which photoregeneration most likely occurs and the molecular origin of the inverted charge flow (e.g., pH dependence). The quantal efficiency of the photoregeneration process appears favorable to study the mechanism in greater detail in even larger cells. When WT-HEK293S cells are regenerated with 9 c Ret, ERC signals are recordable upon 500-nm flash photolysis. 9 c -Ret regenerates a visual pigment, presumed to be isorhodopsin (peak ≈ 483 nm), which would broadly overlap with the 70-nm band stimulus. That ERC signals can be recorded from a “natural” analogue pigment provides evidence to support a role for the ERC in investigation of a wide variety of analogue visual pigments that are known to have unique properties. For example, certain locked analogues are known to prevent energy uptake by preventing isomerization . Others block the bleaching sequence at discrete spectral states, for example, 9-cis-desmethyl-retinal blocks the thermal bleaching pathway at Meta-I . Investigation of analogue pigments can now be extended to the vectorial charge flows that are orthogonal to the chromophore plane. Similarly, analogue retinals could regenerate opsins with engineered side-chain mutations in the ligand-binding pocket to investigate environmental interactions that shape chromophoric properties and activation behavior. A major challenge in structure–function studies of visual pigments has been the preparation of sufficient mutant protein in expression systems for biophysical or biochemical analysis. The ERC method improves detection sensitivity by 10 7 –10 8 -fold, allowing measurements of conformational activation in an ensemble of regenerated rhodopsin molecules in the physiologically intact environment of a single fused giant cell . In applying the ERC to mechanistic studies of conformational activation, a number of rhodopsin mutants were screened at sites of potential proton exchange reactions in the membrane environment of the protein. The time course of the expression ERC R 2 signal overlaps with the temporal scale of Schiff base deprotonation and Meta-II formation, as well as the related proton uptake into the cytoplasmic face of the pigment and the conformational transition to the biochemically active Meta-II b state . Since known mutants can affect various steps in this activation process, a quantitative structure–function investigation on opsin mutants was initiated in this ERC study. A mutation in the PSB + -H environment (D83N) on the second α helix was found to perturb the kinetics of the R 2 ERC phase. A mutation on the cytoplasmic face of the third α helix (E134Q), at a residue known to be a gatekeeper to both proton uptake and the related generation of the transducin docking environment, results in loss of most of the ERC R 2 signal except for the fast initial process. The R 2 signal is well resolved in cellular ERC measurements and overlaps temporally with critical events leading to biochemical activation of rhodopsin. WT rhodopsin R 2 relaxation kinetics are an easily accessible aspect of the total ERC signal and are invariant to intensity or wavelength of stimulation as found in these experiments. Therefore, the R 2 relaxation should be a reliable parameter to evaluate for understanding conformational dynamics. A quantitative analysis of R 2 relaxation in WT rhodopsin should serve not only to begin characterization of the state complexity of charge motion on the millisecond time scale, but also serve for comparison of mutant pigments that might be affected in some aspect of R 2 electrical state transitions. WT rhodopsin R 2 relaxation was first characterized by fitting sums of exponentials to a set of WT ERC waveforms and generating an ensemble of time constants (τ a,b ). We assumed that the ensemble of time constants would represent the kinetic aspects of the signal even with any slight heterogeneity (e.g., due to cell size) that might affect only the fastest aspects of the relaxation . Treating the entire time constant data set as an ensemble is reasonable because the assignment of a value to τ a or τ b is somewhat arbitrary given that the short time constant may not have been weighted or fit in a particular ERC signal and a longer time constant was then assigned to τ a . In fact, there is some overlap in the assignments . Also, analysis of an ensemble of ERC signals is more likely to identify additional complexity for quantification than can be obtained in fittings to single ERC waveforms. A histogram was generated from the ensemble of the total WT time constants data set . The histogram is not consistent with a single distribution of time constants. While there was a prominent peak around 4 ms, there is significant weighting outside this band leading to a skewed distribution with additional peaks being apparent. To characterize the distribution of time constants in the R 2 relaxation, we fit sums of Gaussians to the (skewed) histogram of the entire τ a,b ensemble. We attempted fits of one, two, three, and four Gaussians to the data. With this data set, a reliable fit of the sum of three Gaussians was obtained that was independent of bin width. Three Gaussian peaks were centered around 4.1, 12.45, and 26.4 ms, and the errors in the fitting results are shown in Table . The natural conclusion from this analysis is that there are a minimum of three distinct electrically active states in the WT R 2 relaxation at room temperature in WT rhodopsin. We speculate that the underlying molecular basis of the three fitted time constants are likely to represent processes related to Schiff base deprotonation, proton uptake, and α helical movements associated with conformational activation. However, other interpretations are possible. For example, the capacitative, or AC-coupled nature of the ERC could reflect forward and reverse movements of charges (e.g., protons, sidechains), in particular molecular environments, leading to a series of coordinated charge separation and recombination events, each satisfying a zero-time integral expected for pure capacitative components . We have not demonstrated a net DC component to the ERC. Further investigation will be necessary to examine these hypotheses and to determine the uniqueness of the time constants determined. We anticipate that dissection of the ERC R 2 signal can be achieved with mutations at protein side chains likely to participate in charge transfer events. ERC signals from D83N rhodopsin (regenerated with 11 c Ret) appear slightly simplified in comparison with WT. The R 2 signal appears WT in nature, but shorter in comparison to the broad stretched relaxation of the WT pigment. D83N ERCs were subjected to R 2 relaxation time constant analysis, and the sum of two Gaussian distributions was required to best fit the total time constant histogram. Peaks were identified at 3.67 and 10.67 ms, and the errors in the fitting are shown in Table . These times appear comparable to the fast and medium time constants in the WT pigment that was measured under identical conditions. The third and longest time constant of the R 2 relaxation in WT (26.4 ms) is missing in the D83N pigment. We suspect this is likely to represent a distinct property of the D83N pigment since R 2 relaxation in D83N ERCs have a different waveform compared with WT. In summary, D83N loses the R 1 signal during primary bleaches and its R 2 relaxation loses the “stretched exponential” characteristic of the WT R 2 relaxation. The D83N mutation is known to slightly blue-shift the absorbance of rhodopsin, which is consistent with an alteration of the Schiff base environment in the ground state pigment, resulting from the isomorphic loss of the protonated carboxyl group of aspartate . However, D83N pigment in detergent has the capacity to form Meta-II and to activate transducin, but these properties have not been examined under rapid time-resolved conditions . Weitz and Nathans 1993 concluded that the D83N mutation leads to a more favored formation of Meta-II compared with WT rhodopsin by analysis of difference spectra. The D83 sidechain is located in the immediate environment of the protonated Schiff base and is a highly conserved residue in G-protein–coupled receptors . Therefore, it could play an as yet unrealized role in rapid activation processes. Fahmy et al. 1993 found a shift in carboxyl signals in the WT Meta-II/rhodopsin infrared difference spectra that was lost in the D83N mutant. They suggested that the D83 sidechain undergoes an increase in hydrogen bonding during Meta-II formation, although they could not explicitly rule out a transient deprotonation/reprotonation reaction on the path to Meta-II. Ganter et al. 1988 , Ganter et al. 1989 suggested that the formation of Meta-I from lumirhodopsin was associated with carboxyl groups in the membrane environment undergoing such reactions. This is an area where ERC recordings are likely to be useful because of the extended bandwidth and profound relative sensitivity compared with most spectroscopic techniques. The apparent loss of the R 1 signal is particularly interesting because this signal is believed to originate from charge separation of the protonated Schiff base from its counterion during isomerization . It may also be that later thermal transitions contribute to the inward R 1 charge flow and that the D83N mutation may prevent these states during primary extinction, where we usually see R 1 signals. Since the recording conditions are otherwise constant between WT-, D83N-, and E134Q-expressing cells (e.g., similar access resistance and similar ranges of membrane capacitance), it is unlikely that the loss of the R 1 signal is purely due to changes in the measurement under whole-cell voltage clamp . The loss of R 1 signal in the D83N pigment leads us to consider that side-chain interactions in the ligand binding pocket near the Schiff base could contribute to R 1 . The loss of R 1 in the WT pigment after primary extinction may simply mean that the spatial order of such interactions is not rapidly achieved under the conditions of these experiments. Finally, the apparent simplification of the R 2 relaxation in D83N may indicate that the path to Meta-II is slightly different with respect to the WT pigment, even though Meta-II ultimately results. Further experiments will be necessary to investigate these ideas as we use a variety of approaches to dissect the R 2 relaxation into quantifiable biophysical processes. ERC signals from E134Q rhodopsin have little complexity in comparison with WT and demonstrate no R 1 and a brief R 2 relaxation always well fit by a single exponential. When R 2 relaxation time constant analysis was applied to the E134Q pigment, a single Gaussian was needed to fit the time-constant histogram. The value of the peak was 4.4 ms, which was comparable within error to WT fast phase. The timing of the fast phase did not appear to change when the cytoplasmic pH was held at different constant values over the range from 6.0 to 8.0. The E134 sidechain is known to be essential to proton uptake into the cytoplasmic face of rhodopsin during normal Meta-II formation given that the E134Q mutant binds zero protons in comparison with the two adsorbed by the WT pigment . The sites of binding of the protons are unknown, but the E134 sidechain could titrate one proton. The loss of the medium and slow ERC time constants and the stretch beyond 50 ms in the WT R 2 relaxation suggest that these components might represent proton movements or other charge motions gated by proton uptake. E134Q, when regenerated with 11 c Ret in the dark, is capable of activating transducin in an enhanced manner in comparison with the WT pigment after light stimulation . The E134/R135 charge pair is known to be an essential switch to transducin docking and activation and is screened from solution once transducin is bound to R* . During photoactivation to R*, a rigid body motion of α helix VI relative to α helix III (the location of E134/R135) has been detected. This results in helix VI being displaced outward from the disk membrane into the cytoplasm, although the magnitude of the vectorial movements are not yet clear . The E134Q mutation promotes movements of the III and VII α helices in the dark that are not affected by light and do not affect the subsequent movement of α helix VI. What results is a partial activation of the receptor in the darkness or constitutive activity . The movements of α helices may be normally coupled, but can still occur independently in the presence of environmental perturbation (e.g., mutation). Since spectral Meta-II formation is not perturbed in E134Q, we suggest that the residual fast ERC signal in this mutant represents the charge motion associated with Schiff base deprotonation. In preliminary experiments, the residual R 2 time constant of E134Q did not demonstrate sensitivity to intracellular pH over the range of 6.0 to 8.0 and might reflect the pH-insensitive component of the ERP related to Schiff base deprotonation . By elimination, the remaining medium and slow time constants of the WT R 2 relaxation could be associated with proton uptake into the cytoplasmic face and the subsequent conformational transformation of the rhodopsin that is gated by this chemical potential (e.g., macrodipole movements of α helices). If so, then these later time constants in the WT pigment should be responsive to changes in intracellular pH or to molecular conditions that prevent the conformational activation of the molecule that allows transducin binding. Further experiments will be necessary to decipher the molecular origin of the charge motions during R 2 and how local environment affects these transitions. We were surprised that no R 1 signals were found in fused E134Q giant cells given that the E134 sidechain resides at the cytoplasmic face of the third α helix remote from the Schiff base environment where R 1 is thought to originate. However, total charge motion in E134Q cells is smaller than in WT cells and the R 1 signal may simply have been lost in noise, as we found for R 1 in single unfused cells expressing WT pigment regenerated with 11 c Ret . However, in a separate cell line of E134Q (E134Q-12) that expresses three to four times more mutant opsin , the R 1 signal was also missing and the R 2 relaxation is still simple and single exponential in character (Brueggemann and Sullivan, manuscript in preparation). In future efforts, the stoichiometry of charge motion in the E134Q pigment will be compared with WT. Given that R 1 is likely to be definitively lacking, a model should be considered wherein the membrane-buried Schiff base environment near the middle of the membrane and the proton uptake environment on the cytoplasmic face of the membrane might communicate on a very rapid time scale during activation. Although we do not yet have a molecular understanding of the simplification of the ERC R 2 signal in D83N or E134Q, such mutant pigments with uniquely different ERC signals constitute a substrate to understand the components of the normal ERC signal in WT rhodopsin. At this point, our efforts have clearly established the feasibility of using the ERC to study time-resolved and electrically active conformational changes during rhodopsin activation in WT and two mutant pigments. Future studies will focus on the molecular mechanism of R* formation and the forces that govern the molecular volume increase of the pigment during the Meta-II a to Meta-II b transition in WT and mutant pigments . The sensitivity of the expression ERC method is greatly improved when compared with other contemporary time-resolved techniques applied to rhodopsin activation and allows measurement of rhodopsin activation in single cells containing about a picogram of visual pigment. The sensitivity of this approach is dependent upon the use of gigaohm-seal, whole-cell patch clamping techniques that are capable of resolving small currents with fast time resolution. The ERC is a conformation-dependent charge motion of the same family as ionic channel gating currents. Gating current analysis of expressed mutant ionic channels has significantly extended knowledge of molecular processes of conformational activation of ionic channels, and for this reason we are applying the same approach to visual pigments that are known to be electrically active proteins. In this work, we apply the expression ERC approach to measure properties of WT rhodopsin and found that the ERC signal results from activation of the ground state of rhodopsin, given the action spectra and bleaching photosensitivity. ERC kinetics are independent of wavelength, as expected for the univariance principle, and rhodopsin can be photoregenerated from the Meta-II state. The major impact of this study resides in our success at recording ERCs from an analogue visual pigment (isorhodopsin) and from two mutant human opsins regenerated with 11 c Ret. This study establishes the feasibility of an ERC structure/function investigation applied to the ligand binding pocket, as probed by artificial chromophores or site-specific mutations, or remote regions of the pigment, as probed by engineered mutations or chemically reactive ligands. The ERP has been similarly used in expressed mutant bacteriorhodopsin pigments to understand proton exchange mechanisms . The similarity of the visual rhodopsin ERC/ERP and the bacteriorhodopsin ERP is striking and suggests that similar underlying mechanisms of conformational transitions may be conserved over evolutionary time. The ERC measures a vector of activation across the membrane plane in a population of oriented molecules under physiologically intact conditions not dissimilar to the environment of activation in a photoreceptor. We anticipate that this approach will lead to further understanding of the energies, forces, and molecular processes of rhodopsin activation on a rapid time scale. | Study | biomedical | en | 0.999997 |
10532962 | Type II sodium/phosphate (NaPi-II) 1 cotransporters belong to a unique class of Na + -coupled cotransport proteins that show no amino acid homology to other known cotransport proteins . They can be further subdivided into two subgroups, type IIa and IIb, based on specific motifs in the COOH-terminal region. Type IIa cotransporters are only expressed in the proximal tubule of the kidney, whereas type IIb are expressed in several tissues, such as lung, small intestine, and testis . Members of these subgroups share an overall amino acid homology of ∼57% . Nevertheless, the currently predicted secondary structure indicates that membrane spanning regions and some interdomain linking regions have considerably higher homology between the two subgroups. This suggests that these regions are responsible for the general functional characteristics that all type II Na + /P i cotransporters share, such as high affinity for P i , Na + dependence, and electrogenicity. Although the transport kinetics of both subgroups are well characterized , nothing is known about functionally important sites and domains within the molecule itself that determine the kinetic characteristics of the type II system, such as voltage dependence, substrate specificity, and substrate affinity. One technique that offers considerable potential for identifying functionally important residues and/or domains is the substituted-cysteine-accessibility method combined with the application of methanethiosulfonate (MTS) derivatives that react specifically with cysteine residues . In particular, charged, and therefore membrane impermeant, MTS derivatives have been used extensively to investigate the topology and structure of membrane-spanning proteins . For example, the topology of the serotonin transporter and the K + -pore Kv2.1 have been studied by substituting several amino acids with cysteines . This technique has also enabled identification of functionally important regions of the glutamate carrier EAAT1 and the sodium/glucose cotransporter . Moreover, the recent findings by Loo et al. 1998 that the accessibility of a substituted cysteine (Q457C) in the putative sugar-translocation domain of SGLT-1 to MTS reagents is dependent on substrate and membrane potential, and their finding of a correlation between pre–steady state charge movements and fluorescence changes of the labeled cysteine strongly supports the notion that ligand- and voltage-induced conformational transitions are responsible for coupling Na + and glucose transport. In the present study, we have adopted a cysteine replacement strategy and substituted 15 selected amino acids with cysteine residues with the aim of identifying sites where the reaction with MTS reagents would lead to a detectable change in transport function. Having no precedent for the selection of residues, we based our choice on the following criteria: (a) residues located between hydrophobic and hydrophilic regions were chosen because these intervening regions could be likely candidates for substrate binding or conformational changes during the transport process , (b) serine or alanine residues, where present, were selected for cysteine substitution to minimize changes in the protein, and (c) residues in the amino or carboxy termini were not mutated because these are most likely located intracellularly. Furthermore, based on the evolutionary tree of Na + -coupled P i cotransporters, both termini are located in quite variable regions , which suggested they are not involved in basic substrate binding or transport processes. We have used the Xenopus oocyte expression system and electrophysiological methods to characterize the results of the mutagenesis. We report that of the 15 mutants assayed, only one (S460C) was sensitive to MTS reagents. We show that this mutant, under normal conditions, behaved essentially the same as the wild-type (WT) protein insofar as its kinetic characteristics were concerned. However, after exposure to membrane-impermeant MTS reagents, the kinetic properties of the chemically modified protein suggested that the native Ser-460 lies in a region involved in voltage-dependent conformational changes during the cotransport process. Moreover, Ser-460 appeared to be neither involved directly in the binding of the first Na + ion, nor the subsequent P i binding, but alkylation of the substituted cysteine at this site led to an inhibition of the final cotransport transition. Mutations were introduced following the Quickchange Site-Directed Mutagenesis Kit manual (Stratagene Inc.). In brief, 10 ng of the plasmid containing the rat NaPi-IIa cDNA were amplified with 2.5 U PfuTurbo ® (Strategene Inc.) DNA polymerase in the presence of 250 nM of primers. PCR amplification was performed with 20 cycles of 95°C (30 s), 55°C (1min), and 68°C (12 min). Next, 10 U of Dpn I were added directly to the amplification reaction and the sample was incubated for 1 h at 37°C to digest the parental, methylated DNA. XL1-blue supercompetent cells were transformed with 1 μl reaction mixture and plated onto LB-ampicillin-methicillin plates. The sequence was verified by sequencing. All constructs were cloned in pSport1 (GIBCO BRL). In vitro synthesis and capping of cRNAs were done by incubating the rat NaPi IIa constructs, previously linearized by NotI digestion, in the presence of 40 U of T7 RNA polymerase (Promega) and Cap Analogue (New England Biolabs Inc.) . Yolk-free homogenates were prepared 3 d after injection (H 2 O or cRNA). Pools of five oocytes were lysed together with 100 μl of homogenization buffer [1% Elugent (Calbiochem) in 100 mM NaCl, 20 mM Tris/HCl, pH 7.6], by pipetting the oocytes up and down . To pellet the yolk proteins, samples were centrifuged at 16,000 g for 3 min at 22°C. 10 μl of the supernatant in 2× loading buffer [4% sodium-dodecyl sulfate (SDS), 2 mM EDTA, 20% glycerol, 0.19 M Tris/HCl, pH 6.8, 2 mg/ml bromphenol blue] were separated on an SDS-PAGE gel, and separated proteins were transferred to a nitro-cellulose membrane (Schleicher & Schuell, Inc.). The membrane was then processed according to standard procedures using a rabbit polyclonal antibody raised against an NH 2 -terminal synthetic peptide of the rat NaPi-IIa cotransporter. The specificity of the antibody has been demonstrated previously . Immunoreactive proteins were detected with a chemiluminescence system (Pierce). Groups of five oocytes expressing C460S or the WT protein were incubated for 5 min in 100 μM 2-aminoethyl MTS hydrobromide (MTSEA)–Biotin. Biotin-streptavidin precipitation was performed as described previously : briefly, after taking a sample for Western blotting, the oocyte homogenate was incubated for 2 h with streptavidin beads and precipitated proteins were eluted with 2× loading buffer at 95 o C for 5 min. Samples were loaded on an SDS-gel and immunoblotted after protein separation. Stage V–VI oocytes were prepared as previously described . Oocytes were incubated in modified Barth's solution (see below). Typically, 10 ng of cRNA in 50 nl of water were injected per oocyte and experiments performed 4–6 d after injection. All standard chemicals and reagents were obtained from either Sigma Chemical Co. or Fluka AG. The MTS reagents, MTSEA, [2-(triethylammonium)ethyl] MTS bromide (MTSET), and sodium(2-sulfonatoethyl) MTS (MTSES), were obtained form Toronto Research Biochemicals and freshly prepared in DMSO. The concentration of DMSO did not exceed 1% and control experiments indicated no effect on transport function by DMSO at this concentration. The solution compositions (mM) were as follows. (a) Oocyte incubation (modified Barth's solution): 88 NaCl, 1 KCl, 0.41 CaCl 2 , 0.82 MgSO 4 , 2.5 NaHCO 3 , 2 Ca(NO 3 ) 2 , 7.5 Tris, pH 7.6, and supplemented with antibiotics (10 mg/liter penicillin, streptomycin). (b) Control superfusate (ND100): 100 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , and 5 HEPES, titrated to pH 7.4 with KOH. For pre–steady state recording, where necessary, isomolar BaCl 2 was substituted for CaCl 2 to reduce contamination from endogenous Ca 2+ -activated Cl − currents that were observed for V > −10 mV, except for experiments involving phosphonoformic acid (PFA), which otherwise complexes with Ba 2+ . (c) Control superfusate (ND0): as for ND100, but with N -methyl- d -glucamine or choline chloride replacing Na + to maintain iso-osmolar external solutions. Solutions were titrated with HCl and KOH to pH 7.4. (d) Substrate test solutions: inorganic phosphate (P i ) was added to ND100 from a K 2 HPO 4 /KH 2 PO 4 stock preadjusted to pH 7.4. For PFA-containing solutions, to take account of this being a trisodium salt, the Na + concentration of the control solution was increased by 9 mM to maintain the same transmembrane Na + gradient. The procedure used for the 32 P-uptake assay has been described in detail elsewhere . 32 P-uptake was measured 3 d after injection of both water- and cRNA-injected oocytes ( n = 5). The standard two-electrode voltage clamp technique was used as previously described . Oocytes were mounted in a small recording chamber (100 μl vol) and continuously superfused (5 ml/min) with test solutions precooled to 20°C. Freshly prepared MTS reagents were applied to the oocyte chamber via the common superfusion manifold or using a 0.5-mm diameter cannula positioned near the cell and fed by gravity. Unless otherwise indicated, the steady state response of an oocyte to P i was always measured at a holding potential (V h ) = −50 mV in the presence of 100 mM Na + . Data were acquired online using DATAC software and compatible hardware and sampled at more than twice the recording bandwidth. Recorded currents were prefiltered using an eight-pole low pass Bessel filter (Frequency Devices, Inc.) that was set to 20 Hz for the steady state and 500 Hz or 2 kHz for the pre–steady state measurements. To increase the signal resolution for pre–steady state measurements, we also employed capacitive transient subtraction. The modified Hill equation was fit to the dose-response data: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I_{{\mathrm{p}}}=I_{{\mathrm{pmax}}}{ \left \left[S\right] \right ^{{\mathrm{n}}}}/{ \left \left \left[S\right] \right ^{{\mathrm{n}}}+ \left \left(K_{{\mathrm{m}}}^{s}\right) \right \right ^{{\mathrm{n}}}{\mathrm{,}}}\end{equation*}\end{document} where [ S ] is the substrate concentration, I pmax is the extrapolated maximum current, K m s is the concentration of substrate S , which gives a half maximum response or apparent affinity constant, and n is the Hill coefficient. Pre–steady state charge movements were quantified by first subtracting records obtained in 3 mM PFA to eliminate endogenous currents. An exponential fitting routine, based on the Chebychev transform was used to estimate relaxation time constants. Difference records were integrated to obtain the charge ( Q ) as a function of transmembrane voltage (V). Q –V data were then characterized by fitting a single Boltzmann function: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}Q=Q_{{\mathrm{hyp}}}+ \left \left({Q_{{\mathrm{max}}}}/{ \left 1+{\mathrm{exp}} \left \left[{-ze \left \left({\mathrm{V}}-{\mathrm{V}}_{{\mathrm{0.5}}}\right) \right }/{kT}\right] \right \right }\right) \right {\mathrm{,}}\end{equation*}\end{document} where Q max is the maximum charge translocated, Q hyp is the steady state charge at the hyperpolarizing limit and depends on the holding potential, V 0.5 is the voltage at which the charge is distributed equally between the two states, z is the apparent valency per cotransporter, e is the electronic charge, k is Boltzmann's constant, and T is the absolute temperature. Fig. 1 shows the location of the residues within the rat isoform of the Na/Pi-IIa protein that we mutated individually to cysteines according to the above criteria. All mutations were confirmed by DNA sequencing and were identical to the WT except for the appropriate base changes. Each of the 15 mutants was expressed in Xenopus oocytes and tested for electrogenic transport activity under whole cell voltage-clamp conditions. For this initial functional assay, oocytes were challenged with a nearly saturating concentration of P i (1 mM) in the presence of 100 mM Na + , pH 7.4. The P i -activated current, measured at a holding potential of −50 mV, was compared with that of oocytes expressing the WT protein, obtained from the same donor frog. As shown in Table , six mutants were found to be still active and gave comparable electrogenic responses to the wild type. To test if alkylation of the cysteine residues by the methanethiosulfonate derivative MTSEA would affect the basic transport function, as indicated by the above electrophysiological assay, we incubated oocytes expressing these six active mutants, as well as the WT protein, in 100 μM MTSEA, and then retested for activity under the same conditions as before. The P i -induced change in holding current, exhibited by the WT as well as five of the active mutants (S318C, S373C, A393C, S532C, and S538C; data not shown), was unaffected by MTSEA ( Table ). In contrast, after incubation in MTSEA, the electrogenic response of mutant S460C showed a significant inhibition during application of 1 mM P i . Moreover, prolonged incubation (up to 30 min) in the standard bath medium did not lead to a restoration of function (data not shown). We also found that after alkylation, 32 P uptake of S460C was completely suppressed (data not shown), which confirmed that P i transport was fully inhibited. To demonstrate that the suppression of electrogenic response was an effect of the alkylation and not simply due to the addition of charge in this region (MTSEA is positively charged), we repeated the experiment with the negatively charged MTS reagent, MTSES. Like MTSEA, incubation in 100 μM MTSES, also induced a positive shift in the baseline current during P i application; i.e., the normal inward current induced by P i was fully suppressed (data not shown). We also incubated oocytes in 100 μM MTSET, which has been reported to be less permeant than MTSEA and we obtained the same suppression of P i response ( n = 3). A representative record is shown in Fig. 2 A. This result suggested that both MTSEA and MTSET were acting extracellularly. Moreover, even at high concentrations (1 mM), all three reagents (MTSEA, MTSET, MTSES) had no effect on the electrogenic WT response (data not shown). This was also confirmed in uptake experiments in which there was no statistical difference in 32 P uptake between WT-expressing oocytes exposed to 1 mM MTSEA, MTSET, or MTSES and control oocytes. In each case, the 32 P uptake was >20-fold higher than that of water-injected oocytes from the same donor frog (results not shown). In contrast to the lack of effect of MTSEA, MTSET, and MTSES on the WT, we did observe a dose dependency of 32 P uptake in WT-expressing oocytes exposed to the membrane-permeant reagent methyl-MTS (Lambert, G., J. Biber, and H. Murer, manuscript submitted for publication). This further supported our conclusion that MTSEA, MTSES, and MTSET were only acting extracellularly. All remaining experiments were performed with MTSEA unless otherwise indicated. As a further confirmation that chemical modification (alkylation) of Cys-460 was involved, we incubated oocytes that had been previously exposed to either MTSEA ( n = 3) or MTSES ( n = 3) in the reducing reagent dithiothreitol (DTT, 10 mM, 15 min) and retested for functional activity. As shown in Fig. 2 B for a representative oocyte expressing S460C, exposure to 1.5 μM MTSEA suppressed the P i -induced response to ∼30% of the initial magnitude, and subsequent DTT incubation restored the P i -activated response almost to the original level. This finding was consistent with dealkylation occurring in Cys-460, which would thereby restore the original Cys residue and cotransporter function. The effect of MTSEA on P i -induced response for S460C was both time and dose dependent and short MTSEA exposures (≤30 s) resulted in large variations (up to 50%) in the amount of suppression of the P i -activated response for oocytes from the same batch. Although the speed of recovery from P i application prevented repeated testing of the P i response after exposure to MTSEA for times shorter than 1 min, application of 100 μM MTSEA, together with continuous application of P i , showed that the suppression of P i -induced inward current was complete within 2 min (data not shown). The optimal concentration range was determined from dose-response data whereby the P i response (1 mM) was tested after successive 2-min applications of increasing concentrations of MTSEA. These data gave an apparent half-maximal concentration of MTSEA = 0.5 μM ( n = 5). MTSEA concentrations up to 100 μM did not result in a further change in the P i response. In all subsequent experiments, therefore, we routinely applied MTSEA at ≥10 μM for 2–3 min. The response to 1 mM P i recorded from oocytes expressing the WT NaPi-IIa under the same conditions with repeated application of increasing MTSEA concentrations decreased by ∼20%. This was within the normally observed rundown limits for NaPi-IIa when superfused for periods exceeding 30 min and was therefore not attributable to MTSEA exposure. Finally, to establish that the loss of transport function by S460C was due to a specific reaction of MTSEA with the Cys-460, we incubated oocytes expressing mutant S460C, as well as the WT protein, in biotin-labeled MTSEA (biotin-MTSEA) and precipitated the protein with immobilized streptavidin (see materials and methods ). Expression of both proteins was confirmed by Western blot of the lysate before streptavidin precipitation. This showed that both were expressed at comparable levels . However, as indicated by the immunoprecipitation shown in Fig. 3 B, only the mutant protein and not the WT could be precipitated after incubation with the biotin labeled MTSEA. The electrophysiological assay used above provided only a basic confirmation that mutant S460C behaved like the WT. Before making a detailed characterization of the effect of MTS reagents on S460C, we examined whether the replacement of Ser-460 with a cysteine altered any of the specific properties of the cotransporter that have been previously identified from steady state and pre–steady state measurements of the WT . We first confirmed that S460C exhibited a dose dependency for the respective substrates (P i , Na + ) that was consistent with the WT. These findings are shown in Fig. 4 A for the P i -activated dose response and B for the Na + -activated dose response, pooled from representative oocytes expressing the mutant S460C. In each case, a set of original records at the substrate test concentrations is given for a representative oocyte. These were indistinguishable from the typical WT responses under the same conditions (data not shown). For both substrate activation data sets, the steady state currents at the test concentration were normalized to the maximum current predicted from a fit to the whole data set for each cell using the modified Hill equation ( ). The P i -activated response was determined at 100 mM Na + and fits to the data gave a Hill coefficient, n Pi = 1.04 ± 0.1 mM and an apparent affinity for P i ( K m Pi ) of 0.08 ± 0.01 mM. The Na + dose response was determined at 1 mM P i and fits to the data gave a Hill coefficient, n Na = 2.4 ± 0 2 mM and an apparent Na + affinity ( K m Na ) of 56 ± 4 mM. These parameters were sufficiently close to the previously reported values for the WT under the same measurement conditions , as well as control oocytes expressing the WT tested in the present study (data not shown), for us to conclude that neither the apparent substrate affinities nor the inferred stoichiometry was affected by the Cys mutation. One further steady state property, which characterizes type IIa Na + /P i cotransport, namely pH sensitivity, was also found to be unchanged in the mutant S460C when compared with the WT expressed in oocytes from the same batch. In 100 mM Na + , a reduction in superfusate pH from 7.4 to 6.2 gave a 55 ± 4% ( n = 5) suppression of the P i -induced response (1 mM total P i ) compared with 58 ± 4% ( n = 5) for the WT. In the absence of P i and presence of Na + in the external medium, type IIa cotransporters exhibit a Na + -dependent slippage (or leak) current. For the WT expressed in oocytes, this is inhibited by the P i analogue and competitive inhibitor for Na + /P i cotransport, PFA . As shown in Fig. 4 C (inset) for a representative oocyte expressing S460C, when challenged with both 0.3 mM P i and 3 mM PFA, a significant suppression of the P i -activated response occurred (trace 2). Moreover, when oocytes were challenged with PFA alone (trace 3), the holding current at −50 mV was reduced. Pooled data from oocytes expressing the WT or S460C from two donor frogs confirmed this behavior . The data were normalized with respect to the near saturating P i response to take account of different expression levels. Whereas, the relative inhibition of the P i response by PFA was similar for both WT and S460C, the latter showed a twofold larger PFA-sensitive current relative to the respective P i -induced response. This finding suggested that the mutation had altered the kinetics of the cotransporter in the slippage mode. Voltage dependence was the final property of type IIa Na + /P i cotransport investigated for the S460C mutant, normally characterized in terms of steady state and pre–steady state behavior. Fig. 5 A shows the steady state voltage dependence of the P i -induced current (left current–voltage plot), which was obtained by subtracting the holding current in the absence of P i from that under saturating P i (1 mM) and 100 mM Na + (pH 7.4). These data indicate that the voltage dependence of the WT and S460C were indistinguishable for V < 0 mV. Moreover, the voltage dependence of the normalized slippage current (right current–voltage plot) for the WT and S460C, using 3 mM PFA as the blocking agent, was essentially unchanged. Pre–steady state charge movements result from voltage-dependent steps in the type II Na + /P i cotransporter kinetics . Fig. 5 B (inset) shows typical pre–steady state relaxations recorded from a representative oocyte expressing S460C. These relaxations were also blocked by 3 mM PFA, as previously reported for the WT . The voltage dependence of the apparent charge movement was determined by integrating the current relaxations that remained after subtraction of the PFA response. This procedure was used to eliminate any charge movements not specifically related to S460C that could result from upregulation of other membrane proteins stimulated by injection of S460C cRNA. As illustrated in Fig. 5 B, the Q–V curve for a representative oocyte expressing S460C saturated at extreme potentials for both ON and OFF charge movements. Boltzmann fits to the mean of the ON and OFF charge ( ) gave a midpoint voltage V 0.5 = −54 ± 5 mV and apparent valency, z = 0.7 ± 0.4 ( n = 5). A representative oocyte expressing the WT protein under the same measurement conditions gave V 0.5 = −50 mV and z = 0.6. The turnover φ at −50 mV of the cotransporter can be estimated from : 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\phi}}}=I_{{\mathrm{p}}}^{-50}{z}/{Q_{{\mathrm{m}}}}{\mathrm{,}}\end{equation*}\end{document} where I p −50 is the P i -induced current at V h = −50 mV. For S460C, substitution of the Boltzmann fit data gave φ = 13.5 ± 2.3 s −1 ( n = 5), compared with φ = 14 s −1 for the WT. These data indicate that neither the voltage dependence of the steady state charge distribution nor the apparent valency of the cotransporter substantially changed after mutagenesis. Moreover, the transport turnover in the cotransport mode was unchanged. A noteworthy result of incubation in MTSEA on the P i response was the reduction of holding current during P i application . We investigated this further by testing the response to 3 mM P i or 3 mM PFA before and after 100 μM MTSEA incubation as shown for a representative oocyte in Fig. 6 . These substrate concentrations were chosen to ensure saturation of the responses. After alkylation, the response to PFA remained unchanged, whereas the P i response was now identical to the PFA response. Moreover, this behavior was found to be consistent for all potentials in the range −80 to 0 mV (data not shown). This result suggested that: (a) alkylation of Cys-460 did not affect the slippage mode, (b) Na + , the cation responsible for slippage current, was still able to bind to the carrier after alkylation, and (c) P i can still bind to the carrier after alkylation, but subsequent cotransport was suppressed. Noninjected oocytes from the same batch showed small changes in holding current with either P i or PFA application, under the same conditions, but which were <10% of the responses recorded from S460C-expressing oocytes. It has been recently reported for SGLT-1 that membrane voltage and substrates can confer an apparent protection to cysteine residues from externally applied MTS reagents . We therefore investigated whether similar effects could be observed with mutant S460C. We found that 10 μM MTSEA applied together with (a) 1 mM P i , (b) 3 mM PFA, or (c) Na + replaced by 100 mM N -methyl- d -glucamine offered no protection since subsequent application of 1 mM P i resulted in full suppression of the P i -activated inward current (data not shown). In each case, the membrane potential was held at −50 mV during the entire protocol and MTSEA was applied only after a steady state holding current had been reached under the specific superfusion conditions. The currently proposed kinetic scheme for type II Na + /P i cotransport predicts that a voltage step will induce pre–steady state relaxations, contributed by the empty carrier and Na + binding/debinding, before reaching the final steady state in the slippage mode. Since this mode appeared to be unchanged by alkylation, we would still expect pre–steady state charge movements to be detectable after alkylation. Fig. 7 shows pre–steady state relaxations recorded from a representative oocyte for voltage steps to five test potentials in the presence (A) and absence (B) of external Na + . As before, the endogenous capacitive charging transient was removed by subtracting the response to 3 mM PFA in ND100. Although MTSEA treatment resulted in a significant apparent suppression of relaxations, there was still a charge movement detectable after the endogenous membrane charging was complete (typically after 1–1.5 ms). Moreover, the relaxations were significantly faster in ND0 solution, which indicated that alkylation had altered the kinetics of the empty carrier. The available signal resolution and low expression levels (steady state currents induced by 1 mM P i were typically 100–150 nA) prevented a full analysis of these relaxations. Nevertheless, single exponential fitting of relaxations induced by large voltage steps indicated that, in 0 mM Na + , alkylation led to an approximately eightfold faster time constant, as shown in Table for three test potentials. Since the relaxations recorded from S460C-expressing oocytes after MTSEA treatment were comparable with the speed of membrane charging, we also confirmed that, under the same perfusion conditions, significant charge movements could not be detected from noninjected oocytes from the same batch once the main capacitive charging was complete. In a final set of experiments, we investigated whether holding potential (V h ) during MTSEA application would also protect against alkylation as reported in the case of SGLT1 . As illustrated by the original recordings from representative oocytes expressing S460C , when MTSEA was applied during a depolarization to +20 mV in ND100 solution, the subsequent response to P i at −50 mV was identical to that obtained when MTSEA was applied at −50 mV . This indicated that in ND100 depolarization to +20 mV did not protect Cys-460. However, when MTSEA was applied in ND0 solution, the magnitude of the P i response was now dependent on V h , whereby less inhibition was observed at V h = +20 mV compared with the response after alkylation at −50 mV. We repeated this protocol for individual oocytes voltage clamped to V h = +20, 0, and −20 mV during MTSEA exposure, and the pooled results indicate a clear voltage dependence of inhibition of response by the MTS reagent in 0 mM Na + . For each oocyte tested, we confirmed that exposure to MTSEA at V h = −50 mV in 100 mM Na + gave complete inhibition . We were unable to determine whether at more depolarized V h further protection of the MTS action was possible because continuous voltage clamping of oocytes at potentials exceeding +20 mV during the MTSEA application period resulted in an irreversible and progressive increase in endogenous leak current. This protocol was also repeated with the anionic MTSES with similar results (data not shown). This confirmed that accessibility to Cys-460 was independent of the charge of the alkylating reagent. In this study, we investigated the effect of membrane impermeant alkylating reagents on mutant constructs of the type IIa Na + /P i cotransporter (rat NaPi-IIa). Amino acid residues, hypothesized to be in functionally sensitive regions according to specific criteria based on the topological scheme for NaPi-IIa , were replaced with cysteine residues. Six of the sites were located intracellularly according to this scheme and might be considered inaccessible to externally applied MTS reagents. Nevertheless, we included them in this study since we could neither fully exclude other candidate topologies , nor exclude the possibility that conformational changes might render these sites accessible. In the present study, we have restricted our investigation to the effects of externally applied MTS reagents since it has been shown that the WT response is also inhibited when exposed to membrane-permeant MTS reagents . Therefore, our finding that external MTSEA did not affect two functional mutants with cysteines predicted to be on the trans side (S373C and A393C) does not exclude the possibility that they are located in functionally important areas. Further investigations using internally applied MTS reagents and cysteine deletions would be necessary to confirm whether or not these residues are functionally relevant. Of the six functional mutants, only P i -induced electrogenic response of construct S460C was suppressed by MTSEA. Since this was reversible by DTT, and the WT protein was insensitive to the externally applied MTS reagents MTSEA, MTSES, and MTSET, these findings suggested that only Cys-460 was strongly implicated as the site of alkylation. Moreover, tracer flux studies confirmed that alkylation had indeed suppressed 32 P uptake. The immunodetection experiments confirmed that alkylation had left the protein intact and furthermore provided additional evidence that Cys-460 was located extracellularly (predicted to be in the third extracellular loop) since S460C, but not the WT, could be streptavidin-precipitated after incubation with biotin labeled MTSEA. An alternative hypothesis to explain the action of MTS reagents on S460C could be that mutagenesis has exposed another cysteine that is accessible for alkylation. Although we cannot fully exclude this possibility based on our present data, this would appear unlikely since, with the exception of augmented slippage current (see below), the basic kinetic properties of S460C are identical to the WT. We expect that the conformational changes that accompany exposure of another cysteine in a functionally sensitive region also result in significant changes in the cotransport kinetics. It has been reported that MTSEA is slightly membrane permeant and this could lead to difficulties in distinguishing between an effect exclusively related to external cysteines and one also involving internal cysteines. In our hands, we observed no change in the WT response after incubation in either of the more charged reagents, MTSET, MTSES, or MTSEA, even up to 1 mM concentrations. In contrast, the membrane-permeant reagent methyl-MTS has been shown to cause a suppression of the P i -induced response of oocytes expressing the WT protein (our unpublished results). This indicated that alkylation of internal cysteines can interfere with transport function. In the present study, the insensitivity of the WT to all three nominally impermeant reagents, together with the specificity of their effect on S460C, led us to conclude that these reagents were only acting from the cis side. Based on steady state and pre–steady state kinetics of the WT rat NaPi-IIa and flounder NaPi-IIb isoforms, we have proposed a kinetic model for type II Na + /P i cotransport depicted in Fig. 9 . This model belongs to the alternating access or gated channel class of cotransporter models proposed for other Na + -dependent cotransport systems, including the sodium–glucose cotransporter . It predicts that accessibility of substrates to their respective binding sites on the cis and trans sides of the membrane relies on a reorientation of the protein, the favored state of which (cis or trans facing) is determined by substrate availability on the respective sides and transmembrane potential. Charge movements (measured as pre–steady state relaxations) induced by voltage steps are predicted to arise from the movement of charges intrinsic to the carrier, as well as the translocation of charged substrates to their binding sites within the transmembrane field. This scheme contrasts with the channel-like substrate hopping model proposed recently in which major molecular conformational changes do not occur and charge movements arise exclusively from movement of charged substrates within a channel-like pore, assumed to contain multiple binding sites. The recent findings of Loo et al. 1998 support an alternating access type model for SGLT-1, whereby conformational changes are responsible for the coupling of Na + and sugar transport. Changes in fluorescence of a rhodamine-labeled cysteine residue and pre–steady state relaxations were correlated, as well as the accessibility of this cysteine to MTS reagents and the steady state charge distribution. In Fig. 9 , at least three modes of operation are possible. In the empty carrier mode, the orientation of the charged empty carrier is favored towards the cis side (state 1) when the membrane is depolarized, which increases the accessibility of Na + ions to a binding site. After the binding of one Na + ion (transition 1 ⇔ 2), the system operates in the slippage mode, whereby one Na + ion forms a neutral complex with the empty carrier and slippage occurs through the protein (transition 2 ⇔ 9) in the absence of P i . Occupancy of state 2 increases the affinity of the protein for P i , which then binds (transition 2 ⇔ 3) in its divalent form , together with two additional Na + ions (transitions 3 ⇔ 4 and 4 ⇔ 5). In this cotransport mode (transition 4 ⇔ 5), reorientation of the fully loaded, neutral carrier to the trans side (transition 4 ⇔ 5) is now favored, and release of the substrates can occur as a result of the low internal Na + concentration. The cycle is completed by a reorientation of the empty carrier (transition 10 ⇔ 1). The P i analogue PFA, which inhibits both slippage and cotransport modes, is assumed to place the system in state 2* when bound. This is consistent with the findings of Busch et al. 1994 , who demonstrated that PFA acts as a competitive inhibitor for P i by shifting the apparent K m for P i without changing the apparent maximum electrogenic transport rate, V max . Since V max is determined by the final Na + binding steps . PFA would be expected not to interact directly with these steps, but rather to compete for occupancy of the P i binding site. Details of kinetics in the trans conformation (states 5–10) are as yet unknown; however, the validity of this model, under zero trans conditions, has been confirmed by simulations in which the main features of steady state and pre–steady state kinetics are adequately predicted . Comparison of the kinetics of S460C and the WT rat NaPi-IIa indicated that the cysteine substitution did not alter the properties of the cotransport mode. The apparent affinities for both substrates, pH dependence, and voltage dependence were all similar to the WT. Moreover, oocytes expressing S460C gave pre–steady state relaxations both in the presence and absence of external Na + , as predicted from the kinetic scheme . Of significance was a 50% increase slippage current for S460C relative to the P i response compared with the WT. Since the turnover in the cotransport mode appeared to be unaffected by mutagenesis, the increased relative slippage current would most likely result from increased turnover in the slippage mode. Furthermore, since the voltage dependence of the slippage mode was the same for WT and S460C, this suggested that mutagenesis had altered the kinetics of transition 2 ⇔ 9 (see , ). Despite this departure from WT behavior, the general similarity of S460C and the WT indicated that the model scheme was also valid for this construct. Two questions arise with respect to mutant S460C: (a) Can the behavior of S460C after alkylation be explained in terms of the above kinetic scheme? and (b) Which kinetic transitions are associated with the alkylated Cys-460? The change in the steady state characteristics after alkylation, whereby saturating P i induced a reduction in holding current that exactly matched that of PFA both before and after alkylation, suggested that site 460 was located in a functionally sensitive region of the molecule. In terms of Fig. 9 , an intact slippage pathway after alkylation indicates that the protein can still cycle around the loop 1 ⇔ 2 ⇔ 9 ⇔ 10 ⇔ 1, as well as occupy state 2* when PFA is bound. Moreover, the identical responses to P i and PFA after alkylation suggest that state 3 (with P i bound) is also intact, but alkylation of Cys-460 prevents one or more of the subsequent transitions leading to the cotransport mode. This behavior would also strongly suggest that P i and PFA bind to the same site. Further support for this interpretation comes from our finding that pre–steady state charge movements, albeit with significantly faster relaxation time constants, were still detectable after alkylation for both the empty carrier and slippage modes. From our recordings, it appeared that alkylation also caused a suppression of the charge movement, even though the magnitude of the slippage current and its steady state voltage dependence remained unchanged. However, we were unable to resolve charge movements at times earlier than 1.5 ms after the voltage step onset and, therefore, one explanation for this apparent discrepancy between the pre–steady state and the steady state data might be that part of the charge movement simply remained undetected. To investigate this further, we modeled the behavior of a four-state scheme comprising states 1, 2, 9, and 10 (see ).The model predicts pre–steady state relaxations similar to those observed before and after alkylation. Analysis of the model indicated that the steady state slippage current remains constant, as observed experimentally, if the ratio of the zero voltage rate constants for the empty carrier (transition 1 ⇔ 10) was held constant (see , ). We simulated effect of alkylation by arbitrarily increasing both zero voltage rate constants for this transition 10-fold, to accord with our finding of faster pre–steady state relaxations in 0 mM Na + . In terms of an Eyring–Boltzmann transition rate model, this would imply that alkylation reduces the height of the apparent energy barrier of this step. Since the apparent valencies for the voltage-dependent steps are assumed to remain the same, the increased rates for the empty carrier conformational change after alkylation would not change the overall steady state charge distribution. Our finding of no difference between anionic and cationic MTS reagents in suppressing the P i response would further suggest that the charge of the alkylated Cys-460 is not in the electric field, and therefore cannot alter the voltage dependence of the empty carrier. This is also consistent with an invariant steady state charge distribution after alkylation. Loo et al. 1998 have reported similar behavior for the Q457C mutant of SGLT-1, whereby the voltage dependence of accessibility of the MTS reagents was also found to be independent of the valency of the reagent. That Cys-460 is associated with the conformational state of the empty carrier is also suggested from our finding that in 0 mM Na + the inhibition of the P i response was dependent on the holding potential during MTS application; i.e., accessibility to Cys-460 was voltage dependent. In the empty carrier mode, membrane potential determines the probability of occupancy of state 1 or state 10. Since such a voltage-dependent change of state implies the movement of charged species within the membrane, the associated conformational changes are hypothesized to alter the accessibility of Cys-460. In 100 mM Na + , we found no protection with holding potential (between −50 and +20 mV) and, similarly, no protection was observed in the presence of either P i or PFA (together with 100 mM Na + ). These findings suggest that once the protein is in state 2 (Na + bound), state 2* (PFA bound), or state 3 (P i bound), Cys-460 is readily accessible by externally applied MTS reagents. Interestingly, in the study by Loo et al. 1998 of the SGLT-1 mutant Q457C, alkylation also resulted in inhibition of the cotransport mode, although glucose binding still occurred, just as our data suggest that P i can still bind to S460C. However, in contrast to S460C, the behavior of this SGLT-1 mutant after alkylation indicated that MTS reagents could only access the cysteine residue in the equivalent of state 2 (Na + bound). Our findings indicate that site 460 is located in a functionally sensitive region of the NaPi-IIa molecule, most likely associated with conformational changes of the empty carrier. As indicated in Fig. 9 , states 10, 1, 2, and 3 remain intact after alkylation. The subsequent transition or transitions, which are altered by alkylation and thereby inhibit the cotransport mode, remain to be identified. Our findings complement those of Loo et al. 1998 for SGLT-1 and provide further support for the notion that conformational changes accompany substrate binding in Na + -coupled cotransporters. | Study | biomedical | en | 0.999999 |
10532963 | Many ion channels found in a variety of cell types exhibit nonlinear conduction properties that arise from voltage-dependent block by extracellular divalent cations. Examples include nicotinic acetylcholine-receptor channels , L-type Ca 2+ channels , N -methyl- d -arginine–receptor channels , histamine-operated cationic channels , and cAMP-activated channels . In vertebrate photoreceptors, extracellular calcium and magnesium also exert a voltage-dependent blocking effect on the cGMP-gated conductance that underlies visual transduction . This phenomenon confers to the current–voltage (I–V) 1 relation of the light-sensitive current a pronounced outward rectification under physiological conditions . A number of detailed investigations have appeared, addressing the mechanisms of blockage of the cGMP-dependent channels in excised membrane patches of vertebrate photoreceptors, and a complex picture has emerged that includes interactions of divalent effects with the cyclic nucleotide concentration and the gating machinery and differences between rods and cones . The binding and unbinding of divalents to the cGMP-activated channels of rods is extremely fast, causing the current through individual open channels to fluctuate at high frequency . Because rapid transitions are filtered out by the membrane impedance, the net result is a reduction of the effective single-channel conductance , which has been suggested to serve the purpose of lowering the dark noise and, consequently, to enhance the detection of faint stimuli . By the same token, the rapidity of the phenomenon makes it challenging to characterize its kinetics. The light-sensitive potassium conductance (g L ) of hyperpolarizing (ciliary) photoreceptors in the retina of the scallop, Pecten irradians , shares significant similarities with that of vertebrate photoreceptors, including activation by cGMP and susceptibility to blockade by l -cis-diltiazem and 3,4-dichlorobenzamil . As in rods, the I–V relation also rectifies in the outward direction because of voltage-dependent blockade by extracellular divalent cations . However, light-activated single-channel currents can still be measured in the presence of Ca 2+ and Mg 2+ without a reduction in their apparent amplitude, indicating a slow block/unblock interaction on the order of milliseconds. In the present report, we have taken advantage of this favorable situation to resolve the blocking and unblocking kinetics of the whole-cell photocurrent, and examine interactions between blocking by divalents and the gating process. These functional observations suggest some constraints on the topological organization of the channel pore and gate. Specimens of Pecten irradians were obtained from the Aquatic Resources Division of the Marine Biological Laboratory and used immediately. Retinas were dissected and enzymatically dissociated, as previously described, to yield solitary ciliary photoreceptors . Cells were plated in a low-volume recording chamber (≈200 μl) continuously perfused with a solution that could be exchanged via a system of reservoirs and manifolds. The composition of artificial sea water (ASW) and of the other extracellular solutions is listed in Table . Junction potential changes were measured and compensated. All experiments were conducted at room temperature (≈22°C). Patch electrodes used for whole-cell recording were fabricated with thin-wall (1.5-mm o.d., 1.1-mm i.d.) borosilicate capillary tubing pulled to a 2–3-μm outer tip diameter and fire-polished immediately before use. The “intracellular” filling solution contained 100 mM KCl, 200 mM K-aspartate or K-glutamate, 22 mM NaCl, 5 mM Mg ATP, 10 mM HEPES, 1 mM EGTA, 100 μM GTP, and 300 mM sucrose, pH 7.3. Electrode resistance, measured in ASW, was 2–4 MΩ. In all recordings series resistance was compensated via a positive feedback circuit in the amplifier (maximal residual error typically <2 mV). Whole-cell currents were low-pass filtered with a Bessel four-pole filter, using a cutoff frequency of 500–2,000 Hz, and digitized online at 2–5 kHz sampling rate by a 12-bit resolution analogue/digital interface board . For single-channel recordings, finer electrodes (tip diameter < 2 μm, 8–12 MΩ resistance) were fabricated with thick-wall glass (1.5-mm o.d., 0.75-mm i.d.) and filled with either normal ASW or high-K ASW; the cutoff frequency and the sampling rate used for unitary current measurements were 5 and 10 kHz, respectively. Voltage and light stimuli were applied by a microprocessor-controlled programmable stimulator (Stim 6; Ionoptix). Flashes and steps of broad-band light (515–650 nm) were provided by a 100-W tungsten-halogen optical stimulator whose output beam was combined with that of the microscope illuminator via a beam splitter prism placed above the condenser, as previously described . Light intensity is expressed in terms of equivalent photon flux at 500 nm, as determined by an in vivo calibration in which responses to broad-band illumination were matched to photocurrents elicited by monochromatic stimuli (500-nm peak, 10-nm half width; Ditric Optics); the intensity of monochromatic light was measured with a radiometer (370; UDT). Calibrated neutral-density filters (Melles Griot) provided controlled light attenuation. During experimental manipulations, the cells were viewed with the aid of a Newvicon TV camera using a near-IR long-pass filter for illumination (λ > 780 nm; Andover Corp.). The infrared illuminator was turned off for several minutes before testing light responses. In vertebrate photoreceptors, the light-sensitive conductance is poorly selective among cations, and exhibits a substantial permeability to calcium ions , which carry a significant fraction of the dark current under physiological conditions . “Blockade” by Ca 2+ reflects the prolonged dwell times in the permeation pathway (presumed to be single file) due to tight binding. In ciliary invertebrate photoreceptors, by contrast, the contribution of divalents to the photocurrent is expected to be negligible because the reversal potential is near E K and changes with a near-perfectly Nernstian dependency upon manipulating [K] o , even in the presence of normal concentrations of extracellular Ca 2+ and Mg 2+ . The experiments illustrated in Fig. 1 were designed to assess in a direct way the permeation of Ca 2+ and Mg 2+ through light-activated channels. In Fig. 1 A, the membrane potential of a photoreceptor was stepped in 10-mV increments for several seconds before stimulating with a flash of light of fixed intensity; V m was returned to −30 mV between trials. In normal ASW, the peak amplitude of the photocurrent decreased in a markedly nonlinear way with hyperpolarization, and at −100 mV a barely detectable inward photocurrent was elicited (left). After switching to a solution lacking both calcium and magnesium (middle), the amplitude of the light responses significantly increased, especially at hyperpolarized voltages. In particular, below the reversal potential, a sizable inward photocurrent could be clearly observed. This effect was fully reversible (right). The I–V relation for the three phases of the experiment, plotted in Fig. 1 B, shows that removal of divalents virtually eliminated the rectification, without any obvious concomitant displacement of the reversal potential, which remained near −80 mV ( n = 3). To provide a more sensitive test, additional measurements were conducted in the presence of elevated [K] o (50 mM), as shown in Fig. 1 C. These conditions were designed to displace V rev to a range in which g L is larger so that the greater slope of the I–V curve improves the signal-to-noise ratio (S/N); in addition, the accuracy of the measurements was increased by changing the holding potential in 2-mV increments to detect any small change in the reversal voltage. As shown in Fig. 1 D, V rev was not significantly affected by the presence or absence of divalent cations (mean shift 0.8 ± 0.3 mV, n = 3), confirming that the permeability of Ca 2+ and Mg 2+ must be negligible. To determine whether both Ca 2+ and Mg 2+ are capable of blocking the channel, we tested each divalent cation individually. Fig. 2 A shows photocurrents elicited at −30 mV by repetitive flashes delivered initially in 0-divalents extracellular solution, and subsequently after introducing either 60 mM Ca 2+ or Mg 2+ . The amplitude of the light response was reduced in both cases, but the effect was significantly greater for calcium (82% ± 3%, n = 5) than for magnesium (31% ± 6%, n = 3). The average normalized I–V curves measured between −60 and +20 mV in the three conditions is shown in Fig. 2 B ( n = 5 per group). Each test was preceded and followed by a control flash at −30 mV; cells that failed to satisfy the criterion of <5% change between the pre- and post-test responses were discarded. It is readily apparent that, although Mg 2+ induced a significant curvature in the I–V relation (especially evident at V m < −40 mV), the outward rectification induced by Ca 2+ was far more pronounced. The greater relative potency of Ca 2+ vs. Mg 2+ was also corroborated by the observation that, when the solution is changed from normal to 0-Ca ASW (by substituting Ca 2+ with Mg 2+ on an equimolar basis), the photocurrent amplitude is increased and the outward rectification is substantially attenuated ( n = 3; data not shown); conversely, a switch from normal to 0-Mg ASW (60 Ca 2+ ) leads to a reduction in the photoresponse ( n = 4). The apparent affinity of Ca 2+ for the channel was determined as illustrated in Fig. 3 . Photoreceptors were voltage clamped at −30 mV and stimulated with repetitive flashes of constant intensity, initially in the absence of all divalents, and then after introducing either 2, 10, 30, or 60 mM Ca 2+ (0-Mg). For comparison purposes, responses were normalized with respect to the maximum amplitude attained during the control trials in 0-divalents. Calcium depressed the light-evoked current in a concentration-dependent way. To quantify the dose dependency of the blockade, data from 12 cells tested with the same light intensity were pooled and plotted in Fig. 3 B. The smooth curve represents a Langmuir function fitted to the average data points by the method of least squares; half-maximal block was attained at ≈16 mM. A Hill function provided no better fit, and the resulting Hill coefficient was not significantly different from 1 (0.91), suggesting that a single calcium ion blocks the channel. Similar measurements were conducted at 0 mV, and the obtained K 1/2 was ≈61 mM for calcium. The corresponding estimate for Mg 2+ would be difficult to obtain because of its lower affinity: 60 mM Mg 2+ only reduced the photocurrent by 12.5% ( n = 2), so that an extrapolated figure would be >>100 mM. The remaining parts of Fig. 3 illustrate the concentration-dependent induction of outward rectification in the photocurrent by extracellular Ca 2+ . In Fig. 3 C, families of light-evoked currents were measured as the holding potential was stepped from −60 to +20 mV in 10-mV increments; the experiment was conducted initially in divalent-free solution, and subsequently repeated in the presence of 10 and 60 mM Ca 2+ (in the same cell). As [Ca 2+ ] o was raised, the size of the currents evoked at the more negative voltages became compressed. The peak amplitude of the responses, plotted in D, clearly illustrates the progressively increasing curvature in the I–V relation. Comparable dose-dependent effects were observed in another photoreceptor tested with 0, 2, and 10 mM Ca 2+ , and 10 additional cells in which the 0-divalent solution was compared with one fixed calcium concentration, in the range of 2–60 mM. To help clarify the site of action of calcium ions, we ascertained possible interactions between blockade and potassium permeation; for example, occlusion of the pore may be alleviated by a knock-off mechanism . We addressed this possibility by determining the degree of photocurrent reduction induced by a fixed [Ca 2+ ] o at a steady membrane potential, as the concentration of extracellular potassium was manipulated. Fig. 4 A illustrates a comparison between the responses elicited by a flash of constant intensity at −60 mV, in 0-divalents extracellular solution and after introducing 2 mM Ca 2+ ; the left traces were obtained in the presence of normal [K] o (10 mM), whereas those on the right were recorded with elevated [K] o (50 mM), which made the photocurrent inwardly directed. The extent of blockade was noticeably greater when measured in high-K solution. The histogram in Fig. 4 B shows the average blockade for cells tested under the two conditions ( n = 3 and 2). This result suggests a site of block by divalent cations that is located within the permeation pathway of the light-dependent channels. Unlike in vertebrate rods, in Pecten ciliary photoreceptors, light-dependent single-channel currents can be resolved in the presence of normal concentrations of extracellular Ca 2+ and Mg 2+ . Fig. 5 A illustrates an example obtained in a cell-attached patch from a ciliary appendage, with the pipette filled with normal ASW and depolarized by 90 mV to produce a suitable driving force. Steps of light lasting for 2 s repeatedly administered at 1-min intervals elicited distinct outward single-channel currents. Because the intensity of the light was saturating, the receptor potential may have approached −80 mV at the peak of the response , bringing the absolute voltage across the patch somewhat positive of 0 mV. At that membrane potential, the effect of divalents is inevitably greatly reduced ; therefore, additional measurements were performed at more negative voltages where blockade is far more substantial. To that end, a pipette solution containing elevated potassium (490 mM, replacing Na on an equimolar basis) was used; the resulting estimated value of E K is near +12 mV, which ensures a sizable inwardly directed driving force on K ions over the voltage range of interest. Fig. 5 B shows that with V p set at −30 mV, light-activated single-channel currents could still be recorded. Application of a hyperpolarizing voltage step in the dark to mimic the receptor potential failed to evoke any openings, confirming that channel activation was not a consequence of the change in V m . Two additional patches tested under the same conditions yielded similar results; when light stimulation was delivered at V p = 0 mV (i.e., with the physiological potential across the patch), unitary currents were again clearly resolved. The following considerations suggest that in Pecten divalent blockade of light-dependent channels must be sluggish: at negative voltages, where blockade is strongest, the unitary conductance (28.6 ± 1.2 pS, n = 3) was not lower than the value previously reported for a more depolarized range . The implication is that the block–unblock interactions between channels and divalent cations must be slower than the recording bandwidth, otherwise the apparent amplitude of the open-channel current would have decreased as the blockade increased. A direct comparison in the presence vs. complete absence of divalents could not be carried out because in the latter case seals were invariably poor, and the resulting noise level precluded accurate resolution of single-channel currents; it was possible, nevertheless, to do so with just 1 mM Mg 2+ in the electrode, and a similar conductance value was again obtained (29 ± 1.2 pS, n = 3). Finally, although the obtained data were insufficient for a quantitative analysis of open times distributions (partly because of the presence of multiple channels with frequent overlapping events), it is clear that at negative potentials channel events were briefer , most likely a reflection of the enhancement of divalent block; however, the currents did not exhibit rapid flickering . This indicates that the kinetics of block/unblock must be at least on a time scale comparable with that of channel gating (several milliseconds). In summary, these results point to the conclusion that the slowness of the blockade (milliseconds) should be sufficient to resolve it temporally and to directly examine possible influences of the conformational state of the channels. A detailed analysis using single-channel recording, however, is not feasible for several reasons. (a) Patch clamping onto the light-sensitive ciliary appendages is very challenging owing to their minute size (≈1 μm). (b) Seal resistance rarely exceeds 2 GΩ, so that S/N is often inadequate for high-resolution measurements; furthermore, most patches contain multiple channels, which greatly complicates the analysis. (c) The need to manipulate the concentration of external divalents would pose the additional difficulty of perfusing the recording patch pipette. (d) Unless a second electrode is used to voltage clamp the photoreceptor under study, the membrane potential across the patch will not only be unknown, but will also change during light stimulation, thus affecting the voltage-dependent block by divalents. Our experimental approach to characterizing the kinetics of the interaction between divalent cations and the light-dependent conductance consisted of recording whole-cell currents under voltage clamp and applying perturbations to the command voltage during sustained activation of the light-dependent conductance: an abrupt change in V m will alter the blockade by divalents, and the resulting reequilibration should manifest itself as a resolvable relaxation, provided its kinetics are sufficiently sluggish. The basic phenomenon is shown in Fig. 6 A: a ciliary photoreceptor was voltage clamped at −20 mV in ASW under continuous illumination and the voltage was stepped to −60 mV, causing an abrupt current jump, as one would expect from the sudden reduction in driving force on K + ; additionally, however, a slower further decrease in current is clearly visible. In Fig. 6 B, a semi-logarithmic plot of the tail shows that this relaxation obeyed a single-exponential time course with a time constant of 19 ms. The first issue to be established is whether the observed relaxation is indeed due to time-dependent changes in the current through light-dependent channels. To this end, experiments were designed to remove other possible confounding factors and optimize the resolution of current transients after perturbations of V m . Under normal ionic conditions, the range of voltages where blockade by external divalents is most pronounced is not far from V rev and the current is necessarily small. To improve the signal-to-noise ratio, we resorted to increasing [K] o fivefold to 50 mM; because the light-sensitive conductance in these cells behaves like a near-perfect K electrode, this manipulation shifts the reversal potential by ≈40 mV in the positive direction . As a result, a significant inwardly directed driving force will exist in the voltage range of interest. Fig. 6 C illustrates the protocol used to examine in isolation the changes in light-dependent current after a voltage perturbation. A ciliary photoreceptor was voltage clamped at 0 mV in high-potassium ASW and stimulated with a sustained step of light, which elicited an approximately half-saturating outward photocurrent. When the response was nearly stable, the holding voltage was stepped to −70 mV, causing a rapid downward peak followed by a plateau . This may reflect, in addition to the changes in the current through the light-activated conductance, contributions by leakage and other non–light-dependent ionic currents, as well as residual capacitative transients. To remove these extraneous factors, a similar voltage step was administered in the dark, and the resulting record (V) subtracted from the previous one. The corrected trace, shown in Fig. 6 , right, reveals the time course of the photocurrent alone. The hyperpolarizing step induced an inward current that is initially quite large, but rapidly relaxed by ≈1 nA to a small-amplitude plateau ( n = 11). Capacitative and non–light-dependent ionic currents were subtracted from all records presented below. The next step was to determine the relationship between these relaxations and blockade by divalents. To this end, the effects of voltage perturbations applied in the presence vs. absence of extracellular Ca 2+ and Mg 2+ were compared. Fig. 7 A demonstrates that after removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current that totally lacked the rapid relaxation. The different time course obtained in the two cases is highlighted in Fig. 7 B, which shows the two normalized superimposed traces in an expanded time scale ( n = 5). Considering the disparate potency of voltage-dependent block by Ca 2+ and Mg 2+ at steady holding potentials , some mechanistic insight can be gained by comparing their respective kinetics by perturbation/relaxation analysis. Fig. 8 A shows the normalized currents evoked by a hyperpolarizing step from 0 to −70 mV during illumination, in a cell that was successively superfused with extracellular solution containing elevated K (50 mM), either devoid of divalents or in the presence of 60 mM Ca 2+ or 60 mM Mg 2+ . As before, in 0-divalents, the photocurrent remained stationary after the voltage perturbation, whereas a conspicuous relaxation occurred in the presence of either divalent cation; the most striking difference, however, is that the time course with calcium (τ = 7 ms) was much faster than with magnesium (τ = 29 ms). In both ionic conditions, the relaxations accelerated as a function of the membrane hyperpolarization: in Fig. 8 B, the voltage was stepped in 10-mV increments between −50 and −80 mV, from a holding potential of 0 mV. It is clear that both the amplitude and the speed of the relaxation are graded with the size of the voltage stimulus, although with Mg 2+ these transients remained significantly smaller and slower. By contrast, in 0-divalents, the currents after each step retained a nearly flat time course (except for some slow creep), and their amplitude changed linearly with voltage. These effects were confirmed in a total of five cells (two tested with a shortened protocol). The range of voltages examined could not be extended further, because the applied stimulus had to be significantly more negative than approximately −40 mV to insure a reasonable driving force, but not too large, otherwise membrane breakdown occurs , a situation that is exacerbated by removal of divalents . The progressive shortening of the relaxation time constant with membrane hyperpolarization in the presence of Ca 2+ is illustrated in Fig. 8 C; each point in the plot represents an average value ( n = 3). The corresponding data for magnesium (not shown) were more complex in that the sum of two exponential functions was often required to satisfactorily fit the data, with the relative contribution of the two components changing significantly as a function of voltage. The dependency of the relaxation parameters on the concentration of the blocking ion is illustrated in Fig. 9 A. A photoreceptor was voltage clamped at 0 mV and stimulated with a step to −80 mV; the test was repeated using different concentrations of Ca 2+ (2, 10, and 60 mM) as the sole extracellular divalent cation. Increasing calcium had two clear effects: (a) it resulted in a smaller steady state current, and (b) it accelerated the time course of the relaxation. The latter result is highlighted by Fig. 9 B, in which the relaxations measured at different [Ca 2+ ] o were normalized and superimposed. Similar observations were made in three cells. Notice that the initial peak amplitude of the transient was also inversely related to the Ca 2+ concentration, owing to the fact that, at the holding voltage of 0 mV, a significant degree of blockade is already present . In Fig. 9 C, the kinetic rates were estimated after pooling the data obtained across different photoreceptors; assuming one-to-one interaction, these can be derived from the standard relations: τ = 1/(α × [D] + β) and K d = β/α, where τ is the measured time constant of the relaxation, [D] is the divalent concentration, and the apparent K d was determined from the reduction of photocurrent amplitude upon introducing the divalents at a steady V m (−80 mV). α are β are the forward and reverse rate constants, respectively. As expected, the “off” rate (□) was independent of [Ca 2+ ] o (≈11 s −1 ), whereas the apparent association rate (▪) increased as a function of the concentration of the blocking ion. The plot also includes the corresponding values obtained with 60 mM Mg 2+ (○; average of n = 3). It is noteworthy that the intrinsic “on” rate constant for Mg 2+ (≈6.8 × 10 2 M −1 s −1 ) was substantially lower than that for Ca 2+ (≈3.7 × 10 3 M −1 s −1 from the fitted line). To demonstrate the relief of block, symmetrical experiments were conducted in which depolarizing steps were applied from a negative holding voltage, with and without illumination. This procedure, however, requires some caution for the following reason: whereas membrane hyperpolarization elicits no active currents, depolarization can trigger several voltage-dependent mechanisms, including Ca 2+ and K channels . In principle, none of these would be expected to pose a problem in that the current subtraction protocol should cancel out any contribution by non–light-dependent processes. However, a recent report in distal photoreceptors of a related species of scallop ( Patinopecten yessoensis ) demonstrated the presence of a transient K current (I A ) whose decay kinetics are modulated by light . Fig. 10 A shows that a similar phenomenon also occurs in Pecten irradians: an isolated photoreceptor was voltage clamped at −80 mV and stimulated with depolarizing pulses of increasing amplitude in 10-mV increments. In the dark, voltage steps more positive than −30 mV elicited an outward current consisting of a transient and a sustained component (left); the inactivating current is carried by potassium and blocked by 4-aminopyridine (4-AP, data not shown). When the protocol was repeated in the presence of steady light (right), the time course of the outward current changed markedly, becoming sustained ( n = 8). This phenomenon can undermine the validity of subtracting currents elicited by depolarizing pulses in the dark and light because spurious relaxations could be artifactually introduced. Blockade of I A by 4-AP is not a viable option, as we have previously shown that the light-dependent K conductance in these photoreceptor cells is also extraordinarily susceptible to blockade by this drug . An alternative strategy is to exploit the steady-state inactivation properties of I A to minimize its contribution: Fig. 9 B shows recordings obtained in the dark in a distal photoreceptor subjected to a 500-ms conditioning prestep to various voltages between −10 and −100 mV, followed by a depolarization to 0 mV. As the conditioning voltage was made more negative, a distinct transient outward current was evoked by the step to 0 mV. In Fig. 9 C, the steady state inactivation data were fitted by a Boltzmann function; the average voltage for half-maximal inactivation was −67 ± 8 mV ( n = 4). To examine the kinetics of unblock of the photocurrent, we used cells screened for a particularly small I A , and restricted the holding potential to −50 mV so that contributions by I A were minimized; unfortunately, at that voltage blockade by divalents is also relatively modest and, therefore, the sensitivity of this test is necessarily reduced. Fig. 11 A shows the results of abruptly depolarizing the membrane to 0 mV during sustained activation of the photocurrent; the procedure was conducted first in normal ASW, and then after removal of Ca 2+ and Mg 2+ . In the presence of divalent cations, the voltage jump produced a rapid step increase in the current, reflecting the increase in driving force on K ions, followed by an outward relaxation. As shown by the semi-logarithmic plot in Fig. 11 B, the relaxation had an exponential time course, with a time constant of 8.5 ms. After removal of Ca 2+ and Mg 2+ , the current became rectangular, directly jumping to the asymptotic amplitude. These observations suggest that the relaxation arises from relief of block by divalent cations ( n = 6). The time-resolved reequilibration of blockade of the light-sensitive conductance by divalent cations, demonstrated in the preceding section, raises the question of whether occupancy of the blocking site(s) is linked to the gating process. One possibility is that the binding sites become available to external divalents only when the light-dependent channels are in the open conformation. A straightforward prediction from this conjecture is that the kinetics of the light response should be affected by the membrane potential imposed at the time of photostimulation. The rationale is that equilibration will only begin as the light-dependent channels gradually open, so that the extent of blockade at different voltages would become fully manifest in the late phase of the photocurrent: the more negative the V m , the faster the apparent decay of the response. This prediction is borne out by the data shown in Fig. 12 . On the left side of Fig. 12 A, photoresponses to a fixed flash were measured in control conditions (ASW), at holding voltages that varied between −60 and −20 mV in 10-mV increments; between trials, the membrane potential was returned to −30 mV. The records were normalized with respect to their peak amplitude. The light response decayed progressively more rapidly as the holding potential was made more negative. To rule out the possibility that the phenomenon may simply be due to a direct effect of voltage on the gating of the light-sensitive channels, the procedure was repeated after superfusing the same cell with divalent-free solution: under these conditions, the flash responses remained virtually superimposable , with a slow time course resembling that of the photocurrent in ASW at a depolarized V m . Shifting the range of voltages tested in ASW by 20 mV in the depolarizing direction, to check for possible effects of surface charge screening, did not alter the differences across the two ionic conditions (not shown). As a simple measure of time course, the response half-width (i.e., the time elapsed between the two crossings of the half-maximal amplitude level) is plotted for the two conditions in Fig. 12 B. In normal ASW, the half-width of the light responses increased progressively with depolarization, approaching the value obtained in 0-divalents, which remained relatively constant. The near invariance of response kinetics in 0-divalents was corroborated in a total of eight cells; the pronounced acceleration of the time course with hyperpolarization in ASW was observed numerous times ( n > 20). An alternative approach to testing whether the blockade by divalents interacts with the state of the channel entails applying conditioning voltage steps that are terminated just before the delivery of a light stimulus. If the occupancy of the blocking site by divalents can only change when the channel is in the open conformation (i.e., after photostimulation), then the voltage pre-pulses should have no effect whatsoever. However, if the blocking site is also accessible in the dark (i.e., with the channels closed), then the prepulse should either enhance or depress blockade, depending on the polarity of the stimulus. Such an effect would be expected to linger, owing to the relatively sluggish blocking/unblocking kinetics; as a result, the rising phase of the photocurrent activated immediately after should be affected. Fig. 13 shows the results of an experiment in which a photoreceptor was voltage clamped at a holding potential of 0 mV. The cell was stimulated with a 200-ms voltage step to −70 mV, which terminated immediately before the delivery of a light flash (L+V). A similar voltage step without the flash was also applied (V) to subtract residual leak and capacitative currents. The corrected record was compared with a photocurrent evoked by an identical flash not preceded by the conditioning voltage step (L), as shown in Fig. 13 B: the time course of the two traces is indistinguishable, irrespective of prepulse ( n = 5). It should be pointed out that contamination of the rising phase of the light response by the subtraction procedure (owing to possible light-induced changes of I A time course) is negligible here for two reasons. (a) The prestep voltage was chosen to lie near the midpoint of the h ∞ curve and its short duration (although >>τ of blockade equilibration) only allows a fraction of the recovery from inactivation that can be attained at that V m (≈60% of the asymptotic level; data not shown). As a result, I A is reduced by ≈65%. (b) The brief flash followed the voltage transition, precluding the development of any significant modulatory effect on the kinetics of I A . The converse experiment was also performed, as shown in Fig. 13 C. In this case, the cell membrane was clamped at a more negative holding potential (−50 mV) and a depolarizing step to +20 mV was applied to determine whether blockade could be relieved before the presentation of the light. Again, as shown in Fig. 13 D, the rising phase of the current recorded when the light was present either alone or preceded by the conditioning voltage step were superimposable ( n = 7). The results of these experiments complement those presented in Fig. 12 and corroborate the notion that the blocking site is only accessible when stimulation induces a conformational change of the light-dependent channels to the open state. A final question concerns the fate of a blocking ion upon cessation of photostimulation. Either the gate has to wait for the divalent to vacate the site before closing (owing to some steric hindrance) or, alternatively, the channel could close with the divalent bound within the pore. In the latter case, the fact that calcium and magnesium are not measurably permeant precludes the possibility of any significant fluxing to the cytosol, and so the ion would remain trapped. Appropriate tests to reveal either phenomenon are conceptually straightforward, but achieving the necessary sensitivity with a low-affinity blocker can be arduous. For the “foot in the door” case, one would expect the blocker to slow down the falling phase of the photocurrent; however, because this time constant is already on the order of hundreds of milliseconds, the unblock kinetics would be unlikely to make any significant contribution. In case trapping occurs, if one induced the channels to close during strong blockade, the response to a subsequent light delivered under conditions of reduced block would be expected to have a delayed onset, as the blocker would have to leave its site before current can flow. Because in Pecten ciliary photoreceptors the rising phase of the photocurrent elicited by a bright stimulus is swift and highly reproducible, the possibility exists, in principle, that this effect may be detectable. The results of such an experiment are shown in Fig. 14 . A photoreceptor was voltage clamped at 0 mV and stimulated every 30 s with a light step lasting 1 s. On alternating trials, the voltage was abruptly stepped to −70 mV during presentation of the light in order to greatly enhance blockade by divalents; the negative V m was maintained for ≈5 s after light termination, before being gradually returned to the holding level of 0 mV. The subsequent light may thus activate channels while still in a blocked state, and, upon opening, blockade would take milliseconds to reequilibrate at 0 mV. Alternating stimuli either not preceded by the trapping hyperpolarization or in which the hyperpolarizing step ended before the light termination provided a suitable control. Two superimposed traces obtained with this protocol are shown in Fig. 14 A: the photocurrent that had been preceded by a trapping voltage stimulus during the previous light stimulus exhibited a slight temporal lag with respect to the control record. This difference is more clearly visible in B, where the rising phase of the response is shown in an expanded time scale; the phenomenon could be reproduced with successive repetitions of the protocol. In the same cell, control trials in which the light was presented alone gave rise to photocurrents with virtually identical kinetics (C). The effect was observed in six of nine cells tested, and the average temporal lag, measured at half-maximal response amplitude, was 3.6 ± 2.2 ms. In the present report, we examined the interaction between the light-dependent K conductance of Pecten ciliary photoreceptors and extracellular divalent cations. Ca 2+ and Mg 2+ confer to the I–V relation of the photocurrent its characteristic outward rectification via a voltage-dependent block mechanism, much like it occurs in vertebrate rods. This causes the light-dependent conductance to increase sigmoidally with membrane depolarization . In the present case, the analysis of the blockade was greatly simplified by two fortunate circumstances. (a) Divalents are negligibly permeant, unlike in other cyclic nucleotide–gated channels (CNGC), as demonstrated by the lack of V rev shift when Ca 2+ and Mg 2+ were removed from the extracellular solution. To estimate the possible extent of any residual permeation by calcium, we used the constant-field equation and gauged how low P Ca / P K would have to be so that the shift in V rev upon removal of calcium will fall below the detection limit of our measurements. Considering the minute incremental changes applied to the holding voltage and the very high signal-to-noise ratio and reproducibility of the recordings obtained , the reversal voltage could be measured with submillivolt precision. A permeability ratio of 1:10 would predict a shift of 8.4 mV; for a ratio of 1:100, the value would still be in excess of 1 mV. For a shift in V rev to go undetected under our experimental conditions, Ca ions would have to be on the order of 200-fold less permeable than potassium. Furthermore, in normal ASW, the I–V relation remained flat at voltages as negative as −100 mV, implying that even large hyperpolarizations fail to relieve blockade by pushing the occluding divalent cations through the pore. This significantly reduces complexities, as one only needs to consider the reversible binding from outside, which is determined essentially by two rate constants, and their voltage dependence. (b) The interaction with the channels appears to be substantially slower than in rods, making it possible to apply certain direct approaches to study block kinetics, such as perturbation/relaxation analysis. We must point out that possible confounding effects of changes in extracellular divalents on the transduction cascade are ruled out, not only because recent measurements with fluorescent indicators confirmed the invariance of [Ca] i in these cells , but also because the light response was previously shown to be unaffected by direct manipulations of cytosolic Ca 2+ , which included intracellular application of high concentrations of buffered calcium, as well as superfusion with calcium-free solution and internal dialysis with 1,2-bis-( o -aminophenoxy) ethane- N,N,N ′ ,N ′-tetraacetic acid . By introducing either divalent cation individually in the extracellular medium and examining both the reduction of the flash response amplitude at a fixed holding voltage and the curvature imparted on the I–V relation, it was found that Ca 2+ blocks the light-sensitive conductance with substantially greater potency than Mg 2+ . Dose-response analysis showed that at a holding potential of −30 mV half-maximal blockade by Ca 2+ is attained in the vicinity of 16 mM, whereas 60 mM Mg 2+ only blocked ≈32% of the current at the same potential. Thus, the affinity is quantitatively much lower that in the case of mammalian rod CNGC heterologously expressed in Xenopus oocytes , and those of amphibian olfactory neurons ; however, the relative blocking potency of these two ions is preserved. The dependency of blockade on calcium concentration (Hill coefficient ≈1) is suggestive of a single ion interacting with a channel. The more substantial photocurrent reduction and outward rectification in 10 mM Ca 2+ , as compared with 60 mM Mg 2+ , indicates that under physiological conditions calcium ions contribute the larger share of the voltage-dependent block of the light-sensitive conductance. The steady state blockade increased dramatically with hyperpolarization; a Woodhull model was applied to analyze this voltage dependency for the case of calcium, using the equation: I X / I = 1/(1 + [X]exp( z δ F V/ RT )/ K X , where I X and I are the photocurrent amplitude in the presence and absence of blocking ion X, respectively, [X] is the blocker concentration, K X is its apparent affinity at 0 mV, V is the membrane voltage, δ is the fractional electrical distance, and F , R , and T have their usual meanings. The analysis suggests that the blocking site is located at δ ≈ 0.57 through the membrane field, measured from the extracellular side. A requirement to justify such an approach is that the voltage-dependent block of the light-activated conductance by divalent cations not be due to an allosteric effect, such as voltage-induced conformational changes of the channel that may alter the accessibility of the binding site. This contention is supported by the following observations: in the first place, in the absence of extracellular Ca 2+ and Mg 2+ , the I–V relation of the photocurrent is essentially linear ; as a consequence, any intrinsic voltage dependency of the gating of these channels is marginal at best. Furthermore, the data shown in Fig. 4 demonstrate that the extent of blockade by calcium is affected by the permeating potassium ions in a manner suggestive of a knock-off phenomenon . This strengthens the notion that blockade is likely to involve occlusion of the channel pore. The kinetics of the block were deduced from the effects of rectangular perturbations of the command potential during activation of the light-dependent channels: after the abrupt jump at the onset of the stimulus, a distinct exponential relaxation of the membrane current was observed, provided that Ca 2+ or Mg 2+ were present in the extracellular medium. For hyperpolarizing pulses, the amplitude of the relaxations increased with the size of the applied voltage stimulus, and correlated with the divalent-induced reduction of the peak photocurrent measured with V m steadily clamped at the same potentials. A quantitative correspondence was also observed between the relative size of the relaxation in the presence of calcium vs. magnesium, and the degree of suppression of the light response induced by either divalent cation at that fixed membrane voltage. These observations lend support to the notion that the relaxations indeed reflect the time course of enhancement of voltage-dependent block in response to membrane hyperpolarization. For depolarizing pulses, only a cursory analysis could be carried out, because of possible contamination of the photocurrent by a light-modulated fast-inactivating K current over most of the voltage range of interest. Pharmacological separation was not feasible because 4-AP, the antagonist of choice to suppress I A , is also an extremely effective blocker of light-dependent channels in these cells . Nevertheless, using a holding potential at which I A is largely inactivated, it could be demonstrated that step depolarization causes the photocurrent to relax in a manner consistent with a relief from blockade. Upon removal of divalent cations, these relaxations disappeared and the current time course became steplike. A striking difference was observed in the kinetics of the relaxations in presence of Ca 2+ vs. Mg 2+ : with Ca 2+ in the bath the time course was substantially faster (average τ ≈ 4.3 ms at −80 mV) than with Mg 2+ (τ ≈ 22 ms). For a simple bimolecular interaction in which the on rate is essentially diffusion limited, one would not expect a faster equilibration with the higher affinity blocker. However, the estimated association rates fall greatly below the diffusion limit, suggesting the presence of additional factors. Considering that the binding site appears to lie deep in the channel, the phenomenon may be explained if the steps leading to access to the site are rate limiting, so that the relaxation time constant becomes dominated by the association rate. One possible contributing factor is that the blocking ion may be required to shed its hydration shell; taking into account the higher hydration energy of Mg 2+ , one may then predict that this ion would equilibrate more slowly. Consistently with this conjecture, estimated on rates were over fivefold greater for calcium than for magnesium. The slow kinetics of the block by divalents deduced from the analysis of the whole-cell photocurrent are compatible with the observation that in these photoreceptors light-activated single channels can be resolved in the presence of normal concentrations of Ca 2+ and Mg 2+ and their I–V relation appears to be linear . If blockade occurred on a faster time scale than that of channel opening or than the recording bandwidth, one would expect either rapid flickering fluctuations or a decrease in the apparent unitary conductance with hyperpolarization . The present results, therefore, contrast with the stronger and faster blockade observed for the CNG conductance of other sensory cells of ciliary origin: both in vertebrate photoreceptors and olfactory neurons omission of Ca 2+ and Mg 2+ is required to measure single-channel openings . In fact, experiments on rod cGMP-gated channels heterologously expressed in Xenopus oocytes and native cAMP-activated channels from olfactory cells have shown that even after lowering extracellular divalent concentration to micromolar levels the fluctuations are too rapid to resolve, unless large depolarizations are imposed to relieve the voltage-dependent block . The slowness of the block/unblock process in ciliary photoreceptors afforded the possibility of directly examining the interactions between blockade by divalent cations and gating of the light-dependent conductance. The results of experiments using two complementary strategies suggest that changes in occupancy of the blocking site require the channels to be in the open conformation, which poses a constraint on the location of the gate with respect to the blocking site. In rod excised patches, Karpen et al. 1993 concluded that Ca 2+ and Mg 2+ may bind similarly to open and closed channels, but other blocking divalents did behave as though their binding site was located more intracellularly than some gating structures. In the present case, the deep estimated location of the divalent cation binding site would conceivably allow for the gating machinery to occupy a more external region of the pore. This, however, diverges from the picture that has emerged for other K + channels that are activated by voltage , where the gate appears to be located near the intracellular side of the membrane . The notion that in CNGCs permeation may be controlled by a more externally located structure has been proposed by Sun et al. 1996 . Furthermore, recent data show that rod channels expressed in Xenopus oocytes are blocked by tetracaine from the intracellular side preferentially when they are in the closed conformation, and that the binding site is approximately half way through the membrane field , again suggesting a different topology of the gate. In Pecten ciliary photoreceptors, the proposition that the divalent-binding site lies more deeply than the gate, together with the demonstration that Ca 2+ and Mg 2+ do not appear to permeate the light-sensitive conductance, raises the possibility that the channels may close with the blocking ion trapped inside. This situation differs with respect to that of rods, where Ca 2+ and Mg 2+ can flux through the pore into the cytosol, and thereby would not remain trapped upon closure of the gate . Trapping was first proposed by Armstrong 1971 in a classic study of squid K + channel block by quaternary ammonium compounds. Subsequently, Miller 1987 elegantly demonstrated the phenomenon in single Ca 2+ -activated K + channels from t tubules reconstituted into planar bilayers, with Ba 2+ serving as an open-pore blocker. Several other instances have been reported, such as N -methyl- d -arginine–receptor channels blocked by various organic antagonists . In the present case, the low affinity blockade posed taxing requirements on the temporal resolution necessary to demonstrate trapping; the situation is exacerbated by spontaneous channel openings in the dark, which, although infrequent, over the lengthy interval interposed between trials (tens of seconds, necessary for dark adaptation) can lead to a gradual loss of divalents remaining in the pore, thus diluting any trapping effect. It is nevertheless noteworthy that a significant fraction of the cells tested exhibited a small but reproducible lag in the rising phase of the light response whenever the preceding photostimulation was accompanied by a hyperpolarizing voltage step designed to promote maximum occupancy of the blocking site. A parsimonious interpretation of this phenomenon is that a blocking divalent cation had remained trapped in the pore. It is unlikely that in these cells blockade by divalent cations may serve the purpose of boosting S/N, as has been proposed for rods: in Pecten photoreceptors, like in other invertebrates, light-sensitive channels are closed in the dark (and background noise is therefore already at a minimum), so that detectability of faint stimuli would not benefit from a reduction of the effective single-channel conductance. Furthermore, because the kinetics of block is not rapid, noise variance of V m during the light response may not be decreased by this mechanism. On the other hand, it is noteworthy that the outward rectification of the photocurrent develops most prominently over the normal operating range of voltages for these cells and may thus be of physiological significance, perhaps as a mechanism for regulating the amplitude of the light response: near rest the block is reduced and the response to dim lights would be optimized, whereas, with the hyperpolarization caused by stronger illumination, partial blockade may avoid excessively large sustained currents. Irrespective of commonalties of purpose across different cells, voltage-dependent divalent block can be an important molecular mechanism that modulates channel function and has recently been described in another K + -selective channel, TOK1, which was cloned from Saccharomyces serevisiae . In view of the functional similarities between the light-dependent channels of Pecten ciliary photoreceptors and those of rods , the mechanisms that underlie the observed nonlinear conduction properties may be of relevance to other cyclic nucleotide–gated channels. | Study | biomedical | en | 0.999997 |
10532964 | In atrial tissue, acetylcholine released by the vagus nerve binds to muscarinic type 2 receptors, activates K ACh channels via pertussis toxin–sensitive G proteins, and slows the heart rate. Upon activation, the heterotrimeric G protein dissociates, allowing the G βγ subunits to directly activate the K ACh channel . K ACh has been shown to be composed of two types of G protein–gated inwardly rectifying potassiumb channels (GIRK1 and GIRK4), 1 associated in a heterotetrameric complex . Recombinant (GIRK) channels expressed in oocytes are also directly activated by G protein βγ subunits . In addition, GIRK channels appear to be activated independently of G proteins. In the absence of agonist, ATP hydrolysis leads to an increase in the mean open time and sensitizes channels to gating by Na + ions . Recently, it was shown that the ATP modification of GIRK channels is mediated via phosphatidylinositol phosphates such as phosphatidylinositol-bis-phosphate (PIP 2 ) . PIP 2 has been implicated in the regulation of the sodium–calcium exchanger , the K ATP channel , the inwardly rectifying ROMK1 and IRK1 channels and other Na + -gated nonselective cation channels . Moreover, PIP 2 appears to be essential for GIRK channel activation by the G protein βγ subunits . Here, using both native and recombinant GIRK channels, we show that Na + as well as Mg 2+ ions gate the ATP- or PIP 2 -modified channels. While the two ions seem to exert their effects at distinct sites on the channel protein, they showed synergistic effects on gating. In the presence of exogenous PIP 2 , G βγ and Na + and Mg 2+ ions showed great synergism in activating the channel. However, in the absence of exogenous PIP 2 , preactivation by G protein βγ subunits sensitized the channel to gating by Na + but not Mg 2+ ions. These data suggest that the synergism between Mg 2+ and G βγ subunits in gating GIRK channels shows a much greater dependence on PIP 2 levels than the synergism between Na + and G βγ . The synergism among ions and G βγ proteins in the gating of GIRK channels implies that variations of the concentrations of these molecules in the local environment of these channels could play an important role in the “fine tuning” of their activity. Recombinant channel subunits were expressed in Xenopus oocytes as described previously . Channel subunit coexpression was accomplished by coinjection of equal amounts of each cRNA (∼4 ng). The human muscarinic receptor type 2 was coexpressed with the channel subunits (∼1.5 ng injected per oocyte). The β-adrenergic receptor kinase (βARK)–PH construct, altered to incorporate the 15 NH 2 -terminal residues of Src for membrane targeting, was generously provided by Dr. E. Reuveny (Weizmann Institute of Science, Rehovot, Israel). cRNA concentrations were estimated from two successive dilutions that were electrophoresed on formaldehyde gels in parallel and compared with known concentrations of a RNA marker (GIBCO BRL). Oocytes were isolated and microinjected as described previously . The oocytes were maintained at 18°C, and electrophysiological recordings were performed 2–6 d after injection at room temperature (20–22°C). The procedure used for isolating cardiac myocytes from chicken embryos has been described previously . In brief, atrial tissue was selected using chicken embryos from eggs incubated 14–18 d. Atrial tissue was incubated for 20–30 min at 37°C in 5 ml of Mg 2+ - and Ca 2+ -free PBS supplemented with 1–2% trypsin/EDTA solution (10×, GIBCO BRL). Isolated myocytes were collected by triturating the digested tissue in 5 ml of trypsin-free solution and stored in a high potassium (KB) solution at 4°C for up to 36 h. The cells were allowed to settle on poly-lysine–coated coverslips in the recording chamber before experiments. General chemical reagents, including GTP and ATP, were purchased from Sigma Chemical Co. PIP 2 (Boehringer Mannheim) was sonicated on ice for 30 min before application. Purified recombinant G protein subunits dimer β 1 γ 7 was kindly provided by Dr. J. Garrison (University of Virginia, Charlottesville, VA). The stock of β 1 γ 7 (0.86 μg/μl) was dissolved in 20 mM HEPES, 1 mM EDTA, 200 mM NaCl, 0.6% CHAPS, 50 mM MgCl 2 , 10 mM NaF, 30 μM AlCL 3 , 3 mM dithiothreitol (DTT), 3 μM GDP, pH 8.0. The final concentration was 20 nM in a solution containing 0.012% CHAPS, and 20 μM DTT. QEHA peptide was kindly provided by Dr. R. Iyengar (Mount Sinai School of Medicine) and was used at a final concentration of 50 μM. Single-channel activity was recorded in the cell-attached or inside-out patch configurations using an Axopatch 200B amplifier (Axon Instruments). All pipettes used in the experiments were pulled using the WPI-K borosilicate glass (World Precision Instruments) and gave resistances of 2–8 MΩ. All experiments were conducted at room temperature (20–22°C). Single-channel recordings were performed at a membrane potential of −80 mV with acetylcholine (ACh, 5 μM) in the pipette, unless otherwise indicated. Single-channel currents were filtered at 1–2 kHz, sampled at 5–10 MHz, and stored directly into the computer's hard disk through the DIGIDATA 1200 interface (Axon Instruments). PCLAMP (v. 6.03; Axon Instruments) was used for data acquisition. To remove the vitelline membrane, Xenopus oocytes were placed in a hypertonic solution for 5 min. Shrunk oocytes were transferred into a V-shaped recording chamber and the vitelline membrane was partially removed, exposing just enough plasma membrane for access with a patch pipette . This procedure increased the success rate of forming gigaseals. The pipette solution contained 96 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES, pH 7.35. The bath solution contained 96 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.35. When high concentrations of Mg 2+ ions (>5 mM) were used in the bath solution, the KCl concentration was reduced accordingly to maintain osmolarity. Gadolinium chloride at 100 μM was routinely added to the pipette solution to suppress native stretch channel activity in the oocyte membrane. For chick atrial cells, the experimental solutions were the same as those used with oocyte recordings, except that the KCl concentration was 140 mM without gadolinium chloride. Free Mg 2+ and ATP concentrations were estimated as described previously . Single-channel records were analyzed using PCLAMP software, complemented with our own analysis routine, as described previously . Parameters used for single-channel analysis include activity of all channels in the patch (or the total open probability, NP o ), the total frequency of opening ( NF o ), and the mean open time ( MT o ), and averages over 5-s bins are displayed. In experiments shown in Fig. 7 , where exogenous PIP 2 was applied throughout the experiment , occasional applications of the same ion as a function of time in the experiment were used as control to ascertain that the synergistic effects described were not due to a time-dependent accumulation of PIP 2 in the membrane patch. Similar precautions were taken in the experiments shown in Fig. 4 . Experiments used to generate the data shown in these two figures were never longer than 14 min (usually 10–13 min). Na + and/or Mg 2+ ions were applied for 30 s. It has been shown previously that Na + ions can stimulate K ACh activity in an ATP-dependent manner in the absence of agonist and internal GTP . The ATP-dependent modification of the channel is thought to work via the production of membrane phosphoinositide phosphates (e.g., PIP 2 ), which interact directly with members of the inwardly rectifying K + channel family . PIP 2 also appears to be essential for G protein regulation of the K ACh channel . To further test for a dependence of the MgATP/Na + activation on G protein gating of K Ach , we designed experiments where G protein–dependent activation of the channel was impaired. As shown in Fig. 1 A, the K ACh channel in an inside-out atrial myocyte patch was activated persistently by 10 μM GTPγS, a nonhydrolyzable analogue of GTP. Activation of the channel by GTPγS was blocked upon perfusion of the QEHA peptide. QEHA is a 27 amino acid long peptide derived from the COOH terminus of the G βγ -sensitive adenylate cyclase 2 isoform. It has been shown to block G βγ activation of several different effectors, including the K ACh channel . QEHA (50 μM) application abolished the GTPγS activation of K ACh in <2 min ( n = 3). After washout, the channel activity remained very low, suggesting the persistence of the QEHA-blocking effect. However, under these conditions, the K ACh channel could be activated by MgATP/Na + (5/20 mM). QEHA coapplication with MgATP/Na + failed to block channel activation, whereas QEHA did block GTPγS-induced activation in the same oocyte patches ( n = 3) (data not shown). Another way we impaired the G protein regulation of the GIRK channels was by coexpressing them in oocytes with a βγ-binding protein. We used the PH domain of βARK (βARK-PH), which specifically binds the βγ subunits of G proteins, and thus acts as a “βγ sink” . In oocytes coexpressing the recombinant channels GIRK1/GIRK4 and the construct βARK-PH, 10 μM GTPγS did not induce channel activity. This suggests that the βARK-PH protein bound the oocyte endogenous G proteins, such that no βγ subunits were available for channel activation . However, in the same patches, MgATP/Na + (5/20 mM) caused a >30-fold increase in channel activity. Summary data revealed that channel activities ( NP o ) before, during, and after GTPγS application were similar, 0.0070 ± 0.0039, 0.0077 ± 0.0037, and 0.0097 ± 0.0045, respectively (mean ± SEM, n = 4). During application of MgATP/Na + , NP o was 0.313 ± 0.221 ( n = 4). In control experiments using inside-out patches from oocytes of the same batch that coexpressed the recombinant channels GIRK1/GIRK4 alone, GTPγS caused great channel activation ( n = 3, data not shown). Similar results were obtained in experiments in which we applied Na + ions with PIP 2 rather than MgATP ( n = 4, data not shown). These results suggest that even when G protein regulation is impaired, Na + ions are still able to activate the channel. Thus, Na + gating of the channel can indeed proceed independently of G βγ gating. Na + ions can gate GIRK channels when membrane PIP 2 levels are maintained (i.e., via hydrolysis of ATP). We next tested under conditions that did not maintain PIP 2 at a constant high level whether Na + ions could gate these channels after G βγ activation. Fig. 2A and Fig. B , show representative and summary data from experiments where Na + ions gated GIRK1/GIRK4 channels after activation by G proteins. Inside-out patches from oocytes expressing these channels showed no channel activity upon application of 20 mM Na + . This result suggested a low presence of PIP 2 in the membrane. However, this PIP 2 concentration was sufficient to allow persistent channel activation by a brief exposure to 10 μM GTPγS. Reapplication of Na + ions produced a more than fourfold increase in the channel activity above the level obtained with GTPγS. It should be noted that the effect of Na + ions on the basal channel activity was variable from patch to patch, presumably reflecting different levels of endogenous PIP 2 at the time of Na + application. Na + ions also gated GIRK channels after stimulation of activity by purified G βγ subunits. In Fig. 2C and Fig. D , Na + ions (20 mM) applied on an inside-out patch did not affect significantly the basal activity of the channel. After washout of the Na + ions, recombinant G β1γ7 was applied on the patch at a concentration of 20 nM, causing a slow channel activation. After washout of G β1γ7 , and as activity stabilized, a second application of Na + ions produced a more than threefold increase in channel activity, above the level obtained with β 1 γ 7 . Combined together, these data suggested that the G protein βγ subunits sensitized GIRK channels to gating by Na + ions. It has been shown that the mean open time ( MT o ) increased in the presence of PIP 2 that is generated by hydrolysis of ATP or exogenous application . In the present experiments, no change in the channel MT o was observed in the different solutions perfusing the patches (data not shown). This suggests that the levels of PIP 2 in the membrane were not altered, and thus could not account for the G βγ -dependent gating effects of Na + ions. It has been shown that Li + ions stimulate GIRK channels modified by ATP to ∼10% the activity level achieved by comparable Na + ion concentrations . However, Li + ions were unable to increase the activity of the channel after activation by GTPγS. In three patches, the mean NP o of the GIRK channel was 0.028 ± 0.013 in control conditions, 0.127 ± 0.035 after application of 10 μM of GTPγS, 0.578 ± 0.15 in the presence of 20 mM Na + ions, and 0.099 ± 0.045 in the presence of 20 mM Li + ions (data not shown). When applied together, Li + ions were also unable to affect the gating of the GIRK channel by Na + ions. This suggests that the gating effect of Na + ions on the GIRK channel activated by G protein βγ subunits is specific to Na + ions. In certain experiments, 5 mM MgATP increased the activity of the GIRK channels in the absence of Na + ions . 5 mM MgATP in the solution corresponds to a free Mg 2+ ion concentration of ∼2.1 mM . This observation prompted us to test whether Mg 2+ ions alone were able to gate the channel that had been modified by ATP. In Fig. 3 , in an inside-out patch from an oocyte coexpressing the channel subunits GIRK1/GIRK4, Mg 2+ ions (10 mM) had no significant effects on channel activity in the absence of ATP. After washout of Mg 2+ , the channel was activated by the combination of MgATP (2.5 mM; corresponding to ∼1.1 mM free Mg 2+ ) and Na + ions (20 mM). MgATP application was maintained and, upon withdrawal of Na + ions, channel activity became comparable to basal levels. Application of Mg 2+ ions (10 mM), in the continuous presence of MgATP (2.5 mM), increased channel activity to levels similar to those obtained with Na + ions (as confirmed by sequential application of 10 mM of each of the ions at the end of the experiment). Withdrawal of Mg 2+ ions caused channel activity to return to basal levels ( n = 3). The MT o of the channel activity was increased from ∼1 to ∼2 ms by the application of MgATP, but was not further modified during the gating by Mg 2+ or Na + ions. Using PIP 2 , we could test the ability of different Mg 2+ concentrations to activate the GIRK channels. Fig. 4 represents normalized activity of GIRK channels for different concentrations of Mg 2+ ions. The NP o for each concentration was calculated in reference to the NP o measured at 1 mM Mg 2+ . Mg 2+ ions could activate the GIRK channels at concentrations as low as 100–300 μM. Maximal activity could be obtained at a concentration of ∼7 mM Mg 2+ . At higher concentrations (e.g., 20 mM), Mg 2+ ions resulted in a decrease of channel activity relative to lower concentrations (e.g., 7 mM). It has been shown that, at high concentrations, divalent cations can trigger aggregation of PIP 2 molecules , a result that could account for the effects of high Mg 2+ concentrations on channel activity. In another set of experiments, we showed that Mg 2+ , like Na + gating, can occur independently of G proteins. Patches excised from oocytes coexpressing the βARK-PH domain and GIRK channels were exposed to PIP 2 (2.5 μM) and subsequently to Mg 2+ ions. In these patches, GTPγS (10 μM) was unable to activate the GIRK channels, giving a NP o of 0.08 ± 0.03, identical to the NP o measured in PIP 2 (0.078 ± 0.02). Mg 2+ ions (1 mM) could increase the channel activity approximately sixfold ( n = 4, data not shown) above the activity measured in PIP 2 , showing that Mg 2+ gating could proceed independently of G βγ gating. These results suggest that when modified by ATP or PIP 2 , GIRK channels become sensitive to either Na + or Mg 2+ ions. Recent work has identified an aspartate amino acid residue as the site of action of Na + ions on GIRK channels, GIRK2 (D228) and GIRK4 (D223) . Moreover, it was shown that Na + sensitivity lies entirely with the heteromeric partners of GIRK1, as this channel possesses an asparagine instead of an aspartate residue at the equivalent position. We used the point mutant GIRK4(S143T) (referred to as GIRK4*) that allows for high levels of activity of homotetrameric GIRK4 channels to test for Na + and Mg 2+ sensitivity. GIRK4* channel activity shows high sensitivity to both Na + and Mg 2+ . Fig. 5 shows that indeed GIRK4*(D223N) loses its sensitivity to Na + ions (20 mM). However Mg 2+ ion (1 mM) sensitivity was intact . Summary data are shown in Fig. 5 B. These data indicate that Na + and Mg 2+ ions act at distinct sites to activate GIRK channels. We observed, particularly at high concentrations (>5 mM), that internal Mg 2+ ions reduced the amplitude of single GIRK channel currents. In Fig. 6 A, the activity of the coexpressed channel subunits GIRK1/GIRK4 from an inside-out patch was recorded at −120 mV. After activation by 10 μM GTPγS, channel activity was recorded in a solution containing 1 mM Mg 2+ ions, showing an approximate amplitude of −3.2 pA. When the solution applied to the patch was switched to one containing 20 mM Mg 2+ ions, the amplitude of the single openings was rapidly reduced to a lower value, approximately −2.5 pA ( n = 5). In Fig. 6 B, the activity of native K ACh channels in an inside-out patch from an atrial cell was recorded at −90 mV. After exposure to 5 μM PIP 2 , the patch was perfused with a solution containing 20 mM Mg 2+ ions, giving an amplitude of approximately −2.2 pA. When the solution applied to the patch was switched to one containing 20 mM Na + and 1 mM Mg 2+ ions, the channel amplitude immediately increased to a value of approximately −3.5 pA. This amplitude was also obtained in control conditions, where 1 mM Mg 2+ ions were present ( n = 5). The reduction in the single-channel amplitude was observed at various voltages. Since it was present at negative potentials (i.e., −80, −90, and −120 mV) where no rectification occurs, it is likely to proceed by a mechanism distinct from that of the rectification phenomenon. Mg 2+ ions at high concentrations also decreased the amplitude of GIRK single channels when applied together with Na + ions (data not shown). Thus, regardless of their ability to gate GIRK channels , Mg 2+ ions at high concentrations (>5 mM) show a clear inhibition on single-channel current amplitudes. These data suggest that the inhibitory effect of Mg 2+ ions on the single-channel amplitude was not dependent on their ability to gate the channel. ATP modification of GIRK channels (native or recombinant) is likely to proceed through changes in the level of membrane PIP 2 in the local environment of the channel . In Fig. 7 A, the activity of recombinant GIRK1/GIRK4 channels was not increased by the application of 2.5 μM PIP 2 alone. Mg 2+ ions (10 mM), applied with PIP 2 , stimulated activity >40-fold. As shown previously , Na + ions (10 mM) were able to gate the channel in the presence of PIP 2 , resulting in activity equivalent to that obtained with Mg 2+ ions. When applied together, in the presence of PIP 2 , Mg 2+ and Na + ions (10 mM each) showed a synergistic effect stimulating channel activity >200-fold. We next tested whether Mg 2+ ions, like Na + ions , can further gate GIRK channels after channel activation by GTPγS, under conditions that do not maintain PIP 2 at constant high levels. Fig. 7 B shows that the basal activity of GIRK1/GIRK4 recombinant channels was not affected by Mg 2+ ions. 10 μM GTPγS increased the activity of the channel approximately fivefold above basal levels. After GTPγS washout, the channel activity was stable and, when applied to the patches, Mg 2+ ions were unable to increase channel activity further. In contrast, Na + ions (10 mM) increased activity by another twofold above the GTPγS effect. When Mg 2+ ions were applied together with Na + ions, no further increase in channel activity above the levels obtained with Na + ions was seen. Thus, G protein activation sensitized the GIRK channels to gating by Na + ions, but not Mg 2+ ions. In Fig. 7 C, we show the effects of Mg 2+ and Na + ions after stimulation of the channel by GTPγS under conditions that kept PIP 2 at a constant high level. As shown earlier, in the absence of Mg 2+ and Na + ions, PIP 2 was not able to increase the basal activity of the GIRK channels. When Mg 2+ ions (10 mM) were applied to the patches in the presence of PIP 2 , a greater than eightfold increase over control or PIP 2 activity levels occurred. Mg 2+ and Na + ions (each 10 mM) in combination could raise channel activity by 50-fold over control levels. We then applied GTPγS and studied the effects of ions on G protein–stimulated channel activity in the continuous presence of PIP 2 . GTPγS was able to activate the channel >14-fold above control basal levels. After washout of GTPγS, channel activity was stable. When Mg 2+ ions (10 mM) were applied to the patches after the GTPγS treatment in the continuous presence of PIP 2 , they could enhance channel activity to levels >100-fold higher than those obtained under control conditions. Thus, in the continuous presence of PIP 2 , this high level of activity was greater than that obtained with Mg 2+ or GTPγS alone or their sum, suggesting synergistic interactions among the three molecules. Finally, when Mg 2+ and Na + ions were applied together, the channel total activity was increased 400-fold compared with control. Similar data were obtained when the G protein β 1 γ 7 subunits rather than GTPγS were used. In three cells, the total channel activity measured as the mean NP o was 0.027 ± 0.023 in control conditions, 0.022 ± 0.02 in the presence of 2.5 μM PIP 2 , 0.12 ± 0.09 in the presence of PIP 2 and 10 mM Mg 2+ ions, and 1 ± 0.55 in the presence of PIP 2 and Mg 2+ and Na + ions. When 20 nM β 1 γ 7 was applied in the presence of PIP 2 , it gave a steady state activity of the channel corresponding to a mean NP o of 0.25 ± 0.12. In the continuous presence of PIP 2 and after stimulation of the channel by βγ subunits, the mean NP o was 1.23 ± 0.23 in the presence of Mg 2+ ions and 2.61 ± 0.24 in the presence of Mg 2+ and Na + ions. It should be noted that the differences in channel activity (mean NP o ) for the same condition applied to the patches may be related to differences in the level of channel expression between different batches of oocytes. Taken together, these data make four points. (a) Mg 2+ ions can gate the channel after modification by PIP 2 . At a concentration of 10 mM, their gating potency is comparable with that of 10 mM Na + ions. (b) When applied together, Mg 2+ and Na + ions show synergistic effects, resulting in levels of activity higher than those induced by each of the ions separately or their summed responses. (c) After activation by GTPγS (in the absence of exogenous PIP 2 or MgATP), the GIRK channels are not further gated by Mg 2+ ions, suggesting an important difference between Mg 2+ and Na + ions in gating these channels. (d) After channel modification by the combination of PIP 2 and GTPγS, Mg 2+ ions do stimulate the GIRK channel activity to higher levels than those obtained with PIP 2 alone, suggesting that PIP 2 renders the βγ-activated channels sensitive to gating by Mg 2+ ions. In the present study, we have shown that Mg 2+ ions at physiological concentrations are additional activators of G protein–gated potassium channels. These K + channels can be activated independently either by the βγ subunits of GTP-binding proteins or by intracellular ions, such as Na + or Mg 2+ ions. Activation by either G protein subunits or ions shows an absolute dependence on the presence of PIP 2 . Specific combinations of these molecules show synergism and suggest differential dependence on the level of PIP 2 for channel activation. This complex dependence of K + channel activity on G proteins, Mg 2+ , Na + ions, and PIP 2 could serve to “fine tune” channel activity during physiological and pathophysiological conditions, where changes in the relative concentrations of these molecules might occur. Previous results from our laboratory showed that intracellular solution containing MgATP/Na + was able to stimulate K + channel activity in the absence of acetylcholine in the pipette, suggesting a G protein–independent mechanism of activation . Subsequently, it was further demonstrated that the ATP dependence of G protein–sensitive K + channels, as well as of other inwardly rectifying channels, involved phosphoinositide formation, particularly PIP 2 . In addition, it was reported that G protein activation of the K + channel showed an absolute dependence on PIP 2 . In the present study, we show that impairment of G protein subunit activation of the channel (by binding and competing away G βγ from the channel with either QEHA perfusion or βARK-PH coexpression) did not prevent the MgATP/Na + stimulation of activity . Thus, we have provided further evidence that Na + ion gating of the channel modified by ATP (or PIP 2 ) can be independent of G protein subunit activation. Mg 2+ ions have been shown to play an essential role in the rectification properties of inwardly rectifying K + channels. Unitary current–voltage relations for G protein–sensitive K + channels become ohmic if the internal face of the patch is exposed to Mg 2+ -free solutions. Inward rectification is restored when Mg 2+ is reintroduced in the bathing solutions . Mg 2+ ions are involved in many other reactions as essential cofactors. Kurachi et al. 1986 showed that G protein activation of the native G protein–sensitive K + channel was absolutely dependent on Mg 2+ , possibly due to the requirement of Mg 2+ for the binding of GTP to the G α subunit . More recently, it has been appreciated that Mg 2+ -dependent processes of ATP hydrolysis (likely to be involved in phosphorylation–dephosphorylation of phosphoinositides) regulate channel activity . Our present data show that Mg 2+ ions, in addition to their involvement in the processes mentioned above, are able to activate the ATP- or PIP 2 -modified G protein–sensitive channel . In the presence of PIP 2 , similar concentrations of Mg 2+ and Na + ions yielded comparable levels of channel activity, suggesting equivalent gating abilities for both ions. Since PIP 2 mimics the MgATP effects on the channel, we have been able to study directly Mg 2+ gating effects. Mutation of the amino acid responsible for Na + -ion activation of GIRK channels did not interfere with Mg 2+ -ion activation. This result strongly suggests that Mg 2+ and Na + ions act on distinct sites to gate the channel. Our data also show that Mg 2+ ions reduced the conductance of the G protein–gated channels in a manner independent of their stimulatory effect on gating. Since this inhibitory effect of Mg 2+ ions on conductance was present at negative potentials (−120, −90, and −80 mV), where no rectification is occurring , it is unlikely that the two processes proceed through a single mechanism. This effect of partial block on channel conductance suggests that Mg 2+ ions act at a site located very near the pore. Chuang et al. 1997 described a chronic inhibition of the IRK3 inward rectifier channel by internal Mg 2+ ions, which is independent of the rectification process and is voltage independent. However, the on and off rates of this inhibition were slow (in the minute range) and no reduction of the single-channel conductance was reported. Under our conditions, the blocking effect of Mg 2+ ions occurred much more rapidly, in the range of seconds . At higher PIP 2 concentrations, the combination of Na + and Mg 2+ ions resulted in a stimulation of channel activity that was greater than the sum of their individual effects, suggesting synergistic interactions of these ions on channel gating . Na + ions gate the K + channel in the presence of hydrolyzable ATP or PIP 2 . In the present study, we show that (shortly after patch excision in solutions that do not replenish or supply PIP 2 ) application of G βγ subunits, but not of Na + or Mg 2+ ions, results in stimulation of channel activity . Our previous study showed that in the absence of PIP 2 in the membrane (e.g., by its complete hydrolysis by exogenous PLCβ 2 ) no gating molecule (e.g., G βγ or Na + ) could activate the channel. In the present experiment under conditions that we do not expect to have depleted PIP 2 , G βγ subunits caused a much greater stimulation of activity than Na + or Mg 2+ ions. This result suggests that the dependence on PIP 2 for channel gating is greater for Mg 2+ and Na + than for G βγ . Under these conditions, we find that Na + ions do stimulate channel activity after preactivation by GTPγS or by purified G βγ subunits . This result suggests that G βγ activation sensitizes the K + channel gating to Na + ions. Moreover, in such experiments, G βγ subunits and Na + ions act synergistically in gating the channel. Interestingly, Mg 2+ ions were unable to gate the channel after channel preactivation by GTPγS. These data underscore an interesting difference in the gating of this channel by ions, namely at low PIP 2 levels G βγ subunits synergize with Na + but not Mg 2+ ions to gate the channel. This difference of the two ions on channel gating is lost at higher PIP 2 concentrations . In such experiments, not only were the synergistic effects of the ions shown in Fig. 7 A reproduced, but also Mg 2+ as well as Na + ions cooperated with G βγ . When applied in combination, all three gating particles showed synergistic effects . We have previously shown that block of the Na + /K + pump activates K ACh in atrial myocytes with kinetics similar to those seen for Na + accumulation resulting from the block of the pump . Thus, it is likely that the effects of cardiac glycosides on cardiac rhythm involve the Na + -sensitive K ACh channels. Under physiological conditions, local variations of [Na + ] i (e.g., during an action potential) and possibly [Mg 2+ ] i could provide a sensitive and fast control of the GIRK channel gating and activity. The synergism among ions and G βγ subunits implies that variations in the local levels of these molecules could have a profound impact on the dynamic range of GIRK channel activity under normal or pathophysiologic states. Channel binding sites for PIP 2 , G βγ , and Na 2+ ions have been identified . We postulate that additional distinct sites exist to completely account for the effects of gating molecules on channel activity. Fig. 8 shows the closed channel state C 0 in the absence of PIP 2 . GIRK channels interact weakly with PIP 2 , and as a result PIP 2 does not directly activate these channels (closed state C 1 ). In the absence of PIP 2 , gating molecules such as G βγ , Na + , or Mg 2+ are unable to activate the channel (closed state C 2 ). However, in the presence of PIP 2 , any of the gating molecules can cause channel activation. We envision two possible mechanisms for the synergistic action of gating molecules to activate the channel. Ions and G βγ subunits maybe exerting their combined effects by synergistic interactions of channel sites with PIP 2 . Published reports have already suggested a stronger interaction of channel with PIP 2 in the presence of either G βγ subunits or Na + ions Alternatively, the gating molecules could be inducing conformational changes, affecting gating directly, independently of PIP 2 interactions. Although PIP 2 is absolutely required for gating molecules to be effective, we have seen that at low PIP 2 concentrations G βγ , unlike Na + or Mg 2+ ions, can still gate the channels. This result suggests a stronger influence of G βγ than of Na + or Mg 2+ ions on channel gating, possibly proceeding in a PIP 2 -independent manner. Further work will be required to distinguish between these possibilities. | Study | biomedical | en | 0.999997 |
10532965 | The inwardly rectifying K + channel, Kir 1.1 (ROMK), plays a critical role in kidney function . Convincing correlative observations strongly support the idea that the product of the Kir 1.1 gene encodes the pore-forming subunit of particular ATP-sensitive K + channels that affect urine-concentrating ability and potassium homeostasis . Indeed, immunodetectable Kir1.1 protein is restricted to the apical membrane of the thick ascending limb, distal tubule, and collecting duct , where these unique K ATP channels are exclusively expressed . Moreover, heterologous expression studies in Xenopus oocytes have revealed that Kir 1.1 exhibits nearly identical single channel conductance and high-open probability kinetics as the native secretory K + channel . Coexpression of an ATP-binding cassette protein, cystic fibrosis transmembrane-conductance regulator, is required to confer ATP and glibenclamide sensitivity on Kir 1.1 and to recapitulate the full repertoire of native channel behavior. In these respects, the kidney secretory K ATP channel has been proposed to exhibit a multimeric Kir 1.1 channel–ABC protein modifier subunit composition, similar to the Kir 6.x/SUR channels in the heart and islet beta cells . A definitive link between the Kir1.1 gene, the renal secretory K ATP channel and kidney function has been established by a familial salt-wasting nephropathy called Bartter's syndrome . Resulting from genetic defects in the renal concentrating mechanism, the disorder is characterized by a constellation of fluid and electrolyte abnormalities, including polyuria, hypokalemia, metabolic alkalosis, and hypotension, that resemble those observed with chronic loop-diuretic administration . In fact, mutations within the major components of the NaCl reabsorbtive machinery in the thick ascending limb of Henle's loop have been linked with this genetically heterogenous disorder . Consistent with an essential role of the secretory K ATP channel in the thick ascending limb of Henle , loss-of-function mutations in the Kir 1.1 gene have been identified in several kindreds affected with Bartter's syndrome. The discovery of disease-causing mutations in Kir1.1a not only provides valuable insights into the role of this channel in health and disease, it also lends illustrative clues about structural determinates of function. Like other members of the inward rectifying class of K + channels, a functional channel is formed by the tetrameric assembly of Kir 1.1 subunits. Each subunit is comprised of two putative transmembrane domains that flank a “P” loop containing the potassium selectivity filter and other determinants of the permeation pathway . The transmembrane core is bounded cytoplasmic NH 2 - and COOH-terminal domains, playing roles in channel regulation , conduction and oligomerization . Mutations within each of these domains have been linked to Bartter's syndrome. Many of the mutations introduce nonsense codons or frameshifts in the NH 2 terminus, producing truncated proteins with obvious loss of function consequences . A mutation in the core domain that alters the permeation pathway has been identified . Other mutations disrupt a regulatory protein kinase A phosphorylation site, Ser 219, presumably by influencing its phosphorylation state or ability to modulate channel activity . While no particular function has been assigned to the extreme COOH terminus of the channel, the link between a mutation that deletes the last 60 amino acids (T332–K333 frameshift) and Bartter's disease suggested an important role for this previously unrecognized domain. As predicted from the Bartter's phenotype, we found that deletion of the COOH-terminal 60 amino acid residues renders channels inactive. In the present study, we have systematically evaluated the possible functional roles of the extreme COOH-terminal domain of Kir 1.1a. By elucidating the mechanism underlying the defect, we discovered that the extreme COOH terminus acts as an obligate determinate of channel gating, maintaining the channel in a stable open state. The present study is focused on the consequences of COOH-terminal mutations in Kir 1.1. To ensure that differences in functional expression result from changes in the open reading frame (ORF) 1 rather than changes in the 3′ untranslated regions (UTR), the 3′ UTR was eliminated from wild-type rat Kir 1.1a and COOH-terminal deletion mutant cDNA constructs. Mutagenesis was performed by overlap extension PCR . Deletion mutants (Kir 1.1a 331X, Kir 1.1a 341X, Kir 1.1a 351X, Kir 1.1a 361X, and Kir 1.1a 366X) were constructed by introducing three stop codons in frame at the appropriate locations. To create Kir 1.1a Δ332–351, a PCR product encoding amino acids 352–391 was ligated to a unique NspV site (see below). All cDNAs were subcloned between the 5′ and 3′ UTR of the Xenopus β-globin gene in the modified pSD64 vector to increase expression efficiency . This vector also contains a polyadenylate sequence in the 3′ UTR (dA 23 dC 30 ). Appropriate cDNA sequence was verified by dye termination sequencing (ABI Prism). To critically test the dominant negative effect of Kir 1.1a 331X, three separate concatemeric cDNA constructs were created. In one construct, four wild-type Kir 1.1a subunits (4wt) were covalently linked together. The other two constructs were comprised of three wild-type subunits joined to one Kir 1.1a 331X at either the NH 2 - (1mut + 3wt) or COOH- (3wt + 1mut) terminal position of the tandem tetramer. Concatemeric Kir 1.1a constructs were generated in four steps. (a) Three separate silent mutations were engineered into Kir 1.1a to create unique restriction sites. Two of these sites, RsrII and KasI were introduced in the 5′ end of the ORF, while the other, Bpu1102I, was placed at the 3′ end. The unique 5′ sites were also introduced into the mutant Kir 1.1a 331X. (b) Two different linker oligonucleotides were independently ligated to the unique 3′ restriction site. Linker-1 encoded the 3′ end of Kir 1.1a from the Bpu1102I site (encompassing the final 14 amino acids of Kir 1.1a), codons for 10 glutamines, and the 5′ ORF of Kir 1.1a containing the unique KasI site. Linker-2 was identical to the first linker, except that it contained the unique RsrII site instead of KasI. (c) Three different dimers were constructed by linking-modified monomers together using the unique KasI site. One dimer contained two Kir 1.1a subunits with linker-2 attached to the 3′ end of the ORF. The second dimer contained an RsrII site at the 5′ end of the ORF, but no 3′ linker. The third dimer was identical to the second except that the 3′ subunit encoded the mutant, Kir 1.1a 331X. (d) To create the tetrameric concatemers, dimers were linked together using the unique RsrII site. One concatemer contained four wild-type subunits artificially linked together (4wt), while the other contained three wild-type subunits ligated to a single mutant (3wt + 1mut). To generate the third concatemer (1mut + 3wt), a similar stepwise approach was adopted. (a) A silent mutation containing an NspV site was introduced into Kir 1.1a at the codons for amino acids 331–332. (b) Linker-3 encoding the unique NspV site, residue 331 of Kir 1.1a, codons for 10 glutamines, and the 5′ ORF of Kir 1.1a containing the unique KasI site was ligated on to the unique NspV-restriction site. (c) A dimer was generated by ligating a modified Kir 1.1a monomer containing linker-2 (as above) to the construct containing linker-3 using the unique KasI site. (d) Two dimers were joined as before to create the tetrameric concatemer with Kir 1.1a 331X at the extreme 5′ position in the ORF, followed by three wild-type subunits (1mut + 3wt). The sequence of each modified monomer was confirmed by dye termination DNA sequencing. Appropriate tetrameric construction was confirmed by restriction enzyme analysis. Moreover, in vitro transcription from each concatemeric cDNA template yielded cRNA transcripts of the correct size. To assess the cellular distribution of Kir 1.1a and Kir 1.1a 331X, the ORF of enhanced green fluorescent protein (EGFP; Clontech) was ligated, in frame, to the 5′ end of the open reading frame of either Kir 1.1a or Kir 1.1a 331X. EGFP (GFPmut1) is a mutated form of the Aequoria victoriae protein, GFP, that contains two amino acid substitutions in the chromophore region (F64L and S65T) and uses preferred human codons. It exhibits single excitation and emission peaks at 490 and 509 nm, respectively, fluorescing 35× more intensely than wild-type GFP when excited at 488 nm . EGFP was subcloned into the modified pSD64 for use as a cytosolic marker. Complementary RNA was transcribed in vitro in the presence of capping analogue G(5′)ppp(5′)G using PstI or SmaI linearized cDNA templates. SP6 RNA polymerase was used in all reactions (mMESSAGE mMACHINE; Ambion Inc.). After DNaseI treatment, cRNA was purified by phenol-chloroform extraction and ammonium acetate/isopropanol precipitation. Yield and concentration were measured spectrophotometrically and confirmed by agarose gel electrophoresis. Female Xenopus laevis frogs were obtained from NASCO. Standard protocols were followed for the isolation and care of X . laevis oocytes. In brief, frogs were anesthetized by immersion in 0.15% 3-aminobenzoate and a partial oophorectomy was performed through an abdominal incision. Oocyte aggregates were manually dissected from the ovarian lobes, and then incubated in a calcium-free ORII medium (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES) containing collagenase (type IA, 2 mg/ml; Sigma Chemical Co.) for ∼2 h at room temperature to remove the follicular layer. After oocytes were washed extensively with collagenase-free ORII, they were placed in a modified L15 medium (50% Leibovitz's medium, 10 mM HEPES, pH 7.4) and stored at 19°C. 12–24 h after isolation, healthy-looking Dumont stage V–VI oocytes were pneumatically injected (PV820 picopump; World Precision Instruments, Inc.) with 50 nl of water containing 0–50 ng of cRNA, and then stored in L15 medium at 19°C. Channel activity was assessed 2–6 d after injection. Laser-scanning confocal microscopy was used to determine the cellular localization of EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X. Whole, unfixed oocytes bathed in ORII solution were visualized through a APOCHROMAT 10× objective lens (NA = 0.45; Carl Zeiss, Inc.) using an LSM410 microscope (excitation with 488 nm line of an Omnichrome series 643 Kr/Ar ion laser; Carl Zeiss, Inc.). Fluorescent emissions were passed through a 515-nm long pass filter. Background autofluorescence exhibited by uninjected oocytes was calibrated to zero by adjusting brightness and contrast settings at a constant pinhole size. These settings were maintained throughout the course of studies. Plasma membrane delimited fluorescence was quantified at equatorial focal sections of oocytes using Image software (National Institutes of Health). Average plasmalemmal pixel intensity was determined for continuous line segments of fixed width (∼3 μm) drawn around the entire circumference of the oocyte. For all images, pixel intensity was within the linear range as assessed by histogram analysis for each cRNA dose. At least six cells from two donors were analyzed for four to five cRNA injection doses. Whole cell oocyte currents were monitored using a two-microelectrode voltage clamp equipped with a bath-clamp circuit (OC-725B; Warner Instruments) as described before . In brief, oocytes were placed in a small Lucite chamber and incubated in either a 5 K [5 mM KCl, 85 mM N -methyl- d -glucamine (NMDG)–Cl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.4] or a 45 K (45 mM KCl, 45 mM NMDG-Cl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.4) artificial solution. When required, barium acetate was added to a concentration of 1 mM. Voltage-sensing and current-injecting electrodes had resistances of 0.5–1.5 MΩ when back filled with 3 M KCl. After a stable impalement was attained, such that both electrodes measured the same membrane potential, pulse protocols were conducted. Stimulation and data acquisition were performed with a Macintosh Centris 650 computer using an ITC16 analogue-to-digital, digital-to-analogue converter (Instrutech Corp.) and Pulse software (HEKA Electronik). Data were filtered at 1 kHz and digitized online at 2 kHz to the hard disk using Pulse and IGOR (WaveMetrics, Inc.) for later analysis. Single channel properties were assessed 2–6 d after injection in the on-cell configuration with the patch-clamp technique . In these studies, the vitelline membrane was removed from oocytes after hyperosmotic shrinking . All single channel recordings were performed under symmetrical [K + ] conditions (150 mM KCl, 1 mM CaCl 2 , 5 mM HEPES, pH 7.4). Patch-clamp electrodes, pulled from borosilicate glass , had resistances of 1–5 MΩ. Single channel currents were measured with an patch clamp amplifier (Axopatch 200A; Axon Instruments), digitized at a sampling rate of 47 kHz using a digital data recorder (VR-10B; Instrutech Corp.), and stored on a videotape. Data were acquired and analyzed using the Acquire and TAC family of programs (Bruxton Corp.). Data were replayed, filtered with an eight-pole Bessel filter (900; Frequency Devices Inc.) at a cut-off frequency of 1 kHz, and sampled at at least five times the filtering frequency. A 50% threshold criterion was used to detect events. Open and closed dwell-time histograms (logarithmic time scale, 10 bins/decade) were constructed from 15–60-s records and fit to exponential distributions using the maximum likelihood method . The single channel current magnitude was estimated by fitting Gaussian distributions to the current amplitude histograms or by measuring the amplitudes directly from analogue current traces. Inward slope conductance was assessed from such current measurements at −120 to −40 mV. Kir 1.1a and Kir 1.1a 331X coexpression experiments were analyzed using binomial probability theory, as first described for voltage-gated K + channels by Mackinnon 1991 . Assuming mutant (mut) and wild-type subunits are coexpressed with equal efficiency and randomly assemble into a complex containing n subunits, binomial theory predicts that ( n + 1) different populations of channels will be formed with finite probabilities that are prescribed by the relative amounts of the two subunits [F mut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)]. Two ideal cases were initially considered. For these models, it was also assumed that wild-type and mutant subunits oligomerize with equal efficiency and that any channel population is either fully active or fully inhibited. In the case of a complete dominant-negative effect in a tetrameric channel, the active channel population will be described by the probability of forming channels that are exclusively comprised of wild-type subunits, or (1 − F mut ) 4 . For a negative effect requiring two mutants within a tetramer, the active channel population will be described by the probability of forming channels that have less than two mutant subunits, or (1 − F mut ) 4 + 4 F mut (1 − F mut ) 3 . After considering these two possibilities, a modified probability equation, I / I o = (1 − k F mut ) 4 , was developed to describe the Kir 1.1a 331X dominant negative effect. This model maintains the fundamental properties of a binomial distribution (i.e., random assembly of n subunits determined by F mut ), but accounts for deviations in mutant channel oligomerization efficiency or partial current inhibition by a single mutant subunit. Curve fitting for analysis of the dominant negative effect was performed using IGOR. The correction factor, k , was obtained by fitting normalized macroscopic current ( I / I o ) as a function of F mut with the modified probability equation, using a nonlinear, least squares, iterative algorithm (Levenberg-Marquardt). Statistical evaluation of all data was performed with the GB-Stat™ v 5.0.6 for Macintosh statistics package (Dynamic Microsystems, Inc.). Where applicable, the pooled Student's t test was used to compare test groups. All data are given as mean ± SEM. As a first approach to examine the functional consequences of COOH-terminal truncation, wild-type Kir 1.1a and Kir 1.1a 331X cRNAs were independently injected into Xenopus oocytes, and macroscopic currents were measured using two-microelectrode voltage clamp. As predicted from the link to Bartter's syndrome, truncation of the extreme COOH terminus of Kir 1.1a abolishes channel activity . Oocytes injected with Kir 1.1a cRNA-expressed large, weakly inward rectifying macroscopic currents, typical of the wild-type channel . In contrast, no currents above the background could be detected in oocytes injected with Kir 1.1a 331X cRNA. The mean Ba 2+ -sensitive macroscopic current at −90 mV was −0.11 ± 0.05 μA ( n = 6), compared with −17.95 ± 2.87 μA ( n = 12) for the wild-type channel. Consistent with the macroscopic data, no significant activity, except for occasional endogenous stretch-activated channel openings , was detected in oocytes injected with the mutant cRNA . To determine the function of the Kir 1.1a COOH-terminal domain, we elucidated the mechanism causing the defect in this particular Bartter's mutant. Obviously, the magnitude of macroscopic Kir 1.1a current is a product of the number of channels in the membrane, the open probability of the channel, and the single channel conductance. Truncation of the extreme COOH-terminal 60 amino acids of Kir 1.1a can abolish channel activity by reducing any of these quantities, alone or in combination. The number of channels in the membrane may be reduced by a global structural alteration, loss of an essential oligomerization domain, or a defect in membrane trafficking or plasma membrane stability. Alternatively, the mutant channels may be expressed in the plasma membrane in a nonconductive or nonfunctional conformation. The potential mechanisms underlying the Kir 1.1a 331X defect implicate different functional roles for the extreme COOH-terminal domain, therefore we explored each. An oligomerization defect or global structural mutation can be easily tested by coexpressing the mutant with the wild-type channel. If the mutant protein is synthesized, folded correctly, and capable of oligomerization, the macroscopic current in oocytes coinjected with wild-type and mutant cRNA is predicted to be less than oocytes injected with wild-type alone. Fig. 2 B summarizes the results of such a study. Coexpression of Kir 1.1a 331X with the wild-type channel reduced Ba 2+ -sensitive macroscopic current by 62 ± 5%, demonstrating that the mutant is capable of exerting a negative influence on the wild-type channel ( n = 12, Kir 1.1a 331X; n = 18, Kir 1.1a; P < 0.005). To ensure that the Kir 1.1a 331X effect is specific and not due to the competition of mutant cRNA for the translational machinery, the experiment was repeated with an unrelated dominant negative Kir channel, Kir 3.1-AAA. Kir 3.1 was made nonconductive by replacing the key Gly-Tyr-Gly K + -selectivity sequence (amino acids 145–147) with three Ala residues . Kir 3.1, a G protein–gated Kir channel, is not thought to oligomerize with Kir 1.1a. As shown in Fig. 2 B, Kir 3.1-AAA exhibited no influence on Kir 1.1a expression. This result indicates that the translation of Kir 1.1a cRNA is not inhibited when a second transcript is coinjected. Instead, the dominant negative effect of Kir 1.1a 331X is due to the oligomerization of mutant and wild-type subunits. To gain further insight into the dominant negative mechanism, increasing doses of mutant cRNA were coinjected with a constant amount of wild-type transcript . Because Kir 1.1a channels are known to be tetrameric in structure , coinjection should produce five discrete populations, containing zero to four mutant subunits. Assuming random assembly, a binomial probability distribution defined by two parameters, F mut and n , determines the probability of obtaining each channel population. F mut , the mutant fraction of the total cRNA injected [F mut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)], is the probability of incorporating a Kir 1.1a 331X subunit. For a tetrameric channel, the number of subunits, n, is equal to 4. The relative frequency of functional populations determines the resultant macroscopic current density. The predicted relationships for two specific dominant negative models are shown in Fig. 3 . Consider the situation when incorporation of one or more mutant subunits abolishes channel activity. The relative current is determined by the probability of assembling four wild-type subunits into a tetramer. In this case, the macroscopic current can be predicted by the relationship, I / I o = (1 − F mut ) 4 . If two or more mutants must be incorporated within a tetramer to inhibit channel activity, an additional term, the probability of forming a channel with one mutant and three wild-type subunits [4 F mut (1 − F mut ) 3 ], is added to the equation above (line B). As shown in Fig. 3 , neither ideal model could adequately describe the Kir 1.1a 331X dominant negative effect. Instead, to characterize this particular dominant negative mutant, the data was fit to a modified probability equation, I / I o = (1 − k F mut ) 4 , where the coefficient, k , is a correction factor that can take into account variations in oligomerization efficiency or partial current inhibition. The intermediate model (line C) required a k factor of 0.6 to accurately describe the data, suggesting that Kir 1.1a 331X has a reduced oligomerization efficiency (∼60% compared with wild type) or that a single mutant subunit only partially inhibits channel activity. In the former case, the probability of incorporating a mutant is lower than predicted from the ratio of cRNA injected. In the latter instance, the measured I / I o reflects not only the wild-type population, but also the relative frequency of channels that contain a mutant and carry a diminished current. Because the two possibilities have different implications for the function of the extreme COOH-terminal domain, the source of the intermediate model was resolved by the experiment described below. To critically test the effects of incorporation of a single mutant into a Kir 1.1a channel and determine the origin of the intermediate model, three different tetrameric concatemer cDNAs (4wt, 3wt + 1mut, and 1mut + 3wt) were engineered. By creating tandem concatemers, the functional consequences of incorporating a single mutant subunit within a tetrameric channel can be assessed in a manner that is independent of oligomerization efficiency. As described in methods , four monomeric Kir 1.1a cDNAs were artificially linked together with codons for 10 glutamine residues. Glutamine linkers have been successfully used in previous studies to link potassium channel subunits together and are thought to possess little secondary structure and exert minimal influence on channel function. As shown in Fig. 4 , we validated this approach by examining the functional characteristics of 4wt and the effects of Kir 1.1a 331X monomers on concatenated channels. To confirm that covalent linkage of subunits does not perturb channel structure or function, we compared the biophysical properties of wild-type and concatenated Kir 1.1a. Injection of the 4wt tandem tetramer cRNA ( n = 22) into oocytes produced K + channels that exhibited weak inward rectification and voltage-dependent block by extracellular Ba 2+ (not shown) identical to wild-type Kir 1.1a . Moreover, covalent linkage of wild-type subunits had no effect on single channel properties ( n = 7 each). These results are summarized in Fig. 4D and Fig. E . Both wild-type Kir 1.1a and the 4wt concatemer had identical open probability (0.93 ± 0.01 vs. 0.94 ± 0.004, respectively) and single channel conductance (37 ± 2.47 vs. 39.6 ± 1.26 pS, respectively). Like the wild-type channel, the kinetics of the concatemer are best described by single open (Kir 1.1a, τ o = 20 ± 0.87 ms; 4wt, τ o = 18 ± 1 ms) and closed times (Kir 1.1a, τ c = 1.1 ± 0.03 ms; 4wt, τ c = 1.05 ± 0.04 ms). These data clearly demonstrate that the tandem tetramer precisely recapitulates wild-type channel function. To ensure that the four concatenated subunits contribute to channel formation, the 4wt concatemer was coexpressed with either the dominant negative Kir 1.1a 331X or an unrelated gene product, CD4. The results of these studies are summarized in Fig. 4 C. As predicted if the four concatenated subunits form functional channels, 4wt concatemer currents were not affected by coexpression of the dominant negative mutant, Kir 1.1a 331X ( n = 11). These observations confirm that channels are indeed formed from a single protein rather than portions of multiple concatemers. Having demonstrated that concatenation of subunits does not affect channel function, we tested the functional effects of Kir 1.1a 331X within a tandem tetramer. Incorporation of a single mutant within the concatemeric tetramer, at either the NH 2 - or COOH-terminal positions (1mut + 3wt or 3wt + 1mut, respectively) completely suppressed macroscopic channel activity. These results are summarized in Fig. 5 . In contrast to the 4wt concatemer (−2.56 ± 0.34 μA, n = 25), the 1mut + 3wt and 3wt + 1mut concatemers exhibited no measurable currents above background . The complete dominant negative effect was confirmed at the single channel level. Except for occasional endogenous stretch-activated channel openings in some patches, no significant activity could be detected in on-cell patches of oocytes injected with either mutant-containing concatemer . Because Kir 1.1a 331X exerts a complete dominant negative effect when constrained by the covalent linkage of a concatenated tetramer, it is clear that the intermediate dominant negative model results from an ∼40% reduction in oligomerization efficiency. The role of the extreme COOH terminus in governing the efficiency of subunit assembly is consistent with both the recessive nature of Bartter's disease and two previous reports that implicated the COOH terminus in tetramerization . While this domain influences Kir 1.1a oligomerization efficiency, these results collectively demonstrate that COOH-terminal truncation has more important functional consequences downstream of channel assembly. To test the role of the COOH-terminal domain (amino acids 332–391) in determining membrane trafficking and plasma membrane stability, NH 2 -terminal enhanced green fluorescent protein fusion proteins of Kir 1.1a and Kir 1.1a 331X were constructed and expressed in oocytes. This strategy was favored over an antibody binding assay because incorporation of an external epitope (Flag or AUI) abolished Kir 1.1a function. As shown in Fig. 7 , NH 2 -terminal attachment of EGFP had no effect on the single channel conductance (γ = 39 ± 3 pS) or high open probability kinetics of ( P o = 0.91 ± 0.01) of Kir 1.1a ( n = 4). The lack of EGFP-dependent functional effects offers compelling evidence that channel structure is not altered by the NH 2 -terminal fusion protein. As shown in Fig. 8 , whole, unfixed oocytes injected with EGFP fusion protein cRNA were examined using laser scanning confocal microscopy. Uninjected oocytes exhibited a low basal autofluorescence. EGFP was widely distributed throughout the cytoplasm ( n = 6). In contrast, both EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X were localized along a distinct zone circumscribing the oocyte ( n = 6 in each injection dose). The circumferential fluorescence pattern was maintained in sequential z-plane optical sections through the entire oocyte, consistent with predominant plasma membrane expression. Fluorescence intensity was proportional to the amount of fusion protein cRNA injected, and no significant difference could be detected between EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X at any injection dose . These data support the premise that both channels are expressed on the plasma membrane with equal efficiency. Comparison of macroscopic conductance with plasma membrane delimited fluorescence intensity in oocytes injected with either EGFP fusion protein ( n = 5) further strengthens this conclusion. The results of these studies are summarized in Fig. 8 C. The macroscopic conductance was directly proportional to fluorescence intensity in oocytes injected with EGFP-Kir 1.1a, as predicted if circumferential fluorescence intensity is proportional to the number of channels in the plasmalemma. Consequently, the observation that the circumferential fluorescence distribution and intensity of the Kir 1.1a 331X mutant is indistinguishable from the wild-type channel provides evidence that the mutant channel is also primarily expressed on the plasma membrane. Collectively, these results suggest that the entire extreme COOH terminus of Kir 1.1a (amino acids 332–391) is not necessary to direct Kir 1.1a plasma membrane targeting and stability in the Xenopus oocyte expression system. Subsequently, loss of function in the Bartter's mutant, Kir 1.1a 331X, is not due to a reduction of the number of channels in the plasmalemma. Instead, mutant channels exist in a nonconductive or inactive conformation. This conclusion was confirmed by examining a series of COOH-terminal truncated mutants as described below. Having found that the extreme COOH terminus is involved in maintaining channel activity, we delimited the minimal domain that is required for functional expression in the hopes of revealing the mechanism underlying the Kir 1.1a 331X defect. In these studies, the channel was gradually reconstructed by sequentially adding portions of the extreme COOH terminus back to Kir 1.1a 331X . Addition of amino acids 332–351 was sufficient to rescue minimal channel function, while deletion of this domain in isolation (Kir 1.1a Δ332–351) resulted in the loss of channel function. The results of these studies are summarized in Fig. 9 B. Oocytes injected with either Kir 1.1a 341X or Kir 1.1a Δ332–351, like Kir 1.1a 331X, exhibited no Ba 2+ -sensitive currents above background ( n = 6–11). In contrast, Kir 1.1a 351X–injected oocytes displayed significant weakly inward-rectifying, Ba 2+ -sensitive macroscopic currents ( n = 10, P < 0.001). Current density increased further with the addition of 10 more residues . The addition of five more residues (Kir 1.1a 366X, n = 10) failed to produce a further significant increase in channel activity. Collectively, these studies define amino acids 332–351 as the critical COOH-terminal domain within the extreme COOH terminus that is absolutely required for maintaining channel activity. By identifying an active channel retaining a defect, Kir 1.1a 351X, the mechanism underlying the role of the COOH-terminal domain could be uncovered. The nature of the defect was elucidated by comparing the single channel properties of Kir 1.1a 351X ( n = 5) to the wild type ( n = 4). Aberrant gating behavior was revealed in long duration (∼20 min) recordings, where P o was continuously monitored in 15-s intervals. The results from two representative recordings are shown in Fig. 10 A. In contrast to the sustained high open probability kinetics of the wild-type channel, Kir 1.1a 351X channels exhibited bursts of channel activity, “active gating,” interrupted by sojourns in a long-lived inactive mode ( t inactive = 5.12 min). As a consequence of the long-lived inactive state, the open probability of the Kir 1.1a 351X was significantly reduced compared with the wild-type channel . During the active gating mode, Kir 1.1a 351X channels exhibited the same single channel conductance (γ = 40 ± 2 pS), open and closed times (τ o = 20 ± 1.2 ms; τ c = 1.2 ± 0.1 ms) as wild-type channels , indicating that the COOH-terminal deletion does not alter the conduction pathway. Truncating the extreme COOH terminus of Kir 1.1a reduces channel activity by dramatically increasing the probability of exhibiting an inactive gating mode. This phenotype is unique to Kir 1.1a 351X; the open probability and single channel conductance of Kir 1.1a 361X and Kir 1.1a 366X are not statistically different from the wild-type channel . Although Kir 1.1a 361X and Kir 1.1a 366X gating kinetics were unchanged from the wild type, the macroscopic current amplitude of the wild-type channel was significantly greater than these COOH-terminal deletion mutants. This may reflect a defect in channel trafficking or stability in the plasma membrane, or a significant silent channel population. Nevertheless, these studies define amino acids 332–351 as the domain within the extreme COOH terminus that is absolutely required for maintaining Kir 1.1a channels in the open state. The identification of disease-causing mutations in the Kir1.1a channel provides insightful clues about important functional domains that have previously escaped attention. For instance, a particular Bartter's-linked mutation, a frame shift (T332–K333) that replaces the last 60 carboxyl-terminal amino acids (332–391) with an entirely different 34-residue domain , implicated the extreme COOH terminus as an obligate functional element. As predicted by this hypothesis, truncation of the COOH-terminal domain (Kir 1.1a 331X) produced a complete loss-of-function phenotype. By elucidating the mechanism underlying the defect in Kir 1.1a 331X, we discovered that the extreme COOH terminus acts as an obligate determinant of channel gating, maintaining the channel in a stable open state. This conclusion is supported by several lines of evidence. Because Kir 1.1a 331X is efficiently expressed on the plasma membrane, loss-of-function must be a consequence of an inactive conformation rather than defective surface expression. By characterizing a series of progressively smaller truncations, a minimal COOH-terminal domain that is sufficient to rescue function was defined (332–351), and a discrete gating defect was uncovered. Mutant channels that are truncated at the extreme boundary of the required domain (351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. Upon spontaneous recovery from inactivation, 351X acquired an active-gating mode that exhibits precisely the same conductance and kinetic properties as the wild-type channel. Because these biophysical attributes reflect the unique signature of K + permeation through the Kir 1.1 pore , the mutational effect of 331X must result from aberrant gating rather than a permanent or profound distortion of the conduction pathway. Collectively, these data demonstrate that the extreme COOH-terminal domain is required to maintain channels in the open state; channels lacking this modulatory structure appear to be locked in a closed conformation. It should be pointed out that our result with the 331X mutant in oocytes differs from a recent report of Schwalbe et al. 1998 , who investigated the natural T332-K333 frameshift mutation in Sf9 insect cells. These investigators reported that channels bearing the T332 frameshift mutation are predominantly localized in an intracellular compartment of Sf9 cells, presumably the Golgi. This may reflect that the plasma membrane expression of the Kir 1.1 channel is dependent on the host cell type . Alternatively, the entirely different 34-residue domain that is introduced by the frameshift mutation may prevent proper trafficking to the cell membrane. While we cannot rule out a role for this domain in channel trafficking, we found in Xenopus oocytes where the channel is efficiently expressed that the COOH-terminal domain normally acts as an essential regulator of gating. Presently, we cannot state with any certainty how partial truncation of the COOH terminus alters channel structure and how this favors the occupancy of a quiescent state; however, several mechanisms can be considered. For instance, the mechanism by which intracellular pH and PKA regulate Kir1.1a activity provides some insight into the modulatory role of the carboxyl terminal domain. Kir1.1 activity is dependent on PKA-mediated phosphorylation of three serine residues (S44, S219, S323) located within the NH 2 - and COOH-terminal domains , whereas a drop in cytoplasmic pH, sensed at K80 , inhibits the channel. Because dephosphorylation and cytoplasmic acidification induce a state that resembles the gating conformation conferred by the 351X mutation, it is tempting to speculate a common or coupled gating pathway. In this view, the extreme carboxyl terminal domain may directly influence physiologic regulation of the channel in one of two different ways. The domain could directly interact with the phosphoacceptor and pH-sensing sites, similar to the way the NH 2 -terminal domain in cyclic nucleotide–gated channels interacts with the cyclic nucleotide–binding domain . Alternatively and more likely (see below), the domain may affect the actual gating machinery, as has been postulated to explain the “molecular brake” function of the cytoplasmic PAS domain in the HERG channel . The recent observation that pH-dependent channel closure coincides with a structural rearrangement of the NH 2 and COOH termini provides credence for this latter idea. Indeed, the COOH-terminal structure (C308) that undergoes the pH-dependent allosteric change appears to lay in close proximity to the modulatory domain that we have identified (332–351). While the actual mechanism underlying the COOH-terminal regulatory function remains to be elucidated, our data chiefly point to one particular model. As supported by direct protein–protein interaction studies in Kir1.1 and other inwardly rectifying potassium channels , the dominant negative behavior of 331X revealed that the COOH-terminal domain (332–391) not only controls open-state occupancy, but is also an important determinant of subunit oligomerization. Such overlapping functions suggest that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening. Accordingly, we propose that disruption of the intersubunit contacts within the COOH-terminal domain alters the quaternary structure of the tetramer in a way that perturbs the stability of the open pore, comparable with the way that oxygen induces an allosteric conformational change in hemoglobin . Considering the similarities in the primary structure of the COOH-terminal domain within the inward-rectifying potassium channel family, the modulatory function of this structure may be more widespread. Within the affected COOH-terminal domain of Kir 1.1 (332–391), the distinct region that is absolutely required for channel function (332–351) exhibits the highest degree of amino acid conservation, sharing ∼30% identity with other members of the inward-rectifying channel family. Interestingly, Drain et al. 1998 recently identified a structural determinant of ATP-dependent gating in a distantly related K ATP subunit, Kir 6.2, that lies within this moderately conserved domain. In view of this observation, the extreme COOH-terminal domain may act on conserved gating structure in the inward rectifying potassium channel family. In summary, we have elucidated the mechanism underlying the functional defect of a particular Kir 1.1a mutant that has been linked with the familial salt wasting nephropathy, Bartter's syndrome. These efforts revealed that a previously unrecognized domain, the extreme COOH terminus, acts as an important modifier of channel gating. | Study | biomedical | en | 0.999998 |
10539974 | Sodium–calcium exchangers constitute a family of ion counter transporters that play a prominent role in cellular Ca 2+ homeostasis . These integral membrane-spanning proteins are generally thought to serve as Ca 2+ efflux mechanisms that are driven by the Na + electrochemical gradient. Although present in many (and possibly all) tissues of the body, Na + –Ca 2+ exchange has been most extensively characterized in cardiac and neuronal tissue. Three unique transporter gene products have been identified in mammals (NCX1, NCX2, and NCX3) and two have been shown to undergo alternative splicing . Within the NCX1 subfamily, 12 alternatively spliced isoforms have been identified, whereas a single version of NCX2 has been cloned to date. The existence of three splice variants of NCX3 has been demonstrated . The cardiac Na + –Ca 2+ exchanger, NCX1.1, remains the most extensively characterized isoform with respect to structure, function, and regulation . The consequences of alternative splicing of Na + –Ca 2+ exchangers are largely unknown. One possibility is that expression of different exon combinations could provide a mechanism for directing Na + –Ca 2+ exchangers to appropriate cellular locations. For example, in kidney, the exchanger may reside exclusively in the basolateral membrane of renal cells . Alternatively, or perhaps coincidentally, splice variants could exhibit altered functional and/or regulatory properties appropriate for different cellular environments and Ca 2+ homeostasis requirements. Support for this notion comes from our recent demonstration of significant functional differences in the regulatory phenotypes of alternatively spliced isoforms of the Na + –Ca 2+ exchanger from Drosophila . These splice variants, CALX1.1 and CALX1.2, differ by five amino acids in a region analogous to the alternative splice site of mammalian exchangers . A preliminary report indicates that functional differences exist between Na + –Ca 2+ exchangers found in cardiac and renal tissue with respect to modulation by voltage, [Ca 2+ ] i and phosphorylation . These investigators have also demonstrated preferential expression of splice variants containing the A exon in hippocampal neurons, and the B exon in astrocytes and C 6 glioma cells . Furthermore, a comparison of exchangers expressed in Xenopus oocytes revealed that the AD, but not the BD splice variant, was regulated by cAMP-dependent protein kinase . In an effort to gain a better understanding of the transport and regulatory consequences of alternative splicing among Na + –Ca 2+ exchangers, we selected two versions of NCX1 that differ only in terms of which of its two, mutually exclusive exons (i.e., A and B) is expressed: NCX1.4 (exons AD) and NCX1.3 (exons BD). Since exon A splice variants appear to be expressed predominantly in excitable tissues (e.g., neurons, heart, skeletal muscle), whereas exon B isoforms are more widely distributed (e.g., kidney, liver, lung, astrocytes) , we reasoned that any observed phenotypic differences might provide insight into how Na + –Ca 2+ exchange is tailored to the specific Ca 2+ handling requirements of different tissues. Thus, we expressed NCX1.3 and NCX1.4 in Xenopus laevis oocytes and obtained current measurements of Na + –Ca 2+ exchange using the giant excised patch-clamp technique . By using a variety of experimental protocols, we were able to clearly resolve exon-specific influences on the Na + i - and Ca 2+ i -dependent regulatory mechanisms of NCX1. Kidney and brain samples were collected from adult dogs, rapidly frozen in liquid N 2 and stored at −70°C. Total RNA was subsequently isolated by the method of Chomczynski and Sacchi 1987 , as modified by Quednau et al. 1997 , and splice variants of NCX1 were cloned via reverse transcriptase–PCR (RT-PCR) as described . Complementary DNA was amplified in a total volume of 50 μl containing: 1× TaqPlus Precision buffer (Stratagene Inc.), 200 μM dNTP, 5% DMSO, and 25 pM each of reverse phase–purified, canine NCX1.1 forward and reverse primers that span its alternative splicing region (see below). The reaction mixture was heated to 95°C for 5 min and cooled to 80°C before addition of 2.5 U of TaqPlus Precision Polymerase. Amplification was carried out in 35 cycles of denaturation (30 s at 94°C), annealing (60 s at 55°C), and extension (2 min at 72°C), followed by an additional 10 min of extension at 72°C. Forward primer: 5′-TGGAGGTGAAGGTATTGCGA-3′ ; reverse primer: 5′-TCTGCATACTGATCCTGGGT-3′ (corresponding to amino acids 861–855). Products from the RT-PCR procedure were purified using GeneClean (Bio101) and subcloned into the pCR-Script vector (Stratagene Inc.) according to manufacturer's instructions. Splice variants of NCX1 were identified by dideoxy sequencing. Clones with exon compositions AD (brain) and BD (kidney) were digested with XhoI and KpnI, and the resulting cassettes repaired into a full-length, canine NCX1.1 clone in pBluescript II SK(+) (Stratagene Inc.). The resulting constructs were designated NCX1.4 (exons AD; brain) and NCX1.3 (exons BD; kidney), according to the nomenclature of Quednau et al. 1997 . Complementary DNAs encoding NCX1.3 and NCX1.4 were linearized with HindIII (New England Biolabs Inc.) and cRNA synthesized using T3 mMessage mMachine in vitro transcription kits (Ambion Inc.) according to the manufacturer's instructions. After injection with ≈5 ng of cRNA encoding NCX1.3 or NCX1.4, oocytes were maintained at 16°C in solution B minus BSA (see below). Electrophysiological measurements were typically obtained from days 4–6 after injection. Xenopus laevis were anaesthetized in 250 mg/liter ethyl p -aminobenzoate (Sigma Chemical Co.) in deionized ice-water for 30 min. Oocytes were then removed and washed in solution A containing (mM): 88 NaCl, 15 HEPES, 2.4 NaHCO 3 , 1.0 KCl, 0.82 MgSO 4 ; pH 7.6 at room temperature (rt). The follicles were teased apart and the oocytes transferred to 5 ml of solution A containing 80 mg collagenase (Type II; Worthington Biochemical Corp.) and incubated for 45–60 min at rt with gentle agitation. The oocytes were washed several times in solution B containing (mM): 88 NaCl, 15 HEPES, 2.4 NaHCO 3 , 1.0 KCl, 0.82 MgSO 4 , 0.41 mM CaCl 2 , 0.3 mM Ca(NO 3 ) 2 , 1 mg/ml BSA (Fraction V; Sigma Chemical Co.); pH 7.6 at rt, and then transferred to 5 ml of 100 mM K 2 HPO 4 , pH 6.5 at rt, containing 1 mg/ml BSA. After incubation at rt for 11–12 min with gentle agitation, the oocytes were washed several times in solution B at rt. Defolliculated stage V–VI oocytes were selected and incubated at 16°C in solution B minus BSA until injection the following day. Na + –Ca 2+ exchange current measurements were obtained using the giant excised patch clamp technique, as described previously . Borosilicate glass pipettes were pulled and polished to a final inner diameter of ≈20–25 μm and coated with a Parafilm™:mineral oil mixture to enhance patch stability and reduce electrical noise. To expedite removal of the vitelline layer before membrane patching, oocytes were shrunk slightly by incubation in a solution containing (mM): 100 K-aspartate, 100 KOH, 100 MES, 20 HEPES, 5 EGTA, 5 MgCl 2 , pH 7.0 at rt, for ≈15 min at rt. After removal of the vitelline layer by dissection, oocytes were placed in a solution containing (mM): 100 KOH, 100 MES, 20 HEPES, 5 EGTA, 5–10 MgCl 2 , pH 7.0 at rt (with MES), and GΩ seals formed via gentle suction. Membrane patches were excised by progressive movements of the pipette tip. Rapid solution changes (i.e., ≈200 ms) were accomplished using a custom-built, computer-controlled, 20-channel solution switcher. Hardware (Axopatch 200a; Axon Instruments) and software (Axotape) were used for data acquisition and analysis. For outward current measurements, pipette (i.e., extracellular) solutions contained (mM): 100 N -methyl-glucamine–MES, 30 HEPES, 30 tetraethylammonium (TEA)-OH, 16 sulfamic acid, 8.0 CaCO 3 , 6 KOH, 2.0 Mg(OH) 2 , 0.25 ouabain, 0.1 niflumic acid, 0.1 flufenamic acid; pH 7.0 at 30°C (with MES). Outward Na + –Ca 2+ exchange currents were elicited by switching from Li + i - to Na + i -based bath solutions containing (mM): 100 [Na + Li]-aspartate, 20 MOPS, 20 TEA-OH, 20 CsOH, 10 EGTA, 0–9.91 CaCO 3 , 1.0–1.5 Mg(OH) 2 , pH 7.0 at 30°C (with MES or LiOH). Magnesium and Ca 2+ were adjusted to yield free concentrations of 1.0 mM and 0–30 μM, respectively, using MAXC software . For inward current measurements, pipettes contained (mM): 100 Na-MES, 10 EGTA, 20 CsOH, 4 Mg(OH) 2 , 20 TEA-OH, 10 HEPES, 0.25 ouabain, 0.1 niflumic acid, 0.1 flufenamic acid, 0.002 nifedipine, pH 7.0 with MES. Inward currents were activated by switching between Ca 2+ -free and Ca 2+ containing Li + -based bath solutions described above. All experiments were conducted at 30 ± 1°C. All data reported are mean ± SEM, unless indicated otherwise. Our aim was to determine which aspects of the ionic regulatory profile of NCX1 can be directly attributed to expression of its mutually exclusive exons, A and B. We chose the splice variant pair NCX1.4 (exons AD) and NCX1.3 (exons BD) and characterized their ionic regulatory phenotypes under a variety of conditions. Inspection of the aligned sequences of exons A (encoding 35 amino acids) and B (encoding 34 amino acids) in Fig. 1 reveals substantial similarity between NCX1.3 and NCX1.4. 13 identities are found and the exons are ≈63% similar with respect to conservative substitutions, with the greatest similarity occurring towards their NH 2 termini. Charge reversal occurs at two positions, substitution of charged for neutral residues is observed at six, polar residues coincide with hydrophobics at three, and the overall electric charge of exon A is −2, whereas exon B is +1. Exon B encodes a cysteine residue at position 585 , whereas no cysteines are found in any other exons of the alternatively spliced region of NCX1. Although the specific contribution of dissimilar amino acids between NCX1.3 and NCX1.4 has not been determined, the net effect of interchanging exons A and B is substantially altered ionic regulatory behavior. We examined the [Na + ] i dependence of peak and steady state outward Na + –Ca 2+ exchange currents mediated by NCX1.4 and NCX1.3 to obtain estimates of Na + transport affinities, as well as the rate and extent of I 1 inactivation. Fig. 2 shows representative current traces obtained in response to the rapid (i.e., <200 ms) application of 10–100 mM Na + i to the cytoplasmic surface of the patches in the continuous presence of 1 μM regulatory Ca 2+ i . Transport Ca 2+ o in the pipette was constant at 8 mM. After each current activation event, patches were allowed to recover for 32–48 s in Li + i -containing solution plus 1 μM Ca 2+ i before delivery of the next Na + i pulse. With increasing [Na + ] i , the isoforms exhibited similar increases in peak current and in the extent of current inactivation, characteristic of Na + i -dependent, or I 1 , inactivation . In response to the application of 100 mM Na + i at 1-μM regulatory Ca 2+ i , the rate of inactivation of NCX1.4 was marginally faster than that observed for NCX1.3 (0.29 ± 0.03 s −1 , n = 18 vs. 0.22 ± 0.03 s −1 , n = 11, respectively, NS). However, the major difference between the splice variants was in steady state current levels produced in response to changes in [Na + ] i . Whereas NCX1.4 exhibited a [Na + ] i -dependent increase in steady state current levels, similar to that observed with the cardiac Na + –Ca 2+ exchanger, NCX1.1 , steady state currents mediated by NCX1.3 were mainly insensitive to changes in [Na + ] i over the concentration range examined (10–100 mM). However, this behavior could be abolished by treatment of the patch with α-chymotrypsin , a procedure known to deregulate Na + –Ca 2+ exchangers . After proteolysis, the Na + i dependence of NCX1.3 became hyperbolic, similar to that observed for peak currents with NCX1.4. Thus, ionic regulation alters the apparent Na + i affinity of steady state Na + –Ca 2+ exchange currents for NCX1.3. Fig. 3 summarizes the Na + i dependence of peak and steady state outward Na + –Ca 2+ exchange currents derived from pooled data obtained with NCX1.3 and NCX1.4 in the presence of 1 μM regulatory Ca 2+ i , as above. Currents were normalized to the values obtained at 100 mM Na i + . For both isoforms, peak currents progressively increased with increasing [Na + ] i . Estimates of exchanger affinity for Na + i based on peak current measurements provided nearly identical values of K d (33 ± 5 mM, n = 4 vs. 31 ± 4 mM, n = 6, for NCX1.3 and NCX1.4, respectively). For comparison, the peak current-derived Na + i affinity of the cardiac exchanger, NCX1.1, is ≈27 mM . With respect to the Na + i affinity derived from measurements of steady state currents, however, a K d value of 16 ± 1 mM ( n = 6) was obtained for NCX1.4, whereas the NCX1.3 isoform appeared to be nearly Na + i independent over the concentration range examined. Thus, a K d value could not be estimated. However, after treatment with α-chymotrypsin, a K d value of 26 ± 1 mM (three determinations from two patches) was estimated for NCX1.3, similar to that of peak currents for both exchangers. Fig. 4 illustrates the Na + i dependence of the NCX1 isoforms in terms of their ratios of steady state to peak currents, or F ss values . This fraction is a sensitive measure of the extent of I 1 inactivation and can provide insight into the stability of the I 1 inactive complex. Both splice variants exhibit a decrease in F ss with increasing [Na + ] i , typical of I 1 -inactivated Na + –Ca 2+ exchangers, and reflecting entry into the I 1 state from the three Na + i -loaded configuration of the exchanger . However, for [Na + ] i ≥ 25 mM, the decrease in F ss is more pronounced for NCX1.3 than for NCX1.4. For example, F ss values calculated from currents acquired in response to the application of 100 mM Na + i were 0.24 ± 0.03 and 0.07 ± 0.01 ( n = 20 and 25 determinations from 14 patches) for NCX1.4 and NCX1.3, respectively. Note that for NCX1.3, the extent of I 1 inactivation is ≈90% at high [Na + ] i , whereas at 10 mM Na + i it is ≈20%. This substantial inactivation of NCX1.3-mediated transport is likely to be responsible for the peculiar nature of the Na + i dependence of its steady state currents, as shown in Fig. 3 . That is, the increase in steady state current in response to Na + i appears to be largely offset by the extensive inactivation that occurs as [Na + ] i is raised. Consequently, steady state Na + –Ca 2+ exchange currents mediated by NCX1.3 do not exhibit a hyperbolic response to rising [Na + ] i unless this regulatory mechanism is eliminated by proteolysis with α-chymotrypsin. Fig. 5 illustrates the current–voltage (IV) 1 relationships for NCX1.3 and NCX1.4. Outward currents were activated by switching from 100 mM Li + i -containing perfusing solution to 100 mM Na + i , and 1 μM regulatory Ca 2+ i was present throughout. The IV relationship was determined before (a) and during (b) exchange current activation, and the former values were subtracted from the latter. From a holding potential of 0 mV, 40-ms voltage steps, in 10-mV increments, were applied from −100 to +100 mV, with a return to the holding potential between each step. Pooled data shown in Fig. 5 (bottom) are from three NCX1.3 and four NCX1.4 patches, with currents normalized to the values obtained at 0 mV. Note that a reversal potential is not observed under these conditions as the pipette solution does not contain Na + o . The exchanger isoforms exhibited similar IV relationships to that observed for the cardiac exchanger, NCX1.1 . We did not observe significant differences in the voltage dependency of the kidney exchanger, in contrast to an earlier report . Fig. 6 illustrates the effects of removal and reapplication of regulatory Ca 2+ i on outward Na + –Ca 2+ exchange currents mediated by NCX1.3 and NCX1.4. Currents were elicited by applying 100 mM Na + i , with 1 μM Ca 2+ i present before activation. Regulatory Ca 2+ i was then removed and reapplied in the middle of the current trace in the continuous presence of 100 mM Na + i . For NCX1.4, Ca 2+ i removal led to rapid and nearly complete inhibition of outward exchange current, with a half-time for current decay of 0.52 ± 0.05 s ( n = 5). Similarly, steady state levels were rapidly restored when Ca 2+ i was reapplied, with a half-time of 0.49 ± 0.14 s ( n = 5). For comparison with NCX1.1, the equivalent protocols yield half-times for loss and reacquisition of steady state current levels of 10.8 and 7.5 s, respectively . Thus, steady state Na + –Ca 2+ exchange currents for NCX1.4 exhibits considerably faster responses to this experimental maneuver than those observed for the cardiac exchanger. In contrast, with NCX1.3, half-times for loss and restoration of steady state current after removal and reapplication of Ca 2+ i could not be determined due to the negligible steady state currents generated even in the presence of regulatory Ca 2+ i . Fig. 7 shows representative traces that illustrate the dependence of outward Na + –Ca 2+ exchange currents mediated by NCX1.3 and NCX1.4 upon [Ca 2+ ] i . Regulatory Ca 2+ i , at the indicated concentrations, was present for 32–48 s before, during, and after current activation with 100 mM Na + i . For both isoforms, exchange currents are augmented by Ca 2+ i . In particular, NCX1.4 behaves similar to the cardiac exchanger, NCX1.1, in that regulatory Ca 2+ i not only stimulates exchange activity, but also alleviates I 1 inactivation. At 10 μM Ca 2+ i , Na + i -dependent inactivation is nearly eliminated and the current recording adopts a square appearance. With NCX1.3, however, I 1 inactivation is still prominent at 10 μM Ca 2+ i , in sharp distinction to both NCX1.4 and NCX1.1. That is, regulatory Ca 2+ i is not only incapable of alleviating I 1 inactivation for NCX1.3, it appears to inhibit outward current generation. These relationships are illustrated graphically in Fig. 8 . The pooled data shown in Fig. 8 illustrate the regulatory Ca 2+ i dependence of peak and steady state outward currents mediated by NCX1.3 and NCX1.4 in response to the application of 100 mM Na + i . Currents were normalized to the values obtained at 1 μM regulatory Ca 2+ i . Both exchangers initially exhibit an increase in peak outward current as regulatory Ca 2+ i is raised . For NCX1.4, data were fit to the Hill equation over the [Ca 2+ ] i range 0–3 μM, providing a K d value of 0.2 ± 0.06 μM. Beyond 3 μM Ca 2+ i , however, deviation from simple hyperbolic behavior became evident, and further increases in [Ca 2+ ] i often led to decreased peak outward currents . This is presumably due to competition between Ca 2+ i and Na + i at the intracellular transport site, a phenomenon previously documented for NCX1.1 and two alternatively spliced isoforms of the Drosophila Na + –Ca 2+ exchanger, CALX1 . With NCX1.3, this behavior was more pronounced and regulatory [Ca 2+ ] i > 1 μM mediated a pronounced decrease of exchange current. This reduction in current is also evident in the representative traces shown in Fig. 7 . Thus, we could not derive a meaningful estimate of K d for NCX1.3, but visual inspection suggests that at [Ca 2+ ] i < 1 μM, both isoforms are comparably stimulated by regulatory [Ca 2+ ] i . With respect to steady state currents , NCX1.4 again exhibits a smooth increase of outward current levels with increasing regulatory [Ca 2+ ] i , and similar to that observed for NCX1.1. This reflects the progressive alleviation of I 1 inactivation by Ca 2+ i . In sharp contrast, the results obtained with NCX1.3 indicate that, at most, I 1 inactivation is barely influenced by regulatory Ca 2+ i and no obvious Ca 2+ i dependence is discernible. The effects of regulatory Ca 2+ i on F ss , the ratio of steady state to peak currents are shown in Fig. 8 (bottom). For NCX1.4, a U-shaped Ca 2+ i dependence was obtained. The value of F ss initially declines from 0–0.3 μM Ca 2+ i , and then rises as Ca 2+ i is increased beyond 0.3 μM. This behavior reflects the observation that, in the absence of Ca 2+ i , outward currents are small and show relatively little inactivation. As Ca 2+ i increases, however, peak current progressively rises, causing F ss to decrease at intermediate [Ca 2+ ] i . Finally, as [Ca 2+ i ] is further increased, the extent of I 1 inactivation is reduced to near-zero levels and F ss returns to higher values. These characteristics of F ss are typical of the cardiac exchanger, NCX1.1 . In contrast, Na + i -dependent inactivation of NCX1.3 is essentially insensitive to regulatory Ca 2+ i and an L-shaped relationship is obtained. That is, the progressive alleviation of I 1 inactivation observed with NCX1.4 and NCX1.1 does not occur for the kidney Na + –Ca 2+ exchanger. The observation that NCX1.3 appears to be inhibited at higher concentrations of regulatory Ca 2+ could occur if this exchanger had a higher affinity for Ca 2+ at the intracellular transport site. Consequently, lower concentrations of Ca 2+ could compete for Na + i and reduce current magnitude. To test this possibility, we examined inward Na + –Ca 2+ exchange currents for both NCX1.3 and NCX1.4. Pipettes contained 100 mM Na + and inward currents were activated by applying different Ca 2+ concentrations (0.1–100 μM) to the cytoplasmic surface of the patch. Typical inward current recordings are shown in Fig. 9 . We did not observe any major differences for inward Na + –Ca 2+ exchange currents produced by NCX1.3 and NCX1.4. The apparent affinities calculated for Ca 2+ activation of inward Na + –Ca 2+ exchange currents were 9.0 ± 1.9 μM (mean ± SD, n = 5 patches) for NCX1.3 and 8.1 ± 1.4 μM (mean ± SD, n = 3 patches) for NCX1.4. These values are similar to that reported for NCX1 . Furthermore, this indicates that the inhibitory effects of higher regulatory Ca 2+ concentrations observed for outward currents from NCX1.3 are unlikely to be due to greater competition at the intracellular transport site. Fig. 10 illustrates representative current recordings obtained for paired-pulse experiments conducted at two different regulatory Ca 2+ concentrations (0.3 and 10 μM Ca 2+ i ). In each case, currents were activated by 100 mM Na + i and transported Ca 2+ o in the pipette was constant at 8 mM. Regulatory Ca 2+ i , at the indicated concentration, was present throughout the entire paired-pulse trials. For NCX1.4, the second test pulse is substantially reduced after a 4-s interval at 0.3 μM Ca 2+ i , whereas the two pulses are nearly identical in magnitude at 10 μM Ca 2+ i . This behavior illustrates the ability of Ca 2+ i to accelerate exit from, and/or reduce entry into, the I 1 inactive state, and is typical of NCX1.1 . With NCX1.3, however, I 1 inactivation is only weakly affected by regulatory Ca 2+ i , and substantial inactivation is observed for paired-pulses even at 10 μM. This difference is shown graphically for representative data over a range of regulatory [Ca 2+ ] i 's for a 4-s inter-pulse interval. The parameter chosen to evaluate recovery, (I peak − I ss , pulse 2)/(I peak − I ss , pulse 1), was used so that recovery values would fall between 0 and 100%. That is, steady state currents are subtracted so that only the portion of current that inactivates is analyzed in terms of its recovery. This behavior was confirmed in a total of four patches each for NCX1.3 and NCX1.4. The NCX1 gene encodes a variety of alternatively spliced Na + –Ca 2+ exchangers, several with unique tissue distributions. Although the physiological significance of this diversity is unknown, it may reflect different requirements for the maintenance of Ca 2+ homeostasis in various cell types. Thus, we examined the ionic regulatory properties of two splice variants of NCX1: NCX1.3 and NCX1.4. These particular exchangers were selected for two reasons. First, NCX1.3 is a prominent splice variant in kidney, whereas NCX1.4 is abundant in brain. Second, these isoforms differ only in terms of expression of the mutually exclusive exons A (NCX1.4) and B (NCX1.3), with expression of the D cassette exon common to both. Therefore, our results provide direct insight into the functional role(s) of the mutually exclusive exons of NCX1. From structure–function considerations, our results point to prominent interactions between the alternative splicing region and other domains subserving the Na + i - (i.e., I 1 ) and Ca 2+ i - (i.e., I 2 ) dependent regulatory processes. These observations also provide a foundation towards understanding the physiological behavior of these transporters in their native environments. Sodium-dependent, or I 1 , inactivation of Na + –Ca 2+ exchange current describes the ionic regulatory process that occurs in response to the cytoplasmic application of Na + i . The phenomenon manifests as a rapid (i.e., ≈200 ms) rise in outward exchange current to a peak value, followed by a relatively slow (i.e., ≈30 s) decay to steady state levels of activity. This mechanism has been characterized extensively in giant excised patch experiments , as well as in intact myocytes . Experimental and kinetic modeling studies indicate that entry into the I 1 state proceeds from the three-Na + i –loaded configuration of the exchanger, and that exit from I 1 is a first-order process . Furthermore, structure–function analyses have provided evidence that the XIP region, at the NH 2 terminus of the large intracellular loop of the exchanger, plays a pivotal role in this process. Specifically, mutations within the XIP domain of the cardiac exchanger, NCX1.1, and the Drosophila Na + –Ca 2+ exchanger, CALX1.1, can accelerate or eliminate the I 1 inactivation process . Calcium-dependent, or I 2 , regulation describes the stimulatory (e.g., NCX1.1) or inhibitory (e.g., CALX1.1) effect of micromolar [Ca 2+ ] i on both inward and outward currents associated with the forward (i.e., Ca 2+ efflux) and reverse (i.e., Ca 2+ influx) modes, respectively, of Na + –Ca 2+ exchange. Like I 1 , this process has also been well documented in giant excised patch clamp studies using both cardiomyocytes and Xenopus oocytes expressing NCX1.1 and CALX1 splice variants . However, the results obtained from intact myocytes pertaining to the operation of the I 2 regulatory mechanism have been controversial. For example, stimulation of Na + –Ca 2+ exchange current by submicromolar [Ca 2+ ] i has been reported by Kimura et al. 1987 , Noda et al. 1988 , and Miura and Kimura 1989 , whereas a large Ca 2+ i -independent component of whole cell Na + –Ca 2+ exchange current was observed by Matsuoka and Hilgemann 1994 . Studies using Chinese hamster ovary cells expressing NCX1.1 have also demonstrated enhancement of Na + –Ca 2+ exchange activity in response to Ca 2+ i , an effect that was absent for a deletion mutant shown to lack I 2 regulation . Structure–function studies of NCX1.1 have identified a portion of its large cytoplasmic loop that functions as the regulatory Ca 2+ i binding site . An analogous region is conserved in all cloned Na + –Ca 2+ exchangers and, in particular, for CALX1.1, despite the fact that this exchanger is inhibited in response to Ca 2+ i . Moreover, we have demonstrated limited interconversion of Ca 2+ i -dependent regulatory phenotypes between NCX1.1 and CALX1.1 in a chimeric exchanger study . However, there is no coherent understanding, at present, of the physiological role of I 2 regulation. Virtually all mechanistic information presently available concerning ionic regulation of Na + –Ca 2+ exchangers comes from outward current measurements of exchange activity in giant, excised patches . Although this represents the reverse (i.e., Ca 2+ influx) mode of transport, it is the most effective means available to allow resolution of mechanistic details of Na + –Ca 2+ transport and regulation. Before recognition of the existence of alternatively spliced isoforms of Na + –Ca 2+ exchangers, structure–function studies aimed at delineating the bases of ionic regulation of NCX1 examined the consequences of deleting substantial portions of its large cytoplasmic loop . In particular, two of these constructs (i.e., Δ240-679 and Δ562-685) eliminated the alternative splicing region, as well as appreciable flanking sequences. With Δ562-685, I 1 regulation was ablated, whereas Δ240-679 was associated with loss of both I 1 and I 2 regulation . However, given the extent of the structural changes associated with these constructs, it is difficult to evaluate how elimination of the alternative splicing region specifically contributed to the functional consequences. With a third, chimeric exchanger construct (essentially equivalent to NCX1.3), preliminary electrophysiological characterization revealed functional Na + i - and Ca 2+ i -dependent regulation . We have confirmed these results and extend the characterization of NCX1.3 to demonstrate substantial functional differences between it and the brain- (i.e., NCX1.4) and cardiac- (i.e., NCX1.1) derived exchangers. Qualitatively, NCX1.4 behaves in a similar fashion to NCX1.1 in terms of its Na + i and Ca 2+ i dependencies and I 1 and I 2 regulatory profiles. The main difference we observed between NCX1.1 and NCX1.4 lies in the rapidity with which removal and reapplication of regulatory Ca 2+ i influences steady state Na + –Ca 2+ exchange currents . The response of NCX1.4 to this maneuver is at least 10× faster than that of NCX1.1 . Possibly, this attribute of NCX1.4 provides it with the ability to respond appropriately to frequency-encoded signaling in neuronal tissue. In all other regards, NCX1.4 appears similar to the cardiac exchanger. Notably, both NCX1.1 and NCX1.4 share expression of the A exon in their respective alternative splicing regions. Although it is tempting to speculate that exon A alone is responsible for the observed similarities between the cardiac and brain isoforms, the contributions of the cassette exons, alone or in combination, has yet to be evaluated. However, what is clear is that profound differences in regulatory profiles are associated with expression of the A or B exons. The ionic regulatory behavior of NCX1.3 is considerably different from both NCX1.1 and NCX1.4. Specifically, I 1 inactivation appears to be much more prominent for this exchanger. This inactivation process resulted in steady state currents that were inhibited ≈90% compared with peak current values. Furthermore, regulatory Ca 2+ i was incapable of alleviating this inhibition, in contrast to the behavior of NCX1.1 and NCX1.4. Finally, we observed a unique behavior of NCX1.3, which suggested that regulatory Ca 2+ i exhibits both stimulatory and inhibitory effects . This is unlikely to reflect differences in competition between Ca 2+ and Na + at the intracellular transport site, as both NCX1.3 and NCX1.4 show similar Ca 2+ affinities for inward currents. Consequently, the expression of the B exon adds a novel aspect to the ionic regulatory phenotype of NCX1.3. Characterization of regulatory phenotypes has only been undertaken for a few members of the Na + –Ca 2+ exchanger family (i.e., NCX1.1, NCX1.3, NCX1.4, CALX1.1, and CALX1.2). For example, we have shown that alternatively spliced CALX1 exchangers, which differ by five amino acids, show marked differences in their I 1 and I 2 regulatory properties . Although the characterization of regulatory phenotypes and primary structural determinants subserving these processes is advancing, the physiological significance of I 1 and I 2 regulation is not well understood. It remains the case that the most persuasive evidence suggesting a physiological role for these regulatory mechanisms is the fact that alternative splicing produces changes in their properties. Nevertheless, it is interesting to speculate on the physiological consequences of these regulatory differences. At present, there is no evidence to suggest that these differences exist for the purpose of localizing the exchangers, although this possibility has not been examined. For example, although NCX1.3 is preferentially localized to the basolateral membrane within discrete regions of the nephron , the role, if any, of alternative splicing in this process is not known. We hypothesize that ionic regulatory mechanisms tailor exchange activity to the Ca 2+ homeostatic requirements of individual tissues. For example, Ca 2+ transients in cardiac muscle from large animals (e.g., dog, human) occur within the frequency range of ≈0.5–3 Hz. Frequency-encoded signaling within neuronal tissue, however, occurs considerably faster (e.g., 300 Hz), whereas Ca 2+ reabsorption in the kidney is likely to be a less dynamic process. As a Ca 2+ efflux (and possibly influx) mechanism, the activity of the Na + –Ca 2+ exchange system must be able to keep pace with this wide range of Ca 2+ flux requirements. If ionic regulation contributes to the ability of Na + –Ca 2+ exchangers to accommodate these various cellular Ca 2+ flux rates, then the activity of exchangers in a physiological setting may be governed, in part, by the time-averaged consequences of ionic regulation, in addition to the thermodynamic influences of the Na + and Ca 2+ electrochemical gradients. | Study | biomedical | en | 0.999996 |
10539975 | The phylogenetically ubiquitous Cl − channel proteins of the ClC family are responsible for a multitude of physiological functions in organisms as varied as mammals, elasmobranchs, yeast, and green plants . In humans, these channels are intimately involved in electrical excitability of skeletal muscle and neurons, in epithelial electrolyte homeostasis, and in cellular volume regulation. The ClC channels make up the only known molecular family of voltage-gated Cl − channels, but very little is understood about their molecular architecture or about the roles their conserved sequences play in gating or permeation. Our ignorance of ClC mechanisms stems from the molecular asymmetry of the Cl − permeation pathway, a necessary consequence of the unusual construction of these channels, in which each subunit of a homodimeric complex forms an independent pore . This architecture demands that the pore is lined by protein residues scattered throughout the primary sequence rather than in localized “hot spots” . In this sense, ClC channels differ fundamentally from many well-studied ion channels, such as voltage-gated Na + , K + , and Ca 2+ channels, cyclic nucleotide–gated channels, inward rectifiers, neurotransmitter-activated channels, and connexins, which are all built on a “barrel-stave” plan with the ion conduction pore formed by repetition of a transmembrane subunit around an axis of four-, five-, or sixfold symmetry . Just as the symmetry of these familiar channels has contributed greatly to their tractability in sequence-function studies, its absence seriously hinders efforts to discern molecular features of ClC channels by the classical, but indirect, means of mutagenesis and functional analysis. Recently emergent genome sequences have revealed in prokaryotes many homologues of ion channels hitherto considered strictly eukaryotic . The biological functions of these bacterial genes are entirely unknown. Nevertheless, membrane proteins from prokaryotes have been uniquely successful in heterologous overexpression, and so we were inspired to search for prokaryotic members of the ClC family. This study examines sequences of nine putative ClC channel genes from bacteria and archaea and pursues one of these from Escherichia coli, which can be overexpressed and functionally reconstituted as a Cl − channel. This prokaryotic ClC channel, which we name “EriC,” forms a functional homodimer, a result implying that dimeric quaternary structure is general to the ClC family. All chemicals were reagent grade. 36 Cl was purchased from DuPont NEN as a 0.5–1 M HCl solution and was neutralized with NaOH. Working stock solutions were 50–60 mM NaCl, 26–31 μCi/ml. 3 H-Glutamic acid was also obtained from DuPont NEN. Dowex 1×4–100, from Sigma Chemical Co. was converted to the glutamate form and stored in water. E . coli lipids (polar lipid extract) were obtained from Avanti Polar Lipids. Glutaraldehyde was Grade I from Sigma Chemical Co. Searches for ClC homologues were performed using BLAST 2.0 or WU-BLAST2 at the following sites on the indicated protein database: National Center for Biotechnology Information (NCBI; nonredundant data base), Swiss Institute of Bioinformatics (SIB; completed bacterial genomes and EMBL prokaryotes), The Institute for Genomic Research (TIGR; microbial genomes). Initial searches using ClC-0, ClC-3, or ClC-7 as query retrieved eight prokaryotic sequences with high statistical scores for similarity to ClCs; further searches using prokaryotic sequences as query retrieved one additional sequence ( Methanococcus ). Oligonucleotides corresponding to flanking regions of the E . coli ClC gene yadQ were used to PCR-amplify the gene from E . coli genomic DNA. The PCR product was blunt-end ligated into the ZeroBlunt vector (Invitrogen Corp.) and subcloned into pASK90 , downstream from the tetracycline promoter. The sequence of the open reading frames of the products of two independent PCR reactions matched the sequence in the E . coli TIGR database. A hexahistidine tag was added immediately after the initiating methionine codon using PCR mutagenesis, and the resulting construct, pEriC, was used in all experiments. All SDS-PAGE experiments were run under reducing conditions (2% β-mercaptoethanol) using standard Laemmli solutions . Proteins were visualized using Coomassie Brilliant Blue R-250. E . coli JM83 cells were transformed with pEriC, grown overnight at 37°C, and diluted 50-fold into room-temperature Terrific Broth , containing 100 μg/ml ampicillin. Cells were grown at room temperature (23–25°C) to A 550 of 1.0, and then induced overnight by addition of 0.2 mg/liter anhydrotetracycline (added from a 0.2 mg/ml stock in dimethylformamide). Cells were harvested and kept at 4°C during all subsequent steps. After washing with Buffer A (95 mM NaCl, 5 mM KCl, 50 mM MOPS-NaOH, pH 7.0) and resuspension at 50 ml per liter culture, the cells were disrupted by sonication in the presence of leupeptin (1 μg/ml), pepstatin (1.4 μg/ml), PMSF (0.17 mg/ml), and β-mercaptoethanol (6 mM). Debris was discarded after centrifugation (8,000 g , 25 min), and membranes were collected from the resulting supernatant (110,000 g , 45 min). The membranes were resuspended in Buffer B (95 mM Na-P i , 5 mM KCl, pH 7.0), supplemented with 250 mM sucrose and then stored at −80°C. Membranes were extracted for 2 h (at a concentration equivalent to A 280 = 20 in 0.5% SDS) in Buffer BK (95 mM K-P i , 5 mM KCl, pH 7.0) containing 15 mM dodecylmaltoside (DDM) 1 . Insoluble material was removed by centrifugation (110,000 g , 45 min). For binding, 1/10 volume of Buffer E (400 mM imidazole-HCl in Buffer BK, 1 mM DDM) was added and the pH was adjusted to 7.8. This extract was incubated overnight with Ni-NTA beads (0.25 ml beads/liter culture; QIAGEN Inc.). The beads were transferred into a column and washed with ∼40 vol wash buffer (40 mM imidazole-HCl, 95 mM K-P i , 5 mM KCl, 1 mM DDM, pH 7.8) until A 280 < 0.02. EriC was then eluted with Buffer E, pH 7.0. The 40-mM imidazole present during binding and wash steps suppresses the binding of contaminating E . coli proteins and thereby increases the purity (while decreasing the final yield) of the EriC preparations. Protein concentration was measured from the absorbance at 280 nm, using a mass extinction coefficient (∈ = 0.85 cm 2 /mg) calculated from the sequence , or with the Coomassie Plus protein microassay (Pierce), calibrated using purified EriC dialyzed free of imidazole. Some experiments used the bacterial K + channel protein KcsA, for which expression and purification were carried out as described . Reconstituted vesicles were formed by combining EriC with lipid-detergent–mixed micelles, and then removing detergent by gel filtration. Except where noted, all reconstitution procedures and flux assays were performed at room temperature. E . coli lipid was dried under N 2 in a glass tube, resuspended in pentane, and redried. The lipid was suspended at 20 mg/ml by sonication in Buffer R [450 mM KCl, 20 mM morpholino ethanesulfonic acid (MES)-NaOH, pH 6.2], 34 mM 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate (CHAPS) was added, and the suspension was sonicated again. After a 2-h incubation, EriC was added to the desired concentration (0.09–5 μg/mg lipid), along with enough Buffer R to bring the lipid concentration to 10 mg/ml. In control samples, equivalent volumes of Buffer E were added in place of EriC. After 20 min, detergent was removed and vesicles were retrieved using Sephadex G-50 spin columns as follows. Columns (1.5-ml bed volume) equilibrated with Buffer R were prespun in a clinical centrifuge at 1,000 g , 15 s to remove excess solution. A 95-μl sample of the reconstitution mix was applied to each column, and vesicles were collected by centrifuging 700 g , 1 min. Recovery from the spin varied in the range 100–150 μl. The samples were frozen in a dry ice/acetone bath and stored overnight in a frost-free freezer at −20°C. Before use, samples were removed from the freezer, cooled in a dry ice-acetone bath, thawed at room temperature, and then sonicated 5–10 s in a cylindrical bath sonicator. Influx of 36 Cl − against a concentration gradient was assayed by a three-step procedure, essentially as described . First, extravesicular Cl − was removed by spinning reconstituted vesicles (100 μl) through Sephadex G-50 columns equilibrated in Buffer F (400 mM sorbitol, 20 mM MES-NaOH, pH 6.2). Second, 36 Cl − uptake was initiated by adding 5 μl of the working 36 Cl − stock to 100 μl of each vesicle sample (final extravesicular concentrations: 2.4–2.8 mM NaCl, 1.2–1.5 μCi/ml 36 Cl − ). Finally, after the desired time of uptake, 36 Cl − trapped inside the liposomes was measured after applying the sample to a Dowex-glutamate column (1.5-ml bed volume) and eluting into a scintillation vial with of 1.5 ml 400 mM sorbitol. Just before the assay, Dowex columns were prewashed with 400 mM sorbitol–5 mg/ml bovine serum albumin (2 ml) followed by 400 mM sorbitol (2 ml). To test for conductive release of 36 Cl − , valinomycin (1 μg/ml) was added after uptake had reached steady state. In some experiments, the internal anion was varied by replacing KCl with the potassium salt of the desired anion in Buffer R. In other experiments, external test anions were added from stock solutions in Buffer F immediately before addition of 36 Cl − . EriC-reconstituted liposomes were prepared as described above with the following variations. Proteoliposomes were formed in Buffer L (20 mM KCl, 20 mM K-glutamate, 20 mM MES-KOH, pH 6.0), and stock solutions of 36 Cl − and 3 H-glutamate were added to the vesicles (final concentrations of 2 and 7 μCi/ml, respectively). Intra- and extra-vesicular solutions were equalized by freezing and thawing the samples twice, and then sonicating the suspension 5–10 s. Passive equilibrium-exchange efflux was initiated by diluting the loaded vesicles into Buffer L lacking radioactive tracers. After 40–60 min, intravesicular content was determined by spinning through Buffer L–equilibrated G-50 columns as above. Separate experiments under these conditions demonstrated: (a) that insignificant efflux of Cl − occurs in protein-free liposomes, (b) that full equilibration of Cl − is achieved with EriC-containing liposomes, and (c) that negligible efflux of glutamate occurs for liposomes with or without EriC. The crucial measurement in this assay is the fraction of the intraliposomal volume that is inaccessible to external Cl − . This parameter is determined from the fractional trapped Cl − space, f , measured by double-label counting of internal and external tracer concentrations (dpm per gram lipid and per cubic-centimeter solution, respectively, shown in ): 1a \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f=\frac{ \left \left[^{{\mathrm{36}}}{\mathrm{Cl}}^{{\mathrm{-}}}\right] \right _{{\mathrm{in}}}}{{\mathrm{{\theta}}} \left \left[^{{\mathrm{36}}}{\mathrm{Cl}}^{{\mathrm{-}}}\right] \right _{{\mathrm{ex}}}}{\mathrm{,}}\end{equation*}\end{document} where θ, the total intravesicular volume (cm 3 /g lipid), is measured from 3 H-glutamate ( ): 1b \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\theta}}}={ \left \left[^{{\mathrm{3}}}{\mathrm{H}}\right] \right _{{\mathrm{in}}}}/{ \left \left[^{{\mathrm{3}}}{\mathrm{H}}\right] \right _{{\mathrm{ex}}}}{\mathrm{.}}\end{equation*}\end{document} For vesicles that have been diluted infinitely, the Cl − -inaccessible fraction, f 0 is ( ): 2a \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{0}=f{\mathrm{.}}\end{equation*}\end{document} For practical purposes, the samples were diluted not infinitely, but 10-fold, and f 0 was correspondingly normalized, by : 2b \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{0}={ \left \left(f-0.1\right) \right }/{0.9{\mathrm{.}}}\end{equation*}\end{document} This assay can be used to estimate the fraction, s , of EriC protein that is functionally active as a Cl − channel. The analysis assumes that the protein distributes randomly according to a Poisson distribution into spherical liposomes of uniform size, and that Cl − is retained only in those liposomes that contain no EriC molecules. As the mass, m E , of EriC reconstituted in a fixed mass, m L , of liposomes increases, this Cl − -inaccessible fraction will decrease according to Goldberg and Miller 1991 : 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{ln}}f_{0}=-{36{\mathrm{{\pi}N}}_{0}{\mathrm{{\theta}}}^{2}sm_{{\mathrm{E}}}}/{M_{{\mathrm{E}}}m_{{\mathrm{L}}}{\mathrm{{\sigma}}}^{3}}\end{equation*}\end{document} where N o is Avogadro's number, θ is the intravesicular volume (cm 3 /g lipid), M E is the molecular mass of the EriC channel (51,000 g/mol per subunit), and σ is the surface area of lipid in a bilayer (cm 2 /g). Glutaraldehyde-mediated cross-linking of EriC was performed at room temperature. EriC (6 μg) was diluted to 27.5 μl in Buffer X (150 mM NaCl, 50 mM Na-P i , 1 mM DDM, pH 7.0), and glutaraldehyde (0.5 μl, 18 mM final concentration) was added. The reaction was quenched with 19 mM Tris for 10 min. Sodium dodecylsulfate (SDS)–loading buffer was added, and the sample was analyzed by SDS-PAGE. In some experiments, we varied concentrations of protein (5–500 μg/ml), DDM (1–5 mM), or glutaraldehyde (14–160 mM). Samples were chromatographed at 1 ml/min on a Superdex 200 column (10 × 300 mm, 24 ml bed volume; Pharmacia LKB Biotechnology Inc.) equilibrated in buffer G (100 mM KCl, 50 mM Na-P i , 1 mM DDM, pH 7.0). Elution profiles were monitored at 280 nm. EriC was dialyzed overnight against Buffer U (155 mM KCl, 95 mM K-P i , 1 mM DDM, pH 7.0) containing 5 mM dithiothreitol (DTT). For control experiments, KcsA protein was dialyzed into Buffer U lacking DTT. To ensure proper blank subtraction, for each dialyzed sample, the solution outside the dialysis cell at equilibrium was collected as the blank. Samples and buffer blanks were loaded into charcoal-Epon two-sector cells. Sedimentation velocity was analyzed using an Optima XL-A analytical ultracentrifuge (Beckman Instruments, Inc.) at 44,000 rpm, 20°C; scans at 280 nm were taken every 5 min. Data were fit to the modified Fujita-Macosham function using Svedberg 5.01 software. Protein partial-specific volumes, calculated from amino acid composition , were 0.743 and 0.724 cm 3 /g for EriC and KcsA, respectively. For the past few years, it has been appreciated that ClC channels are represented in microbial genomes . By sequence identity, the prokaryotic ClCs lie in the twilight zone for homology modeling (∼15–25%), but the statistical scores obtained from BLAST alignments indicate a high probability that the prokaryotic proteins are true ClC homologues . Manual scrutiny of the prokaryotic sequences supports this conclusion. Several stretches of ClC sequence are invariant among the known eukaryotic family members, and these appear throughout the entire membrane-spanning region, ∼400 residues, and only in this region. In these regions, the prokaryotic proteins display high sequence identity to their eukaryotic counterparts . The roles that these conserved sequences play in channel function have not been experimentally established; however, certain of these, when mutated, lead to changes in both gating and permeation , as though they either directly mediate these functions or are necessary for proper folding and assembly of ClC channels. We cloned one of the above ClC genes into an E . coli expression vector and added an NH 2 -terminal hexahistidine sequence for large-scale production and straightforward purification of EriC, a putative ClC-type channel product of the yadQ gene. After an overnight room-temperature induction of E . coli bearing the pEriC plasmid, a protein band of ∼38 kD, absent in uninduced controls, was observed on SDS-PAGE of bacterial membrane fractions . This protein was detergent-extracted from the bacterial membrane fraction and purified on a Ni-chelate column. The apparent molecular mass of ∼38 kD is smaller than that calculated from the sequence (51 kD), but the integrity of the full-length protein is indicated by two observations. First, robust interaction with the metal-affinity column indicates that the NH 2 -terminal hexahistidine tag is present; second, an EriC construct carrying an eight-residue, COOH-terminal 1D4 epitope , showed a similar apparent molecular mass, detected on immunoblots (data not shown). Typical yields of purified EriC are 0.5–1 mg/liter culture. EriC is unambiguously a ClC-family protein at the level of primary sequence, but does it function as an ion channel? To assess this protein's ability to catalyze passive transmembrane flux of 36 Cl − , we reconstituted EriC into liposomes and performed a concentrative influx assay . Vesicles were loaded with a high concentration (450 mM) of Cl − , and 36 Cl − influx was measured at low (2.5 mM) external Cl − . The resulting Cl − gradient will polarize any Cl − -selective liposome membrane (positive potential inside), and 36 Cl − will accordingly accumulate inside the liposome. Liposomes that have failed to incorporate Cl − channels or have sprung nonselective leaks will not accumulate 36 Cl − in this assay. The results of Fig. 3 demonstrate that EriC is a Cl − -selective channel; tracer Cl − accumulates into EriC-containing vesicles with a time constant of ∼4 min. Permeabilization of the liposomes to K + with valinomycin causes rapid loss of Cl − , a result demonstrating that the movement of 36 Cl − is conductive, not electroneutral. The final level of tracer rises in a saturating fashion with the amount of EriC reconstituted, as the population of channel-containing liposomes increases. Control protein-free vesicles or vesicles reconstituted with KcsA, a bacterial K + channel, fail to transport 36 Cl − , although the latter vesicles accumulate 86 Rb + under similar conditions . It is desirable to know if the observed flux activity is due to a major, minor, or minuscule fraction of the purified EriC protein. However, the observed influx kinetics are inadequate to quantify activity in our preparations because the absolute rate of Cl − uptake catalyzed by a single EriC channel is unknown. Instead, we employ an assay that does not require knowledge of absolute flux rates, but merely relies on the assumption that any liposome permeable to Cl − will equalize internal and external concentrations of this ion at thermodynamic equilibrium. Vesicles are loaded to equilibrium with two tracers: 36 Cl − , which is permeant exclusively to EriC-containing liposomes, and 3 H-glutamate, a trapped volume marker that is impermeant to all liposomes on the experimental time scale. The loaded vesicles are then diluted into tracer-free solution of identical ionic composition, and 36 Cl − is allowed to flow out (in exchange for unlabeled Cl − ) until equilibrium is achieved. Any vesicle containing one or more functionally active EriC molecules releases 36 Cl − and retains impermeant glutamate; vesicles devoid of EriC channels will trap both 36 Cl − and 3 H-glutamate. Glutamate is thus used as an internal standard to measure the total intravesicular volume to which the Cl − -accessible volume can be compared. As EriC is reconstituted at increasing protein/lipid mass ratio, the fraction of Cl − -impermeable volume, f 0 , falls exponentially from a protein-free value of unity , as demanded by a Poisson distribution governing the random incorporation of N E EriC channels into N L liposomes . The absolute value of intravesicular volume, θ, does not change systematically over this range of protein concentration (data not shown), which roughly spans the range of 0.2–4 channels per liposome. The protein concentration dependence of f 0 quantitatively obeys the expectations of , where the crucial fit quantity, the fraction of active protein, is s = 1.9. Since we have based our analysis on a utopian model in which protein molecules distribute perfectly into uniform spherical vesicles, it is perhaps not surprising that we reach a physically impossible conclusion of 190% activity. As discussed below, several assumptions in this analysis are expected to lead to uncertainty in the estimation of EriC activity, so we can easily rationalize this absurd value. The important point of this calculation is that a major fraction of the purified EriC protein—perhaps 100%—is responsible for the functional activity detected in the assay. The concentrative uptake assay was used in two ways to gauge the ionic selectivity of EriC. In the first set of experiments, internal Cl − was replaced with various test anions. Permeant anions support concentrative uptake, while impermeant anions do not. Of the anions tested , only Cl − , Br − , and NO 3 − are permeant by this criterion. In the second set of experiments, we added test ions to the external solution to see which of these would collapse the liposome membrane potential and thereby impede influx. In this assay, SCN − , and to a lesser extent I − and F − score as permeant in addition to Cl − , Br − , and NO 3 − . The discrepancy observed with SCN − is not disconcerting; SCN − both blocks and permeates some eukaryotic ClCs and, if acting in such a fashion here, would also inhibit influx. In other words, the flux assay in which the test anion is applied externally does not distinguish a permeant ion from a strong blocker. In any case, the two assays taken together demonstrate (a) that EriC-mediated fluxes are highly selective for anions over cations, and (b) that among anions the selectivity sequence is roughly similar to that found for ClC-0 and ClC-1 : Cl − , Br − > NO 3 − > I − , F − >> H 2 PO 4 − , glutamate − . It is worth noting, however, that these selectivity assays are performed under ionic conditions and with methods very different than in electrophysiological measurements, and that no single interanionic selectivity sequence applies to all eukaryotic ClC channels. The most extensively studied eukaryotic ClC channels, ClC-0 and ClC-1, are both homodimers, a quaternary structure underlying their double-barreled behavior . But is this dimeric characteristic merely an idiosyncrasy of these two ClCs, which populate the same muscle-type subfamily, or is it a general property of the entire ClC family? Since EriC is evolutionarily distant from the eukaryotic ClC subfamilies, its quaternary structure would provide valuable insight into this question. We used three different techniques to assess the oligomeric state of EriC in detergent micelles: cross-linking, gel filtration, and velocity sedimentation. Glutaraldehyde, a nonspecific cross-linking agent, has been used convincingly to report the oligomeric state of several membrane proteins . Reaction of glutaraldehyde with EriC gives a clean result . Within 10 min of reaction, the 38-kD band is quantitatively converted into a band migrating at ∼90 kD; no conversion to higher mass species occurs over the next 60 min. The possibility of intermolecular cross-linking is largely eliminated by the control experiment showing no cross-linking of EriC dispersed under denaturing conditions in SDS. In addition, variation of the protein-detergent ratio over two orders of magnitude failed to alter the cross-linking, a result that rules out artifactual dimerization resulting from multiple protein molecules sharing the same micelle . Moreover, similar results were obtained with EriC reconstituted at very low density (0.09 μg/mg lipid), where most liposomes have either one EriC channel or none. Cross-linking was robust to varying glutaraldehyde or protein concentrations; while the rates of cross-linking increased with these variables, the overall pattern, particularly the absence of high aggregates, was unchanged. To complement the cross-linking experiments, we analyzed EriC by gel filtration chromatography and compared its migration to a reference membrane protein of known size, the K + channel KcsA, a 74-kD homotetramer . EriC runs slightly ahead of KcsA on a Superdex gel filtration column . This result suggests that the EriC channel is substantially larger than its monomer molecular mass of 51 kD. Since these gel filtration results cannot distinguish dimers from higher-order oligomers, we also analyzed velocity sedimentation profiles of EriC and again used KcsA as a membrane protein size standard. Qualitatively, EriC sediments more rapidly than KcsA . A fit of the sedimentation data to a Fujita function , which determines both the sedimentation and diffusion coefficients and hence molecular mass, yields 92 kD for KcsA and 126 kD for EriC. These estimates are both ∼25% higher than the formula size of tetrameric KcsA and dimeric EriC, a result easily rationalized by bound detergent. A value of 126 kD for EriC would be difficult to reconcile with a trimer or higher oligomer. We attempted to carry out equilibrium sedimentation experiments in neutral-density detergents, to eliminate rigorously the contribution of bound detergent to the measured mass of the protein . These experiments failed, however, since EriC was unstable in all neutral-density detergents tested, as indicated by rapid aggregation accompanied by high-order cross-linking (data not shown). Nevertheless, the three independent lines of evidence presented here argue powerfully that the functionally active form of EriC used for reconstitution is a homodimer. At the current early stage of understanding the chemistry of integral membrane proteins, prokaryotes have served as the singular bearers of high-resolution structural information about ion channels. Porins, K + channels, and mechanosensitive channels from bacteria have been expressed at high levels in E . coli as a prelude to crystallization and structure determination . No eukaryotic ion channel has been successfully expressed in any heterologous systems at levels high enough even to contemplate crystallization. It is therefore encouraging that the ClC family of Cl − channels is represented widely in prokaryotes. We have overexpressed a prokaryotic ClC channel, EriC, the product of one of the two ClC genes in E . coli . The biological role of this channel is unknown, but the present results make it clear that it is in fact a ClC-type Cl − channel. The purified protein promotes the passive, conductive flux of Cl − and other anions across liposome membranes, and the ionic selectivity of this effect is reminiscent of vertebrate ClC channel electrophysiology. We attempted but failed to observe single-channel current fluctuations induced by EriC in planar lipid bilayers; this negative result is disappointing but not surprising in light of the extremely low conductances of many eukaryotic ClC channels . In the liposome flux assay, the time scale (seconds) of EriC-catalyzed 36 Cl − release is substantially lower than expected (milliseconds) for a channel of conventional unitary current (1 pA) open all the time. In the absence of direct electrophysiological information, we have no way of knowing whether the low fluxes reflect low channel current, low open probability, or both. From a biochemical standpoint, perhaps the most important property to quantify for any new protein preparation, even more important than purity or yield, is functional competence. This measurement is difficult for uncharacterized ion channels reconstituted into liposomes, and our value implying 190% activity is certainly disconcerting. We have used a limiting Poisson method of “counting” the fraction of liposomes that carry no channels in a sample containing N L liposomes and N E EriC channels, a fraction s of which are functionally active. If the insertion of channels into a uniform population of liposomes is random, then this fraction, f 0 , must obey a Poisson distribution ( ): 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{0}={\mathrm{exp}} \left \left({-s{\mathrm{N}}_{{\mathrm{E}}}}/{{\mathrm{N}}_{{\mathrm{L}}}}\right) \right {\mathrm{.}}\end{equation*}\end{document} We estimate f 0 directly from the fraction of Cl − -impermeable liposomes, and N E is known. The experimental samples consist of a known mass of lipid corresponding to a fixed liposome surface area, and the total intravesicular volume is directly measured. If the liposomes were uniform spheres of known lipid surface density, this information would be sufficient to determine N L and hence to derive a functional activity value s . However, estimation of the number of liposomes is subject to several sources of error. First, the estimated value of activity varies with the cube of the bilayer surface area per mass of lipid, σ, in . The reconstituted liposomes used here are formed from a phosphatidylethanolamine-rich, undefined, complex mix of lipids. Molecular surface areas of phosphatidylethanolamines vary substantially (55–72 Å 2 ) depending on hydrocarbon chain, lipid composition in mixed bilayers, and other factors ; if used in these would lead to an approximately twofold range of estimated activity values (average 190%, range 140–320%). Second, large unilamellar liposomes such as these are not expected to be spherical in general; ignoring this kind of nonideality always overestimates activity in the trapped-Cl − assay since any departure from a spherical shape increases the surface-to-volume ratio. For example, if the liposomes were on average approximated by a squat cylinder (height = radius), this effect alone would lower the activity estimated above into the range 80–180%. Unfortunately, we have no experimental data on liposome morphology, so we cannot rigorously quantify the expected errors in our estimate of s . Nevertheless, this uncertainty provides a plausible rationale for an activity value that is “impossibly” high. Another source of ambiguity arises from the assumption that the liposomes are uniform in size, which is certainly false ; however, calculations indicate that this effect is relatively minor, producing errors of <20% in either direction, depending on the size distribution of the liposome population. Together, the magnitude of these uncertainties dictates that we cannot estimate the value of s to better than two- to threefold accuracy. Nonetheless, the key inference of the experiments—that the flux activity we observe is mediated by a nontrivial fraction of the purified EriC—is preserved. The hydrodynamic properties of EriC further argue that a major, not a minor, fraction of the protein is active; in both gel filtration and analytical centrifugation experiments, purified EriC behaves as a properly folded, monodisperse macromolecule. EriC behaves, as do the muscle-type channels ClC-0 and ClC-1 , as a homodimer in both micellar and bilayer environments. None of the three methods used to assess quaternary structure is in itself rigorous. Nevertheless, a dimeric structure for EriC is strongly supported by the consistency of these three independent, complementary techniques. Gel filtration and velocity sedimentation behavior argue that Eric is larger than a monomer; velocity sedimentation additionally indicates that it is smaller than a trimer; chemical cross-linking marks it as a dimer. These results underscore the structural homology between EriC and the eukaryotic ClCs and thereby identify homodimeric architecture as a general hallmark of the ClC family rather than a peculiarity of the muscle-type subfamily. ClC channels carry out many crucial biological functions, but conventional mutagenesis studies have provided only limited glimpses of ClC molecular architecture. Since EriC is functionally active as a Cl − channel and may be obtained in milligram quantities, this protein is an excellent candidate for future structural studies. | Study | biomedical | en | 0.999995 |
10539976 | Voltage-gated ion channels are remarkably sensitive to membrane potential changes. Large charge movements in the channel protein couple changes in membrane potential to the channel opening process. In the skeletal muscle sodium channel and the Shaker potassium channel, the size of this “gating charge” movement is seen to be equivalent to the displacement of 12–14 elementary charges entirely across the membrane . The large charge movement makes sense for the sodium channel, where the resulting high sensitivity helps to establish a firing threshold for excitable cells. For transient potassium channels such as Shaker, the high sensitivity is important to allow channel activation during threshold depolarization, enabling these channels to control repetitive firing rates in neurons . On the other hand, high voltage sensitivity in delayed-rectifier potassium channels is arguably less important: these channels need only be activated by the large excursions of action potentials, and indeed the presence of large charge movements can be detrimental. As pointed out by Hodgkin 1975 , gating charge movements serve as a capacitive “load” on the action potential, increasing metabolic requirements and decreasing the velocity of action potential propagation in excitable cells. The various voltage-gated potassium channels contain four α subunits; many of these subunits show differences in their S4 regions that are suggestive of differing amounts of gating charge. The S4 region is thought to be the primary voltage-sensing region ; in the Shaker family it has seven positively charged residues and in the other potassium channel families it has between four and six. Some mutations in Shaker that reduce the number of basic residues reduce the apparent voltage sensitivity, but other mutations leave the voltage sensitivity apparently unchanged . It should be pointed out that in members of Shaker and other related families, the acidic residues in S2 and S3 are conserved; one of these, the second glutamate in S2, has also been implicated in the voltage-sensing function . Because of uncertainties in past measurements of charge movement based on voltage sensitivity, and to complement the studies in which the magnitude of gating charge has been changed through mutations, we report here measurements of the gating charge in four “native” voltage-gated potassium channel types using improved methods. Gating charge movement is most directly measured by integrating the gating currents that are recorded from membranes having a high density of channels, under conditions in which no ionic current flows through the channels. To then evaluate the charge movement in a single channel, the number of channels contributing to the gating current must be estimated; this has been done for Shaker channels using either fluctuation analysis or radioligand binding . The results of the two methods agree well, yielding values of the single-channel charge of 12–14 e o . An alternative approach is called the “limiting-slope” method, and is analogous to the determination of the Hill coefficient of a binding reaction. It involves measuring the dependence of the channel open probability, P, on membrane potential V. Given a channel that opens with depolarization and has a single open state, then asymptotically, as V → −∞ (and P → 0), the apparent charge, 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}q_{{\mathrm{s}}} \left \left({\mathrm{V}}\right) \right =kT\frac{d{\mathrm{ln}}P \left \left({\mathit{V}}\right) \right }{d{\mathit{V}}}{\mathrm{,}}\end{equation*}\end{document} approaches the total charge movement q T . The limiting-slope method has been applied to Shaker and some mutants and its results are in good agreement with the direct charge measurements outlined above. The limiting-slope method has the advantage that it measures charge that is actually involved in opening the channel. Its asymptotic nature, on the other hand, poses a problem: how can one be sure that q s is evaluated at sufficiently negative V ? Two recent advances now allow this problem to be addressed in a rigorous fashion. The first is the development by Hirschberg et al. 1995 of a method for measuring very small P values at the very negative membrane potentials where one hopes that the asymptotic limit is reached. The method involves measuring single-channel events in membrane patches having large numbers of channels N . For channels having brief open times, the aggregate open probability NP can be measured in such patches down to values of 10 −4 or less; with N on the order of 10 3 , P can be reliably estimated down to values of 10 −7 or less. The second advance is the theory of Sigg and Bezanilla 1997 , which shows that an ordinary gating current measurement can be used as an independent test of the extrapolation of q s values to estimate q T . Let Q̂ Vbe the macroscopic gating charge that is obtained by integrating the gating current, normalized such that Q̂ −∞=0and Q̂ +∞=1. Then, in the case of a channel that is found in its single open state at large depolarizations, the apparent charge q s is related to Q̂ by: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}q_{s} \left \left({\mathrm{V}}\right) \right =q_{{\mathrm{T}}} \left \left[1-{\hat {Q}} \left \left({\mathrm{V}}\right) \right \right] \right {\mathrm{.}}\end{equation*}\end{document} Thus, given experimentally determined values of q s and Q̂ , a one-parameter curve fit can be used to estimate the value of q T . In this paper, we incorporate these advances to the limiting-slope method and apply it to the determination of charge movements in homomeric channels encoded by the Drosophila potassium channel genes Shaker and Shab , and from the rat homologues Kv1.1 and Kv2.1. A comparison of these channel types is interesting in view of their differing amino acid sequences . Shaker and Kv1.1 both have seven basic residues in the S4 region, while Shab and Kv2.1 have five. Shaker H4 with N-type inactivation removed and the W434F mutant were subcloned into the vector pGEM, linearized with Not I and transcribed with T7 RNA polymerase. The final cRNA concentration was 1 μg/μl. Kv1.1 was subcloned into pGEMHE, linearized with PstI and transcribed using T7 polymerase. Shab was subcloned in a modified version of Bluescript for oocyte experiments. Linearization of cDNA was carried out with NotI and transcription with T3 polymerase. The rat clone Kv2.1-Δ7 (DRK1-Δ7), derived from DRK1 was provided by Drs. R. MacKinnon (The Rockefeller University, New York, NY) and S. Aggarwal (Harvard Medical School, Cambridge, MA). This construct has seven point mutations in the putative pore mouth that permit very high affinity binding ( K d ≈ 15 pM) of the channel blocker Agitoxin-1 . Kv2.1-Δ7 in Bluescript was linearized with NotI, transcribed with T7 RNA polymerase and resuspended in water to a final concentration of 0.3–0.5 μg/μl. Xenopus laevis oocytes were harvested under anesthesia and defolliculated by incubation in Ca +2 -free OR2 solution supplemented with 2 mg/ml collagenase 1A for 1 h. Oocytes were incubated at 20°C in a solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 5 HEPES, and injected 1 or 2 d after harvesting with 50 nl of cRNA solution with a Nanostepper (Drummond Scientific Co.) using pipettes of 20-μm opening diameter. Oocytes were used for experiments 1–7 d after injection. In some cases, oocytes expressing Kv2.1-Δ7 were incubated in the presence of Agitoxin-1 at a concentration of 1–3 μM for the period of channel expression. This resulted in an increase in expression of two- to threefold. The Shab channel was expressed in Sf9 cells for the recording of gating currents in whole cells. The Bac-to-Bac™ recombinant baculovirus system (GIBCO BRL) was used. After transfer of the Shab gene to a bacmid vector, Sf9 cells at 70% confluence were transfected with lipofectin in Sf900 medium (GIBCO BRL). The culture medium containing the Shab baculovirus was collected after 48 h of culture and stored at 4°C. After infection, expression of channels in cells was assessed by electrophysiological recording. Sf9 cells were maintained in an incubator at 26°C in 1% CO 2 . Patch-clamp recordings were obtained using standard techniques. All recordings reported here were made in the cell-attached configuration, for maximum patch stability. Oocytes were bathed in a depolarizing solution that effectively zeroed the membrane potential as checked with a two-microelectrode voltage clamp. The bath solution contained (mM): 120 K-aspartate, 20 KCl, 1.8 CaCl 2 , 10 HEPES, adjusted to pH 7.4. Unless otherwise stated in the figure legends, pipettes were filled with a solution of the following composition (mM): 40 K-aspartate, 20 KCl, 1.8 CaCl 2 , 60 N -methyl- d -glucamine (NMDG) 1 -aspartate, 10 HEPES, pH 7.4. Gating currents of Kv2.1-Δ7 were recorded using a similar solution that contained 120 mM NMDG-aspartate and no K + , and included 1 μM Agitoxin-1. The same solution was used for Shaker gating currents without the Agitoxin-1. For gating currents, 20 sweeps were averaged at every voltage. A P/5 subtraction protocol was used to subtract linear capacity and leak currents, using a leak-holding potential of −120 mV. Pipettes were pulled from soft borosilicate glass capillaries (Kimax-51) and had resistance in the range 1.5–3 MΩ. Currents were recorded with an EPC-9 amplifier running the Pulse software (HEKA Elektronik) on a Macintosh computer. In macroscopic current recordings, the linear current components were subtracted by the use of a P/5 protocol and were filtered at 2.5 kHz (−3 dB) with a Bessel filter and sampled at 15 kHz. Single-channel recordings were obtained in the same patches as macroscopic currents, with the same filter cut-off frequency and a sample frequency of 10 kHz. Null-trace averages were used to subtract the linear capacitive and leak currents. All recordings were obtained at room temperature (22°C). Whole-cell gating currents in Sf9 cells were recorded using the following solutions (mM): Pipette: 150 NMDG-HCl, 10 MOPS, 5 EGTA, 1 MgCl 2 , pH 7.2; Bath: 150 NMDG-HCl, 10 MOPS, 10 CaCl 2 , 1 MgCl 2 , pH 6.7. Recording electrodes had a resistance of 0.5–1 MΩ and were coated with Sylgard ® to reduce capacitance. No series resistance compensation was used. The capacitance of the Sf9 cells used for gating current recordings ranged from 20 to 42 pF. The settling time of capacity transients was ∼100 μs. The subtraction protocol was similar to that used for Kv2.1. For macroscopic currents, the voltage dependence of the open probability was estimated from tail current measurements; the size of the tail current is proportional to the channel open probability at the end of the preceding depolarization. We averaged the first millisecond of tail current at −100 mV in the case of Kv2.1 channels. For Shaker and Shab , the first 300 μs of tail currents at −90 mV were averaged and for Kv1.1, 400 μs was averaged. The values obtained were in very close agreement to the more traditional method of measuring P from the steady state currents and normalizing by the instantaneous current–voltage curve. Contamination of the ionic tail currents by gating currents was taken to be negligible because OFF gating current amplitudes were <0.5% of the maximal tail current amplitude. Estimation of the number of channels in a patch of membrane was done by nonstationary noise analysis . The mean and variance were calculated from 50–60 current traces elicited by depolarizing steps to the indicated membrane potential filtered at 5 kHz. Pulses were delivered at 0.5-s intervals. The variance estimation made use of groups of four sweeps, to reduce the influence of drifting baselines and channel rundown. The data were displayed in the form of mean-variance relationships and fitted to the equation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}^{2}=iI-\frac{I^{2}}{N}{\mathrm{,}}\end{equation*}\end{document} where σ 2 is the variance, I is the mean current, i is the single channel current, and n is the number of channels. The maximal open probability was then obtained as P max = I max / Ni . The analysis of the single channel traces to obtain the time-dependent NP values was carried out as follows. The current traces were leak subtracted using a template of averaged null sweeps (sweeps that contain no channel openings), digitally filtered with a Gaussian filter to 1.5 kHz and analyzed with the 50% threshold crossing method with multiple thresholds to allow detection of overlapping channel currents. This produced an idealized record of open and closed events. The idealizations of 100–1,000 sweeps were then averaged, with null traces included. The steady state average value, typically computed from the last 150 ms of the averaged idealization, was taken as the final value for NP . Open and closed dwell time histograms were obtained using a single threshold from sweeps with no overlapping openings and constructed according to the log-binning technique and probability density functions were fitted by the method of maximum likelihood. The apparent charge q s was estimated from experimental data by discretizing in the following way. Given open probabilities P 1 and P 2 obtained at adjacent voltages V 1 and V 2 , and letting V′ = (V 1 + V 2 )/2, we computed q s as: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}q_{{\mathrm{s}}} \left \left({\mathrm{V^{\prime}}}\right) \right =kT\frac{{\mathrm{ln}}P_{1}-{\mathrm{ln}}P_{2}}{{\mathrm{V}}_{{\mathrm{1}}}-{\mathrm{V}}_{{\mathrm{2}}}}{\mathrm{.}}\end{equation*}\end{document} The equilibrium voltage dependence of state occupancies was computed according to : 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{{\mathrm{n}}} \left \left({\mathrm{V}}\right) \right =\frac{{\prod_{j=1}^{n}}K_{{\mathit{j}}} \left \left({\mathrm{V}}\right) \right }{{{\sum^{M}_{i=1}}}{\prod_{j=1}^{n}}K_{{\mathit{j}}} \left \left({\mathrm{V}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where P n is the probability of occupancy of state n, K j is the equilibrium constant for transition from state j − 1 to state j , and the M states are linearly connected. Statistical quantities are given as mean ± SEM To extend the previous studies of Shab channel currents , we first compare Shab channel properties with those of the other channel types. For this study, we have used a Shaker construct having a deletion at the NH 2 terminus to remove fast inactivation, to simplify the determination of steady state open probability. For Kv2.1, we have used a mutant, DRK1-Δ7, that was engineered to bind the pore blocker Agitoxin-1 with high affinity , making possible the measurement of gating currents. Macroscopic currents were obtained in cell-attached patch recordings from mRNA-injected oocytes or in whole-cell recordings from recombinant baculovirus-infected Sf9 cells in the case of Shab channels. Representative current traces are shown in Fig. 2 A. The channel open probabilities P for each channel type approach limiting values between 0.7 and 0.9 for large depolarizations. The voltage-dependence of P for Shaker and Kv1.1 can be described well by the fourth power of a Boltzmann function, as would be predicted by a simple model incorporating four independently acting voltage sensors . The gating charges estimated from these fits are 11.5 and 7.7 e o for Shaker and Kv1.1, respectively; it should be emphasized, however, that such gating-charge estimates are strongly dependent on the particular model used. The corresponding fit to the P–V relationship for Shab channels yields an apparent charge of 6.3 e o , while for Kv2.1 it yields 10.1 e o . Activation time constants τ were estimated from exponential fits to the second half of the activation time course of macroscopic currents . From these fits an estimate of the delay in activation δ was also obtained. Shaker channels show a small activation time constant that is rather weakly voltage dependent with an effective charge of 0.51 e o ; the delay is roughly equal to the time constant and has similar voltage dependence over the positive voltage range. The kinetic features of Kv1.1 channels are nearly identical to those of Shaker , having almost identical activation time constants and delay values, with the main difference being a slightly steeper activation curve. Like Shaker , Shab channels activate and deactivate quickly, with time constants similar to those of Shaker . Shab channels activate, however, with a smaller delay that is more steeply voltage dependent ; this means that at positive voltages these channels show less sigmoidicity in their activation time course than Shaker . This behavior can be explained if the rate-limiting step for activation is less voltage dependent than other steps that determine the delay. The same general phenomenon is seen in Kv2.1, although here the much slower activation is reflected by τ and δ having values that are larger by an order of magnitude. The time course of charge movement of Shaker , Kv2.1, and Shab channels was directly measured in the form of gating currents. Shaker gating currents were obtained from cell-attached patches in oocytes expressing the nonconducting mutant W434F using the same solutions as those used to record macroscopic ionic currents. The voltage dependence of the charge obtained from integration of ON gating currents is similar to that previously reported, as is the decay time constant of ON currents . Kv2.1 gating currents were also measured in cell-attached patches in oocytes, using Agitoxin-1 to block the ionic currents. The general characteristics are similar to those previously reported from the cut-open oocyte recording technique. ON gating current decays are approximately five times slower than Shaker' s (at 0 mV), but show a steeper voltage dependence of the time constant. Taglialatela and Stefani 1993 observed gating current decay time constants that were very close to the ionic current time constants; however, under our recording conditions and with the mutant channel that we are using, the gating current time constants are severalfold shorter than the ionic current time constants. Thus, we observe a gating current time constant that is similar to the delay of ionic currents but is shorter than the activation time constant. This can be explained if the gating current comes predominantly from early voltage-dependent transitions that give rise to the ionic current delay, and the rate-limiting step comes later in the activation process. The Kv2.1 charge movement curve lies to the left of the P ( V ) curve in the voltage axis, as is expected for a channel that has multiple closed states before the open state. The voltage dependence of charge can be fitted to a Boltzmann function with a charge of 2 e o . Shab gating currents were recorded in the whole-cell configuration from baculovirus-infected Sf9 insect cells, with ionic currents eliminated through the use of impermeant N -methyl glucamine (NMG) cations. Fig. 3B and Fig. C , compares the voltage dependence of ON charge and the voltage dependence of the time constant of ON currents in these channels. Shab ON gating currents are less voltage dependent than those of Shaker or Kv2.1, and their kinetics are intermediate. Shab OFF gating currents are, however, very different in having a much faster time course than the others. The slow OFF time course in Shaker channels arises from slow initial closing transitions ; in Shab, these transitions presumably proceed much more quickly. With the high expression obtained with Shaker in oocytes, it was possible to record from patches containing hundreds or thousands of channels. The number N of channels in a patch was estimated by nonstationary fluctuation analysis, using depolarizations to +70 mV. Subsequent recordings using small depolarizing pulses allowed NP to be estimated from the statistics of single-channel events in the same patch. Recordings from a representative patch containing 2,250 channels are shown in Fig. 4 A. The channel openings observed during the small depolarizations were identified as Shaker channels based on their conductance and kinetics. To obtain the steady state open probability from pulsed data sweeps, we first performed an idealization of the single channel events for each sweep, and then averaged these idealized records. This ensemble average represents the product NP of the open probability and the number of channels that contribute to the record. No inactivation is visible in these time courses, and because of the high external K + concentration the degree of slow “C-type” inactivation on these channels is expected to be very small , reducing P by at most 2% at −60 mV . At the most negative voltages (−70 to −55 mV), the value of NP changed by about a factor of 10 for each 5-mV change in the depolarization. No openings were detected during the total 450 s of recording at the holding potential of −80 mV. At −80 mV, the mean burst duration is expected to be ∼1.5 ms so that, were a single opening to occur, it would correspond to NP ≈ 3.3 × 10 −6 . The fact that no events were observed at −80 mV in this patch or in two others having similar numbers of channels implies that at −80 mV P is smaller than ∼10 −9 . Fig. 4 C shows the result of combining the single channel data with macroscopic activation measurements and the value of n obtained from nonstationary noise analysis. The smallest open probability measured in these patches was ∼10 −7 , and the maximum was 0.79. At values of P between 10 −7 and 10 −4 the P ( V ) relation is very nearly exponential and can be well fitted to the relationship 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P \left \left({\mathit{V}}\right) \right =P \left \left(0\right) \right {\mathrm{exp}} \left \left({q_{{\mathrm{l}}}{\mathit{V}}}/{kT}\right) \right {\mathrm{.}}\end{equation*}\end{document} The charge estimate q l from this linear fit to the log-transformed P values represents a local average of q s as defined in ; for these channels it had the value 12.7 ± 0.3 e o ( n = 3). We performed two tests to determine whether these experiments can provide estimates of the apparent charge that approach the true asymptotic value. First, we computed q s from at each voltage and compared its behavior with that of two simple models in which P is given by a Boltzmann function or by the fourth power of a Boltzmann function. The comparison is done by plotting the logarithmic slope variable q s as a function of P , with V being the independent variable . Quite good correspondence is seen with the latter model given a total charge of 13 e o ; were this model correct, our estimated slope q l would be within 4% of the true limiting slope. In the second test, q s ( V ) is compared with the function, which from should yield identical values. Fig. 4 E makes this comparison for the case q T = 13 e o , where it is seen that the voltage dependences are very similar. Most importantly, the charge movement shows very little change below V = ∼70 mV, implying that q s will remain essentially constant below this membrane potential. On the basis of this fit, we extrapolate the q s values to obtain an estimate of 13.0 e o for the total charge q T . Rat Kv1.1 (previously called RCK1) is a potassium channel that does not have fast inactivation. Its general gating characteristics are very similar to those of inactivation-removed Shaker and the amino acid sequence in the S4 region is identical . Not surprisingly, our limiting-slope estimates of gating charge in Kv1.1 are almost exactly the same as those from Shaker channels. Single-channel estimates of NP show 10-fold changes for 5-mV changes in membrane potential . The apparent gating charge q l computed for values of P between 10 −6 and 10 −4 using , was 11.5 ± 1.3 e o ( n = 3). The smallest open probability attained in this measurement was two orders of magnitude higher than in Shaker , due to a lower density of channels in patches. Nevertheless, estimates of q s extrapolate well to a value of q T = 13.0 e o . Because of the low channel density, we were unable to record gating currents from patches containing Kv1.1 channels; therefore, we were unable to provide an independent check on the approach to the limiting charge movement. We conclude that q T is at least 11.5 e o and is probably near 13 e o , as is the case with Shaker . Although no inactivation is apparent in the reconstructed time courses of Fig. 5 B, it is conceivable that slow inactivation or rundown could introduce errors into our estimates for P . We therefore compared the N values obtained from noise analysis before and immediately after delivering the 600 depolarizing pulses to −60 and −50 mV for single-channel analysis of one patch; the values of N were 14 and 12, respectively. If the reduction in the number of channels is caused by inactivation, this indicates that there was at most 14% long-term inactivation. In our experiments, this small amount of inactivation at negative voltages would, if anything, have led to an underestimation of the limiting slope. Previous estimates of the gating charge of the rat Kv2.1 channel and the human homologue, hKv2.1 were in the range 6–7 e o . These were obtained from model-dependent fitting, a methodology which often underestimates the gating charge. As with Shaker , we were able to record from patches containing hundreds of Kv2.1 channels in which we could measure macroscopic currents at depolarizing voltages and resolve single-channel openings at hyperpolarized voltages. Fig. 6 shows recordings from a patch containing ∼1,650 Kv2.1 channels. At the most negative voltages (−45 to −65 mV), the value of NP decreased by a factor of ∼10 per 5-mV decrease in depolarization . No openings that could be assigned to Kv2.1 channels were detected at the holding potential of −80 mV in two experiments where the total time of recording at −80 mV was 118 and 180 s and the number of channels N was 967 and 1,250, respectively. The channel burst duration, extrapolated to −80 mV, is ∼10 ms. Were a single burst of openings to occur in one of these records, this would correspond to the open probability P ≈ 4 × 10 −8 . Therefore, we take the value of P at −80 mV to be less than ∼4 × 10 −8 . The result of combining the single channel data with the values of N and P max is shown in Fig. 6 C. The minimum measured value of the absolute open probability in this patch is ∼10 −7 and the maximum, from noise analysis at 70 mV, was 0.71. At values of P between 10 −7 and 10 −4 exponential fits of the P ( V ) relation yield an apparent charge q s of 12.1 ± 0.5 e o . A fourth power Boltzmann function with 12 or 13 e o total charge (continuous curves) superimposes fairly well on the data when q s is plotted as a function of P . The same charge estimate results in comparing q s with the macroscopic charge movement. The quantity superimposes well on the q s data with the fitted value q T = 12.5 e o . Thus, this channel behaves as expected from and the charge movement in the voltage range below that accessible to our q s measurements is seen to be small. Our best estimate of the total charge for Kv2.1 is therefore 12.5 e o . It is unlikely that this estimate of total charge is in error due to inactivation or missed channel events. Inactivation in the Kv2.1 channels was measured with 500-ms prepulses and was found to be negligible at potentials below −50 mV . Single-channel events at negative voltages show a single open-time component with a mean duration of 8 ms , consistent with the presence of only one open state of the channel. The duration of bursts of openings, ∼15 ms , is essentially voltage independent, always much greater than the detection dead-time of 120 μs; thus, a very small and voltage-independent fraction of events is expected to be missed in the evaluation of NP . In Xenopus oocytes, the expression of Shab channels was low, producing maximal whole-cell currents of ∼10 μA at +40 mV. To obtain higher channel densities and to allow the possibility of whole-cell recording of Shab gating currents, we established a baculovirus expression system using Sf9 insect cells. The currents obtained in cell-attached patches from Sf9 cells infected with the Shab baculovirus have very similar characteristics to those recorded in patches in oocytes. No voltage-dependent currents were observed in whole-cell recordings from five uninfected Sf9 cells. Interestingly, single Shab channels in single-channel patches and in recordings from multiple channel patches show a tendency to open to subconductance states. These events arise from Shab channels since direct transitions are observed between the subconductance and the fully open states. Fig. 8 A shows representative current recordings at −70 mV. Indicated are the fully open state and the substate level at 40% of the full open current level. It is evident that the channel can dwell only in the substate or make transitions to the open state with or without having to traverse the substate level. Subconductance openings are more prominent at negative voltages. At −90 mV, less than one percent of the time is spent in the subconductance level, but 98% of the observed events are subconductance openings. This is illustrated in Fig. 8 B, where all-points histograms of current recordings from multichannel patches at the indicated potentials are shown. The intermediate Gaussian component fitted to the histogram corresponds to the substate and its integral is proportional to the probability of occupancy of the substate. At potentials more negative than −70 mV, the probability of the substate P s is greater than the probability of the fully open state, P . These probabilities are plotted in Fig. 8 C; it is seen that while P increases monotonically, P s increases and then decreases with depolarization. This characteristic of P s , along with its smaller voltage dependence (the apparent charge is only 2.2 e 0 between −90 and −70 mV) is consistent with the sublevel representing an intermediate state in the activation pathway. The voltage dependence of occupancies of the substate and the fully open state can be described by specific schemes. The steady state occupancy curves in Fig. 8 C were computed from the model shown in that figure in which sequential transitions bring the channel to a substate (S), and then the fully open state. In the model, a parallel pathway also allows direct openings without passing through the substate; this provision is necessary because the substate is traversed in only ∼6% of full openings of the channel. The model yields a limiting slope corresponding to a total charge of 7.2 e o for the open state and 5 e o for the substate. Kinetic analysis of the substate was carried out to characterize the transitions near the open and subconductance states at negative potentials. The rate constants for transitions away from the substate were obtained from the dwell-time distribution in the substate and the fraction of transitions occurring in each particular path. For example, at negative voltages, dwells in the substate were seen to be terminated by transitions to the open state, to a closed state, or transitions to a closed state followed, after a delay of ∼50 ms, by channel opening. The rate constants k ij were obtained: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{{\mathrm{ij}}}=\frac{f_{{\mathrm{ij}}}}{{\mathrm{{\tau}}}_{{\mathrm{i}}}}{\mathrm{,}}\end{equation*}\end{document} where τ i is the mean dwell time in state i and f ij is the fraction of all transitions leaving state i that end in state j . Dwell-time distributions in the fully open state were used to obtain rates for transitions to the closed state in the activation pathway identified as C n , and to the substate, using the same method. Values of the rate constants for transitions near the open state obtained in this way are shown in Fig. 8 D, which is presented as an example of a scheme that can account for the kinetic phenomena we observe. In conclusion, both the steady state and kinetic behaviors are consistent with the subconductance level being associated with a state that is a kinetic intermediate and that is reached with less than the full charge movement. For the estimation of total charge movement, we ignore dwells at the sublevel because these reflect an intermediate state. Instead, we make use of only the probability P of the fully open state, which is the predominant state at large depolarizations. Sublevel dwells will not affect the estimation of N by noise analysis because they are brief and very rare at the test voltage of +60 mV. Even ignoring the sublevels, activation of Shab channels is not as voltage dependent as that of Kv2.1. At a holding potential of −90 mV it is possible to observe channel openings, and NP for the fully open state is seen to change only 10-fold for 10-mV depolarizations ; this is about half the sensitivity seen for the other channel types. In constructing idealized traces, we took care not to confuse overlapping sublevel currents with open-state events; such overlapping events were very rare except at relatively depolarized voltages such as −70 mV. The resulting apparent charge estimate, based on P values between 10 −5 and 10 −3 was q l = 7.1 ± 0.3 e o . Plotting q s against P , the asymptotic value appears to be 7.5 e o . Similarly, low apparent charge values were seen in recordings from Shab channels in oocytes, where q l = 7.5 ± 0.3 e o ( n = 3) when evaluated over the range of P values from 10 −6 to 10 −4 . Whole-cell recordings of gating currents in Sf9 cells allow the comparison of macroscopic charge movements with the voltage dependence of q s . The quantity superimposes fairly well on q s values with q T = 7.5 e o and demonstrates that very little charge movement occurs at voltages negative to −90 mV. We therefore take 7.5 e 0 to be the estimate for the total charge in this channel. Sigg and Bezanilla 1997 point out that limiting-slope estimates of charge can be in error when there are multiple open states of a channel. For example, our estimate for Shab charge could be in error if there were an additional open state O′, having indistinguishable conductance, connected to the main open state O through a voltage-dependent transition. For example, Fig. 1 could account for the “missing” 5 e o of charge. To be consistent with the relatively shallow activation curve , the transition from O to O′ would necessarily occur in the voltage range of −40 mV and above. This additional transition will give rise to a large gating-charge movement that occurs at depolarized potentials. A comparison of the voltage dependence of q s with that predicted from Q ( V ) indeed shows a small discrepancy of this sort: a minor component of the gating charge Q ( V ) moves at potentials above −40 mV, where the q s curve is essentially flat. This component is, however, much smaller than that expected from Fig. 1 and therefore is unlikely to arise from an additional open state. The discrepancy between q s and Q ( V ) is in fact similar to the discrepancies seen in Shaker and Kv2.1 channels . Because the discrepancy occurs in the voltage range of channel opening, we believe that it arises from an interaction of permeant ions with the open channel. Effects of permeating ions on activation gating have been studied in sodium channels and potassium channels and effects on Shaker channels have been noted . In most cases, the presence of permeant ions is seen to result in the stabilization of the open state; meanwhile, blocking the ionic current with a toxin or pore mutation has an effect similar to the removal of extracellular ions. We expect that, in the presence of permeant ions, the equilibrium of the final opening transition is shifted toward the open state, tending to increase the cooperativity of channel opening and resulting in a steeper voltage dependence of q s . In our gating-current experiments, permeant ions were either absent or prevented from entering the pore, so the cooperative effect would be absent, yielding the shallower Q ( V ) curves that are observed. We conclude that there is no evidence for a large, additional charge movement that occurs while the channel is open. As a further check for the possible existence of multiple open states, we examined the single-channel kinetics of Shab channels. Fig. 10 A shows recordings from a patch containing a single Shab channel. Over the range of voltages from −30 to +40 mV, the open time distribution showed a single exponential component . Data from multichannel patches also show no evidence for a second open-time component at voltages down to −80 mV. The mean burst duration at −60 mV is 3.7 ± 0.1 ms ( n = 3) and has a very weak voltage dependence corresponding to a partial charge of 0.1 e o . The single-exponential open-time distributions support the idea that Shab has only one fully open state. Thus again, we find no reason to expect multiple open states, and find the simplest conclusion to be that Shab channels have a reduced gating charge of ∼7.5 e 0 . Ideally, this estimate of gating charge should be confirmed with an independent measure in which the total integrated gating current is normalized by the number of channels. Unfortunately, the relatively low expression level of Shab channels has precluded this sort of measurement, which requires either the ability to measure gating currents in a patch membrane or the ability to measure radioligand binding to a single cell. The Shab single-channel closed-time distributions were well described by three exponential components . The fastest closures are most frequent at the largest depolarization of +40 mV. The dwell time in this closed state is essentially voltage independent . This closed state may be the result of block by intracellular components or it could be a conformational state that is entered after the channel has opened, as is the case in Shaker channels . We favor the latter interpretation since the rapid closures are also observed in inside-out patch recordings where the only solutes bathing the internal membrane surface are K + , aspartate, HEPES, and EGTA. Transitions to this fast closed state from the open state accounts for the fact that P does not approach unity at large depolarizations. The value of P max is 0.72, as obtained from noise analysis of macroscopic currents at +50 to +70 mV. From the single-channel recordings, we calculate an open probability of 0.68 at +60 mV, very close to the estimate from noise analysis. Shab channels inactivate on a time scale of seconds and with a half-inactivation voltage of −30 mV . To test for possible inactivation effects in our experiments, we measured the inactivation induced by 500-ms prepulses . The reduction in current due to inactivation during depolarization to −60 mV or below is seen to be at most 5%, which would have a negligible influence in our estimates of the voltage dependence of P . In the experiments described in this report, we have estimated the effective gating charge of four members of the voltage-gated K + channel superfamily. We find that Shaker and Kv1.1 have an effective gating charge of ∼13 e o , as does the Kv2.1 channel. Shab channels, on the other hand, have an effective gating charge of only 7.5 e o . The estimates of gating charge are based on measurements of the channel open probability P at negative membrane potentials where P is very small. The voltage dependence of P provides a lower-bound estimate on the total charge movement ; in all channel types except Kv1.1, the reliability of the gating-charge estimate was confirmed by comparison of the voltage dependence of gating current according to the theory of Sigg and Bezanilla 1997 . We have also determined that the probability of any voltage-independent openings is very small in these channels and that the charge movement is tightly coupled to channel opening in Shaker and Kv2.1. In measuring the open probability, we are confronted with several possible sources of error. First, an artifactually large voltage dependence of P would result if the probability of missing brief channel openings increased substantially as the membrane potential was made more negative. We find, however, that the channel burst durations are weakly dependent on voltage in the range of potentials examined for each of the four channel types so that most bursts of openings are sufficiently long to be detected ( Table ). The measurements in Shaker and Shab channels were the most susceptible to this effect, since the mean burst duration becomes <4 ms at −70 mV. This is nevertheless considerably longer than the minimum detectable event duration, 120 μs at our analysis bandwidth of 1.5 kHz, so that the error is negligible. A second source of error would arise from voltage-dependent inactivation of channels, resulting in an artifactually reduced apparent voltage dependence. The known properties of Shaker channels, and tests for inactivation in Kv1.1, Kv2.1, and Shab channels, however, showed that inactivation effects were negligible under the conditions of our measurements. Finally, our estimates of P to very low values make use of the assumption of a homogeneous population of channels; if a fraction of channels has a shifted voltage dependence, then the estimated voltage dependence of P would be distorted. Heterogeneity in the voltage dependence of single, mutant Shaker channels has been observed , but this is of a form (a positive shift of a minority of the channels) that would have little influence on our estimates; such heterogeneity was not seen in the wild-type truncated Shaker channels studied here . We encountered no evidence for such heterogeneity in the single-channel behavior of the other channel types; if it were present, it would reduce the apparent voltage dependence of P at intermediate voltages, but leave the asymptotic voltage dependence unaffected. We take the relatively good agreement between the voltage dependence of q s and Q̂ as evidence that errors from heterogeneity are small. Logothetis et al. 1993 observed that Shab channels have a low voltage sensitivity, only ∼60% of that seen in Kv1.1 channels. They ascribed this difference to the differences in the S4 regions, where Shab has only five basic residues as compared with seven in the Shaker family channels. They also found a similar reduction in voltage sensitivity in Kv1.1 when the first (R1) and last (K7) basic residues were replaced with the neutral residues found in Shab , consistent with the idea that these two residues contribute to the gating charge in Kv1.1. Our experiments, which provide limiting-slope estimates of charge to much lower open probabilities and which distinguish between the main conductance level and a subconductance state in Shab , nevertheless confirm the view that Shab 's total charge movement is much smaller, only 7.5 e o , compared with 13 e o in Kv1.1. Other evidence from mutation studies is equivocal about the role of residues R1 and K7 in the voltage sensor. Early studies showed moderate effects on the voltage dependence of gating from the neutralization of these residues in Shaker , but large effects from their neutralization in Kv1.1 . More rigorous Shaker mutation studies involving the direct measurement of gating charge show a large decrease in charge when R1 is neutralized, but no effect from the neutralization of K7 . Studies in which cysteines were placed at or near the R1 position of Shaker show that this residue undergoes a moderate, voltage-dependent change in accessibility to externally applied reagents PCMBS and [2-(trimethylammonium)ethyl]methanethiosulfonate , leading Baker et al. 1998 to assign it an effective electrical distance change δ = 0.5. Meanwhile, near K7, no voltage-dependent accessibility change is seen . From these studies, one concludes that, in Shaker , R1 seems to contribute to the gating charge movement, while K7 does not. In view of these results, the large charge movement in Kv2.1 channels, indistinguishable from that in Shaker channels, comes as a surprise. Like Shab , Kv2.1 has only five basic residues in S4, and serves as a counterexample to the requirement of R1 for full charge movement. Instead, this result is consistent with the idea originally proposed by Larsson et al. 1996 that only the central five S4 residues participate in the gating charge movement. Previous estimates of the gating charge of Kv2.1 are much smaller than ours, in the range of 4–6 e o . They were obtained from the steepness of channel activation over the range of open probabilities from 0.005 to 0.1 or from the fit of macroscopic and other kinetic data to a particular sequential gating scheme . The relatively small charge values can be understood from the limited range of P values that were measured by these authors; only at negative potentials where P is smaller than ∼10 −5 is an asymptotic steepness corresponding to 13 e o approached . It should be kept in mind that our experiments made use of a channel with mutations in the outer pore region, but it seems to us very unlikely that these mutations could underlie an increased voltage sensitivity of the channels. Why is the behavior of Shab so different from its mammalian homologue Kv2.1? In these channels, the sequences of S2, S3, and S4 are very well conserved, with identical charged residues. Since the gating charge movement arises from charges within the protein , it seems inescapable that it is the degree of movement of charged groups in the membrane field, rather than the total charge, that is reduced in Shab compared with Kv2.1. It is not difficult to imagine how the degree of movement could be decreased. Charge movement in voltage-gated channels occurs in multiple steps; in Shaker channels, at least two, and probably three or more, sequential, charge-moving conformational changes occur in each subunit . Perhaps in Shab channels one of these steps does not occur, limiting the total charge movement to ∼60% of that of Shaker . Ligand-gated ion channels show a finite open probability even in the absence of agonists. The mouse muscle acetylcholine receptor channel has a spontaneous open probability of ∼5 × 10 −6 , while cyclic nucleotide–gated channels have spontaneous open probabilities on the order of 10 −5 to 10 −3 . The activation of these channels is well described by allosteric models in which the open state is stabilized by the binding of ligands. In the present study, we find that the voltage-insensitive open probability of Shaker channels is very much smaller, <10 −9 ; this suggests an obligatory coupling of charge movement to channel opening rather than a traditional allosteric mechanism. At the most negative potentials studied here, the steepness of the open probability curve does not decrease, and the effective charge computed from that steepness agrees well with other estimates of the total gating charge per channel. Taken together, these results imply that essentially all of the charge movement is obligatorily coupled to channel opening; that is, Shaker channels cannot open unless all of the charge movement has occurred in each subunit. The only exception to this obligatory coupling is a small, weakly voltage-dependent charge movement in Shaker channels that has been recently described by Stefani and Bezanilla 1996 . The same strict coupling between charge movement and channel opening is seen in Kv2.1 channels, where we have estimated the voltage-insensitive open probability to be <4 × 10 −8 , and in sodium channels, where the open probability has been measured down to 10 −7 . The situation is, however, different in Shab channels, where even at −90 mV the probability of occurrence of a subconductance state was the relatively large value of 8 × 10 −3 . This subconductance state appears to represent an intermediate step in channel activation, becoming predominant only at negative potentials. Subconductance states representing kinetic intermediates in channel activation have been observed in Kv2.1 and Shaker channels , but isolated openings to these levels were not detected in our measurements because either their lifetimes were too short or their equilibrium occupancies were even lower than that of the main conductance state, under the conditions we used. As might be expected for the reduced voltage sensitivity requirement of delayed-rectifier potassium channels, Shab channels have a much lower voltage dependence of channel activation. They also have less gating charge movement than the A-type Shaker channel and its homologue, Kv1.1, which also produces transient currents when coexpressed with beta subunits . It is surprising that the homologous mammalian delayed rectifier Kv2.1 does not have a similarly reduced charge movement. It will be interesting to see what is the molecular origin of this difference. | Study | biomedical | en | 0.999996 |
10544192 | It is well established that injection of antigens into the AC of the eye results in a form of immune deviation termed AC-associated immune deviation (ACAID; for a review, see reference 2). This phenomenon was first identified by Kaplan and Streilein 7 , who reported that injection of F1 lymphoid cells into the AC of rat eyes alters the recipient's systemic immune response such that rejection of subsequent allografts from the donor strain used for AC priming is impaired. Since then, ACAID has been demonstrated for several antigens, including viruses, corneal allografts, and tumor cells. The hallmarks of ACAID are suppression of delayed-type hypersensitivity (DTH) and inhibition of the synthesis of complement-fixing antibody isotypes. Antigen introduced into the AC is captured by specialized APCs of the eye that carry the antigen via the blood to the spleen. A deviant form of immunity is then generated which is capable of suppressing Th1-mediated DTH responses and production of complement-fixing antibodies. CD8 + T cells isolated from the spleen of AC-immunized mice can induce ACAID and inhibit DTH responses when transferred into naive syngeneic mice 8 . These findings strongly indicate that AC immunization results in the induction of CD8 + Tr cells that inhibit Th1 cells . Interestingly, since ACAID in this system is induced by soluble antigen, presentation of antigen to CD8 + Tr cells must follow the poorly characterized exogenous pathway of MHC class I–restricted antigen presentation 4 . There is considerable evidence that the immunosuppressive cytokine TGF-β plays a critical role in the induction and/or effector function of CD8 + Tr cells during ACAID 2 . For example, AC injection of antigen induces the generation of T cells in the spleen that produce TGF-β when stimulated with antigen in vitro. Studies by Griffith et al. 9 further indicated that immune tolerance after antigen injection in the eye depends on the death of lymphoid cells by the Fas/FasL system. Although the exact mechanism by which apoptosis of lymphoid cells affects ACAID remains unclear, it was suggested that apoptotic lymphoid cells produce IL-10 which, in turn, induces immune deviation by its effects on APCs 10 . The paper from Sonoda et al. 6 provides another piece to this puzzle, demonstrating that NKT cells are required for the induction of CD8 + Tr cells. NKT cells represent a relatively novel lymphocyte subset distinct from conventional T cells, B cells, and NK cells. NKT cells share receptor structures with both NK cells and conventional T cells (for reviews, see references 11, 12). NKT cells express typical NK cell markers, including IL-2Rβ, members of the NKR-P1 NK cell–activating receptor family, and members of the Ly49 NK cell–inhibitory receptor family. They also express a TCR with an invariant Vα14-Jα281 chain and a polyclonal Vβ8.2 (and to a lower extent, Vβ7 or Vβ2) chain. Approximately 60% of all NKT cells express CD4, and the remaining cells do not express either CD4 or CD8. These cells have a memory phenotype, expressing high levels of the early activation marker CD69, high levels of CD44 (Pgp-1), and low levels of CD62L (L-selectin). NKT cells are abundant in the thymus and represent a major population of lymphocytes in the spleen, bone marrow, and liver, but are rare in lymph nodes. Unlike conventional T cells that recognize peptide antigens in the context of self-MHC class I or class II molecules, NKT cells are specific for glycolipid antigens bound with the MHC class I–like molecule, CD1d (for reviews, see references 11, 12). As CD1d expression is required for the development of NKT cells, CD1d-deficient mice have a marked reduction in the number of NKT cells 13 14 15 . Autoreactivity appears to be common among NKT cells. Many NKT cell hybridomas were able to react with splenocytes, thymocytes, or CD1d-transfected cells 11 . Interestingly, individual hybridomas differed in their reactivity with CD1d-expressing cells, suggesting substantial heterogeneity among NKT cells 16 17 . Recent studies have shown that CD1d can bind with a variety of glycolipids, including phosphatidylinositol derivatives 18 19 and glycosylceramides 20 . However, the physiological significance of these glycolipids for NKT cell function is unclear. When activated through their TCR, NKT cells become cytotoxic and quickly produce a variety of cytokines, including large amounts of IL-4, significant amounts of IFN-γ, and some inhibitory cytokines such as TGF-β and IL-10 11 21 . Although their physiological functions remain unclear, NKT cells have been implicated in immune responses against infectious agents, bone marrow grafts, tumors, and self-antigens 11 12 . The paper by Sonoda et al. 6 provides convincing evidence that NKT cells are required for development of ACAID. AC administration of antigen to NKT cell–deficient CD1d knockout mice did not induce ACAID, unless these animals were reconstituted with NKT cells from wild-type mice together with CD1d-expressing APCs. Since anti-CD1d antibodies inhibited development of ACAID in wild-type animals, direct interaction of the invariant Vα14 TCR with CD1d appears to be required. The question then arises as to what NKT cells recognize in the spleen after AC administration of protein antigen. One possibility is that eye APCs express, as a result of exposure to immunosuppressive cytokines such as TGF-β, high levels of CD1d that activate autoreactive NKT cells. Alternatively, introduction of antigen into the AC may cause expression of specific endogenous glycolipids that activate NKT cells. At what step do NKT cells contribute to the induction of ACAID? Prior studies have shown that CD8 + T cells from the spleen of AC-primed mice can adoptively transfer ACAID to naive animals 8 . The studies of Sonoda et al. 6 further extend these findings by demonstrating that NKT cell–depleted spleen cell populations from AC-primed animals can inhibit DTH reactions after adoptive transfer to naive animals. Therefore, these findings indicate that NKT cells are specifically required for the generation of antigen-specific CD8 + Tr cells. A likely scenario for the induction of ACAID, based on the findings of Sonoda et al. 6 , is presented in Fig. 1 . Antigen inoculated into the AC of the eye is captured by specialized APCs that carry the antigen to the spleen. These eye APCs may have unique features that are critically important for the induction of ACAID, including increased expression of CD1d (perhaps induced by TGF-β in the AC) and altered antigen-presenting properties (perhaps in response to IL-10 produced by apoptotic lymphoid cells). These eye APCs then activate effector T cells, CD8 + Tr cells, and NKT cells. Activated NKT cells produce cytokines (TGF-β is a likely candidate) that stimulate the generation and expansion of antigen-specific CD8 + Tr cells. In turn, these CD8 + Tr cells can inhibit, by production of cytokines (TGF-β and IL-10 are likely candidates), subsequent Th1-mediated DTH responses to the same antigen. There is now compelling evidence that Tr cells participate in many immune responses, including responses against foreign and self-antigens 22 . The findings of Sonoda et al. 6 raise the possibility that activation of NKT cells is a general mechanism for the generation of Tr cells. Prior studies have suggested a role for NKT cells in the regulation of immune responses. Various mouse strains with genetic susceptibility for the development of autoimmune disease, including nonobese diabetic (NOD) mice with a propensity for development of type I diabetes, SJL mice with a susceptibility for development of experimental allergic encephalomyelitis, and several lupus-prone strains, were found to have defects in NKT cell development and/or function 23 24 25 26 . Reconstitution of NOD mice with NKT cells 27 or transgenic overexpression of NKT cells in these animals 28 was able to inhibit development of diabetes. Further, NKT cells isolated from Vα14-Jα281 transgenic mice either induced or prevented murine lupus in an adoptive transfer model 29 , depending on the source and cytokine profiles of the NKT cells that were transferred. Defects in NKT cell development and function were also observed in human patients with systemic sclerosis 30 and in patients with type I diabetes 31 . In addition to their role in the regulation of autoimmunity, NKT cells were shown to influence immune responses against tumors 21 and infectious agents 19 32 33 34 , and to suppress graft versus host disease 35 . In many of these studies it was suggested that NKT cells influence the disease process by production of cytokines. For example, the inhibitory role of NKT cells for the development of Th1-dominated inflammation in several autoimmune diseases was attributed to their production of IL-4, which promotes Th2 immunity. An alternative explanation, not excluded by these studies and supported by the findings of Sonoda et al. 6 , is that NKT cells induce the generation of Tr cells that, in turn, inhibit inflammation. It is unlikely that NKT cells participate in all types of immune regulation controlled by Tr cells. Indeed, Sonoda et al. 6 showed that NKT cells are dispensable for the systemic tolerance induced in response to intravenous injection of antigen. Perhaps other cell types, such as γ/δ T cells and NK cells, can contribute to the regulation of immune responses and induction of self-tolerance. A role for γ/δ T cells as regulators of immune responses has been suggested (for a review, see reference 22). The realization that NKT cells play a critical role in immune regulation and maintenance of self-tolerance raises the possibility of manipulating these cells to modulate immune responses during prophylaxis and therapy. This should be possible with reagents such as the glycolipid α-galactosylceramide that selectively activates NKT cells 20 . Recent studies have shown that administration of this agent to mice can polarize adaptive immune responses towards Th2-dominated immunity 36 37 . | Review | biomedical | en | 0.999998 |
10544193 | Ikaros null and DN mutant mice as well as wild-type control littermates on a mixed C57BL/6J × 129SV background were bred and maintained under sterile conditions in a pathogen-free animal facility at Massachusetts General Hospital. Due to the high morbidity of the Ikaros DN mutant mice, all animals were kept on oral antibiotics. Mice used for the different studies were between 2 and 5 wk old. The genotypes of mice were determined by PCR analysis of the Ikaros locus with primers and conditions described previously 10 . C57BL/6J–Ly5a-Pep 3b –congenic mice used as transplant recipients or donors were obtained from The Jackson Laboratory and bred in the animal facility. Bone marrow (BM) was prepared by crushing femora and tibiae with a mortar and pestle and then passing the suspension through a 70-μm cell strainer to remove bone debris. Spleen cell suspensions and day 14 fetal liver cells were obtained by disrupting the tissue in PBS (plus 5% dialysed FBS) and passing it through a 70-μm cell strainer. The aorta-gonad-mesonephros regions (AGM) and yolk sacs (YS) from 11 d postcoitum embryos were prepared as described previously 22 . In brief, the AGM and YS were dissected in PBS/5% FCS and digested for 1 h at 37°C in 0.125% collagenase (Sigma Chemical Co.). Viable cell counts were based on trypan blue exclusion. The mAbs used for immunofluorescent labeling and the fluorochromes employed are specified elsewhere 17 . In brief, three-color flow cytometry was performed using anti-Ly5b (AL1-4A2) to identify donor-derived cells and antibodies against B220 (RA3-6B2), CD4 (RM4-5), CD8 (53-6.7), TCR-α/β (H57-597), Mac-1 (M1/70), Gr-1 (RB6-8C5), and TER-119. All antibodies were purchased from PharMingen. Marrow and spleen cells were harvested from neonate and 2–5-wk-old mice. Single-cell suspensions from each tissue were prepared in PBS (plus 5% dialysed FBS), counted, and cultured in IMDM containing 1.2% methyl cellulose, 15% FBS, 0.5% BSA fraction V, transferrin, insulin, lipids, α-thioglycerol, 15 U/ml IL-3, 2 IU/ml erythropoietin, and 50 ng/ml kit ligand. Colonies were scored after 2–3 d for CFU-E and after 7–10 d for all other colony types. Erythroid colonies containing at least two other lineages were attributed to colony-forming cell (CFC)-multi. Pure erythroid colonies on day 7–8 were attributed to BFU-E, and colonies containing at least 500 granulocytes (G) and/or macrophages (M) were attributed to CFC-G/M. Purified murine kit ligand was provided by Genetics Institute. The CFU-S 14 content in the BM and spleens of Ikaros mutant and wild-type littermates 2–5 wk after birth was determined by injection of 5 × 10 4 nucleated BM cells or 2.5 × 10 5 spleen cells into the lateral tail vein of lethally irradiated (9.5 Gy; 137 Cs single dose) wild-type recipient mice. Mice were killed 14 d after the injection, and their spleens were fixed in Bouin's solution for macroscopic examination and weighing. Absolute numbers of CFU-S 14 per organ were calculated based on the frequency measurement and the cellularity of the spleen and the BM, assuming that one femur and one tibia represent 10 and 5%, respectively, of total BM. To determine the lineage composition of spleen colonies, single colonies were dissected before fixation, erythrocytes were lysed in 0.4 M ammonium chloride buffer, and cells were stained with a subset of antibodies described previously and subjected to FACS™ analysis. For PCR genotyping, colonies were lysed in DNA lysis buffer, and DNA was prepared as previously described 10 . Irradiated mice injected with PBS alone were included as controls in all experiments. Congenic C57BL/6J–Ly5a-Pep 3b mice were irradiated with a single lethal dose of 9.5 Gy from a 137 Cs radiation source (gamma irradiator; J.L. Shepherd) at 0.95 Gy/min. Cell suspensions containing 10 5 wild-type or 6 × 10 6 mutant BM cells from 2–5-wk-old mice in a final volume of 200 μl PBS were injected intravenously into the lateral tail vein. Recipient mice were maintained on 1.1 g/liter neomycin sulfate (Sigma Chemical Co.) and 10 6 U/liter polymyxin B sulfate (Sigma Chemical Co.) in their drinking water for the duration of the assay. Mice were monitored for survival daily for 35 d. For the competitive assays, lethally irradiated Ly5a mice (9.5 Gy of gamma irradiation) were injected with a fixed amount of 10 5 Ly5a autologous BM cells along with 10 5 –10 7 Ikaros mutant or wild-type BM cells (Ly5b). Donor-derived (Ly5b) hemopoietic contribution was measured from the peripheral blood at different time points starting on day 19 after transplant and from both peripheral blood and hemopoietic organs at the time mice were killed. BM cells were collected from femora and tibiae of 2–5-wk-old mice. Cells were layered at 2.5 × 10 7 cells/ml over 3 ml of sodium metrizoate (Nycodenz; Accurate Scientific) solution (1.077 g/ml) and centrifuged at 1,000 g for 20 min. Low density cells were harvested and incubated with mAbs to CD4, CD8, B220, Mac-1, Gr-1, and TER-119 (all antibodies are rat IgG), followed by goat anti–rat IgG coupled to magnetic beads (Miltenyi Biotec). Lin + cells were depleted by attachment to a magnetic column according to manufacturer's instructions (MACS™; Miltenyi Biotec). For cell sorting and analysis, the lineage-depleted (lin − ) population was incubated again with the lineage antibodies followed by goat anti–rat IgG conjugated to allophycocyanin (Caltag Labs.), normal rat IgG (Caltag Labs.), and antibodies to c-kit (PE conjugate; PharMingen) and Sca-1 (fluorescein conjugate; PharMingen). Cells were then analyzed in a dual laser cell sorter (FACScan™; Becton Dickinson). Cells within the blast forward and side light scatter gate that were negative for the lineage markers were gated electronically and analyzed for or sorted according to expression of c-kit and Sca-1. RNA purification, first-strand cDNA synthesis, and PCR amplification were performed as described previously 15 . PCR primers used were as follows: HPRT F, CAC AGG ACT AGA ACA CCT GC; HPRT R, GCT GGT GAA AAG GAC CTC T; FLK1 F, AGA ACA CCA AAA GAG AGG AAC G; FLK1 R, GCA CAC AGG CAG AAA CCA GTA G; FLK2 F, GGA GGA GGG CAG CTA CTT TGA G; FLK2 R, CTG TTA GCC TTT TTA TTC CAA ACT C; GATA1 F, CAT TGG CCC CTT GTG AGG CCA GAG A; GATA1 R, CGG AGA TAA AGT TCG AGG TAG TCC A; GATA2 F, ACA CAC CAC CCG ATA CCC ACC TAT; GATA2 R, CCT AGC CCA TGG CAG TCA CCA TGC; GATA3 F, ACG TCT CAC TCT CGA GGC AGC ATG; GATA3 R, GAA GTC CTC CAG CGC GTC ATG CAC; SCL F, GTC CTC ACA CCA AAG TAG TG; SCL R, GGC ACC TCA AAG CTT GAC TCT CCA; GMCSF F, GAG GTC ACA AGG TCA AGG TG; GMCSF R, GAT TGA CAG TGG CAG GCT TC; PU1 F, GAG TTT GAG AAC TTC CCT GAG; PU1 R, TGG TAG GTC ATC TTC TTG CGG. In Ikaros null and DN −/− mice, the BM cellularity is decreased to 37 and 18% of wild-type levels , exceeding the reduction expected from the lack of B lymphocytes and their precursors. In normal adult mice, the spleen is the secondary hemopoietic organ, with 80–90% of its population comprised of B and T lymphocytes and 10–20% of the cells being of erythromyeloid origin. In Ikaros null mice, the cellularity in the spleen is reduced to only 62% of wild-type levels, a much smaller reduction than that expected given the lack of B cells and impaired T cell development 11 17 . Ikaros DN −/− mice, which lack both B and T lymphocytes, also show a reduction to only 57% of the cellularity of the wild-type organ . Taken together, these results reveal a significant increase in myeloerythroid cells in the spleens of Ikaros mutants. Such disproportionate changes in hemopoietic populations between BM and spleen are indicative of extramedullary hemopoiesis. We have previously shown 11 that Ikaros null mice have normal hematocrits throughout their life spans. Ikaros DN −/− mice, however, display a drop in hematocrit with age . The hematocrits of 2–3-wk-old Ikaros DN −/− mice are similar to those of wild-type littermates but soon after drop and by 6 wk of age reach a value of <50% of wild-type levels. During this period, Ikaros DN −/− mice also develop extensive infections from opportunistic microorganisms and die 10 . Therefore, the cause of death in these mice cannot be unequivocally ascribed to the lack of an immune system or to hemopoietic failure but rather may be due to both. Thus, in addition to lymphoid defects, Ikaros deficiency has other effects on hemopoiesis. Mice exposed to high doses of whole body irradiation die within 9–18 d from hemopoietic failure unless they are transplanted with hemopoietic precursors and progenitors that provide radioprotection and short-term reconstitution. The radioprotective quality of Ikaros null and DN −/− BM was assayed in strains of mice congenic for the panleukocyte marker Ly5. Both Ikaros null and DN −/− donor mice expressed the Ly5b variant, whereas the transplant recipients expressed the Ly5a allele. Ikaros null and wild type BM cells, when given at a dose of 10 5 cells, radioprotected 100% of the lethally irradiated (900 rads) recipients for at least 30 d after transplant . Animals receiving 10 6 Ikaros null BM had almost 100% donor contribution to the myeloid (Mac-1 + ) lineage 7 mo after transplant . Ikaros DN −/− BM cells provided at doses of 1–6 × 10 6 were capable of only short-term radioprotection at first , with a steady decrease in donor contribution observed between 3.5 and 5 wk after transplant . The hematocrits of Ikaros DN −/− BM recipients also decreased during this time period. 3 wk after transplant, hematocrits of Ikaros DN −/− BM recipients were <50% of wild-type BM recipients, and by day 35 they were down to one-third of wild-type values . All recipients of Ikaros DN −/− BM died by day 35 after transplant from severe anemia. Given the extramedullary hemopoiesis manifested in the spleens of Ikaros DN −/− mice, we examined whether hemopoietic progenitors developed in this secondary hemopoietic site. Splenocytes from these mutants were unable to radioprotect even when provided at a dose of 1.2 × 10 7 cells (data not shown). This observation suggests that there is no shift in the production or expansion of hemopoietic progenitors from the BM to the spleen and that extramedullary hemopoiesis at this site is due to the differentiation of more committed and short-lived erythromyeloid precursors. The LTR potential of Ikaros null and DN −/− BM was analyzed in a competitive repopulation assay 23 24 . Ikaros mutant Ly5b BM cells were injected into lethally irradiated Ly5a recipients along with a constant competitor dose of 10 5 Ly5a marrow. When Ikaros null BM (10 5 ) was transplanted with an equal amount of wild-type competitor marrow, it failed to contribute to myeloid (Mac-1 + ) cells in the BM or the periphery of recipients . In sharp contrast, wild-type BM contributed to 44% of the BM myeloid (Mac-1 + ) populations . Ikaros null BM injected in 10-fold excess (10 6 ) over wild-type competitor gave 17% contribution to the myeloid (Mac-1 + ) lineage in the BM . No significant contribution to the T cell lineage was observed in the thymus or the spleen (data not shown). In comparison, wild-type BM coinjected at a similar dose contributed to >99% of the myeloid and lymphoid (data not shown) populations in the BM and periphery. When 7.5 × 10 6 Ikaros null BM cells were injected alongside 10 5 competitor BM cells, donor-derived contribution to the myeloid lineage increased to 75.3% . This analysis indicates that there is a 30–40-fold decrease in LTR activity in the Ikaros null BM. In a previous study 17 , we reported short-term myeloid lineage contribution by Ikaros DN −/− BM when injected at a ratio of 100:1 over competitor BM. At this ratio, Ikaros DN −/− BM contributed to 52% of myeloid (Mac-1 + ) cells by 19 d after transplant, but by day 27, this contribution declined to 6% and by day 48 to 3% . No donor-derived myeloid contribution was detected 120 and 260 d after transplant . PCR analysis of DNA prepared from the blood, spleens, and BM of recipients also failed to detect donor-derived cells, confirming the lack of LTR activity in Ikaros DN −/− BM (data not shown). Thus, in the absence of Ikaros , LTR activity as estimated in competitive repopulation assays is reduced by 30–40-fold. However, a >100-fold reduction in LTR activity is observed in mice homozygous for the DN Ikaros mutation. Lethal irradiation of normal murine recipients before BM transplant is required to ensure engraftment and detection of donor-derived hemopoietic cells. However, if the hemopoietic system of the recipient is already compromised, then donor-derived hemopoiesis is detectable even in the absence of prior conditioning of the recipient. This phenomenon has been observed with c-kit receptor ( W ) mutant mice 25 . Given the depletion in hemopoietic progenitors resulting from the null and DN Ikaros mutations, we attempted to repopulate unconditioned Ikaros mutants with wild-type BM. A transplant of 10 6 wild-type Ly5a BM cells into unconditioned Ikaros null mice completely repopulated the B cell lineage, consistent with the total lack of B cells and their precursors in Ikaros mutants . Donor repopulation of T cells and some myelocytes was also observed, albeit at low levels, possibly due to competition with endogenous precursor populations . Ikaros DN homozygous mutants and heterozygous littermates were injected with 10 6 wild-type Ly5a BM cells at 12 d of age and subsequently analyzed at 4, 12, and 22 wk after transplant. No detectable donor contribution in any hemopoietic lineages was observed after transplant of wild-type Ly5a BM into unconditioned Ly5b Ikaros DN heterozygotes . In striking contrast, complete donor repopulation of B and T lymphocytes and myeloid cells was observed in unconditioned Ikaros DN −/− mice receiving 10 6 wild-type BM cells , which persisted for the full duration of the experiment. To detect potential hemopoietic contribution by endogenous Ikaros DN −/− mutant cells at levels that may fall below the detection limit of FACS™, PCR analysis of DNA prepared from various hemopoietic tissues was employed . Given a PCR DNA detection limit of 1:1,000 mutant/wild-type DNA , we conclude that host-derived hemopoietic cells were at least 1,000-fold less frequent in unconditioned Ikaros DN −/− recipients after transplant of wild-type BM. Thus, the hemopoietic system of Ikaros null and DN −/− mice is readily repopulated by wild-type HSCs without the need for prior myeloablative conditioning. This repopulation of unconditioned Ikaros mutant mice by wild-type HSCs reflects a severe depletion in the endogenous pool. The more committed myeloid precursor content of Ikaros mutant mice was investigated in a spleen colony-forming assay (CFU-S 14 ) 26 . CFU-S 14 levels in Ikaros null and DN −/− BM were reduced 9.2- and 81-fold relative to wild type . Splenic CFU-S 14 was also reduced by three- and fivefold in Ikaros null and DN −/− mice relative to wild type . The combined limb BM and splenic CFU-S 14 contents of Ikaros null and DN −/− mice were reduced 7.7- and 34-fold relative to wild type. Although these data assume similar spleen seeding efficiencies by wild-type and Ikaros mutant CFU-S, we have not tested this hypothesis. Qualitative effects of the Ikaros mutations on the size and lineage composition of day 14 spleen colonies were also observed. First, spleen colonies derived from Ikaros mutant cells were smaller than those derived from wild-type populations . Second, Ikaros mutant colonies contained 20–40% of nucleated erythroid (TER-119 + ) cells, whereas wild-type colonies consisted mainly (76–91%) of more mature erythroid cells (data not shown). The donor origin of the CFU-S 14 colonies was confirmed by PCR (data not shown). Interestingly, a small number of endogenous colonies was detected in recipients of Ikaros mutant BM and spleen but not in recipients of wild-type populations or in PBS controls. A possible facilitating effect is suggested, perhaps in the form of growth factors produced by the Ikaros mutant cells that promote expansion and differentiation of endogenous progenitors. The CFU-S 14 content of day 10 YS and AGM and day 14 fetal livers of Ikaros DN −/− mutant and wild-type embryos was also measured. The CFU-S 14 content in these late embryonic and early fetal sites of hemopoiesis was drastically reduced in Ikaros DN −/− embryos compared with wild type . Thus, the lack of Ikaros , coupled with interference toward the activity of other family members, affects the production of both fetal and adult hemopoietic progenitors. BM and splenic hemopoietic populations were analyzed for their content of myeloid-restricted in vitro CFCs. The absolute number of precursors giving rise to multilineage colonies (CFC-multi) and G- and/or M-restricted colonies (CFC-G/M) in Ikaros null BM was within the range of wild-type values . In contrast, the absolute number of mature erythroid-restricted clonogenic precursors (CFU-E) was reduced to 30% of wild-type levels . Furthermore, the most immature erythroid-restricted precursors (BFU-E) were reduced to 5% of wild-type numbers. In the spleens of Ikaros null mice, all CFC classes, including CFU-E, BFU-E, CFC-G/M, and CFC-multi, were reduced in absolute number . However, the frequencies of all CFCs, with the exception of BFU-E, were higher than in wild type due to the observed decrease in spleen (and BM) cellularity in Ikaros mutants. A more severe reduction in the absolute number of all CFCs was detected in Ikaros DN −/− mice. BFU-E cells were the most drastically affected in BM and spleen, with absolute numbers <1% of wild type . CFU-E cells were also reduced, but to a lesser extent than BFU-E. A CFU-E reduction to 5% of wild type was noted in the BM and to 25% of wild type in the spleen. In the BM, the absolute number of CFC-G/M cells was reduced to 15% of wild-type levels, reflecting the reduction in BM cellularity. Thus, the CFC-G/M frequency was similar to that of wild-type BM. However, in the Ikaros DN −/− spleen, CFC-G/M numbers were greatly increased (greater than twofold), indicating a dramatic increase in granulomonocytic precursors in this normally lymphoid organ . Nonetheless, no increase in splenic erythroid precursors (BFU-E and CFU-E) was detected, suggesting specific effects of the Ikaros mutation on the expansion of myeloid versus erythroid precursors . Given the BM hypoplasia and abnormal splenic hemopoiesis manifested in both Ikaros mutants, the combined total BM and spleen content of hemopoietic precursors was calculated per mouse . BFU-E cells are reduced 10-fold in the Ikaros null and 30-fold in Ikaros DN −/− mice . These BFU-E reductions are in line with the reductions seen in the most primitive HSC population. However, the total number of the later erythroid precursors (CFU-E) was reduced only three- and sixfold in Ikaros null and DN −/− mice, respectively. Furthermore, in both Ikaros mutants, CFC-G/M cells were reduced to 40–75% of wild-type levels, a less severe decrease than that observed for other CFC classes. Although the numbers of myeloid precursors in the spleens of the Ikaros null and DN −/− mice (with the exception of CFC-G/M) are decreased, the number of terminally differentiated erythroid (TER-119 + ) and myelomonocytic (Mac-1 + /Gr-1 + ) cells is increased in this organ 10 11 . This suggests that the splenic microenvironment is conducive to the rapid differentiation of myeloid precursors to their mature progeny. Hemopoietic cells that lack expression of mature lineage markers (lin − ) and that coexpress Sca-1/Ly6A and the tyrosine kinase receptor c-kit on their cell surfaces (lin − c-kit + Sca-1 + ) are highly enriched in HSC activity in normal mice 27 28 . The HSC-enriched lin − c-kit + Sca-1 + population was detected in Ikaros null BM . In sharp contrast, no c-kit + /Sca-1 + cells were present among the lin − BM cells of Ikaros DN −/− mice . The lack of BM cells with a lin − c-kit + Sca-1 + surface phenotype in Ikaros DN −/− mice is consistent with the dramatic depletion in HSC activity in these mutant mice. Analysis of lin − BM cells from Ikaros null and DN mice revealed a progressive decrease in the cell surface expression level of c-kit . Given the importance of the steel factor/c-kit signaling pathway in the expansion and differentiation of hemopoietic progenitors 29 , we predict that the decreased expression of c-kit in Ikaros mutant hemopoietic cells may underlie some of the progenitor defects. To address the molecular basis of the hemolymphoid deficiencies in Ikaros mutant mice, we analyzed expression of growth factor receptors, signaling molecules, and transcription factors known to be important in the production, maintenance, and differentiation of hemopoietic cells. Lin − BM cells from Ikaros null and wild-type mice were sorted into c-kit hi , c-kit lo , and c-kit hi Sca-1 + populations, and those from Ikaros DN −/− mice were sorted into c-kit hi and c-kit lo populations and subsequently used for reverse transcriptase (RT)-PCR analysis . mRNA expression of the tyrosine kinase receptor flk-2, shown to be required for HSC differentiation along the B cell lineage 30 , was undetectable in all lin − BM populations, including those that are highly enriched for HSC activity, in Ikaros null mice . Expression of a second related tyrosine kinase receptor, flk-1, required for development of the vascular endothelium from the hemangioblast, was also tested 31 . Flk-1 was expressed in the Ikaros mutant lin − populations but at a somewhat reduced level compared with wild type . The mRNA level of the tyrosine phosphatase Shp-1, a downstream effector of the c-kit signaling pathway, was also determined and found not to be significantly different between Ikaros mutant and wild-type populations . Expression of several transcription factors known to play key roles in the development of the hemopoietic system was also determined. Ikaros mutant lin − cells were analyzed for the presence of SCL, GATA-1, GATA-2, GATA-3, and PU.1 transcripts. A small decrease in SCL levels was seen in the Ikaros null c-kit hi and c-kit hi Sca-1 + population . Small changes in GATA-2 but not GATA-1 levels were also seen among the Ikaros null and DN −/− hemopoietic populations, the most dramatic being an increase in GATA-2 levels in the c-kit lo cells in the Ikaros DN −/− BM. These possibly reflect changes in hemopoietic progenitor composition in the lin − compartment. GATA-3 is expressed among the lin − hemopoietic cells, possibly in progenitors undergoing specification along the T cell lineage. GATA-3 was expressed within lin − progenitor populations of Ikaros null mice, which generate T cell precursors. However, it was not detected in Ikaros DN −/− lin − populations lacking T cell differentiation potential, reflecting either lack of the relevant precursor population or lack of expression of the GATA-3 factor in these cells. Finally, levels of PU.1 are reduced in the c-kit hi population of the Ikaros null mutants but elevated in the same population in Ikaros DN −/− BM. The Ikaros mutant lin − hemopoietic populations were also analyzed for expression of mRNAs encoding hemopoietic growth factors and receptors. In the c-kit lo of Ikaros null and in the c-kit lo-med populations of Ikaros DN −/− BM, levels of GM-CSF receptor were significantly elevated relative to wild type, which could be the cause or effect of the increase in GM precursors observed in clonogenic assays. We have previously shown that Ikaros is an essential regulator of lymphoid lineage specification in the fetal and adult hemopoietic system. Ikaros is, however, expressed early during ontogeny of the hemopoietic system and is detected at high levels in mesodermal progenitors in the splachnopleura of the day 8 embryo and within the blood islands of the YS (reference 18 and Georgopoulos, K., unpublished results). In addition, in adult hemopoietic sites, Ikaros is expressed within cell populations that are highly enriched in HSCs 15 32 . A detailed analysis of the entire hemopoietic hierarchy in Ikaros mutant mice is presented here, revealing a role for Ikaros in the production or activity of the self-renewing HSC. These studies also provide evidence for a functional interplay between Ikaros and other nuclear factors, including family members during differentiation of the most primitive of hemopoietic progenitors. Mice homozygous for an Ikaros null or DN mutation show a decrease in BM cellularity that cannot be ascribed solely to the lack of B cell differentiation. In addition, an increase in erythromyeloid precursors is detected in the spleens of Ikaros mutant mice, an observation not made in other strains of mice depleted of lymphocytes. The progressive development of anemia in mice homozygous for the Ikaros DN −/− mutation, manifested as a progressive drop in hematocrit, is also indicative of a failure of the hemopoietic system to supply mature erythrocytes at normal levels. BM progenitors from Ikaros null mice provide short- and long-term contribution to most of the hemopoietic lineages, apart from the lymphoid, when transferred alone into lethally irradiated recipients. However, when measured against wild-type BM in a competitive assay, a severe depletion in both short- and long-term repopulating activities is revealed. Ikaros null BM is unable to contribute to any hemopoietic lineages when competed against wild-type congenic BM at a ratio below 10:1. Even when present in a >10-fold excess, only limited hemopoietic contribution is observed from Ikaros null BM, suggesting a reduced number of HSCs in these mutants. In contrast, wild-type BM, when used at an excess of 10:1 over competitor, repopulates 100% of all hemopoietic lineages tested. Thus, there is a quantitative reduction estimated to be 30–40-fold in the number of HSCs in Ikaros null mice . A greater reduction in HSC numbers is detected in mice homozygous for a DN Ikaros mutation. Ikaros DN −/− BM is unable to provide LTR in a competitive assay, even when used at 100-fold excess to wild-type congenic BM, indicating a severe depletion in stem cell activity below assayable levels. A limited short-term hemopoietic contribution from the Ikaros DN −/− BM was detected in both competitive and radioprotective assays, suggesting a transient expansion in short-term repopulating cells. Wild-type BM cells sorted according to the absence of cell surface lineage-specific markers and expression of c-kit and Sca-1/Ly6A are enriched for HSC activity 28 . This population of cells was absent from the BM of Ikaros DN −/− mice, consistent with the depletion in both short- and long-term repopulating activity in these mutants. The defect in hemopoietic progenitors within Ikaros mutants was also illustrated by the ability of wild-type BM to provide hemopoietic reconstitution to these animals without their prior conditioning. Transplant of wild-type BM cells into unirradiated Ikaros null mice resulted in chimeric animals in which some hemopoietic lineages, such as T, Mac-1 + , and Gr-1 lo cells, all of which are unaffected by the Ikaros mutation, derive from both wild-type and Ikaros mutant precursors. B lineage cells and Gr-1 hi cells, neither of which are produced from Ikaros mutant progenitors, are derived exclusively from wild-type precursors. In Ikaros DN −/− mutants, repopulation by wild-type BM without the need for prior conditioning is observed for all lineages. The ability of normal BM to function in Ikaros mutants suggests that the Ikaros defects are manifest within hemopoietic populations rather than in nonhemopoietic accessory cells of the BM and spleen. Evaluation of the more committed hemopoietic precursor compartment in Ikaros mutant mice reveals amelioration of their phenotypes. CFU-S 14 clonogenic assays were used as a measure of the multilineage erythromyeloid-restricted progenitors. An eightfold reduction in CFU-S 14 activity was detected in Ikaros null mice, whereas the reduction in Ikaros DN −/− mice was 34-fold. In vitro clonogenic assays show that BFU-E, the earliest erythroid-restricted precursor, is reduced by 10- and 30-fold in Ikaros null and Ikaros DN −/− mice, respectively. This reduction in BFU-E may in part explain the decrease in CFU-S 14 , particularly as wild-type spleen colonies are comprised mainly of erythroid lineage cells. However, later stage erythroid precursors (i.e., CFU-E) are not as severely compromised, with only three- and sixfold reductions detected in Ikaros null and DN −/− mice. Lack of Ikaros has an even lesser effect (25 and 50% decrease) on the number of myelomonocytic precursors, indicating their possible preferential expansion relative to BFU-E . The decrease in CFU-S 14 and BFU-E activity in Ikaros mutant mouse strains is smaller than the estimated 30–100-fold decrease in LTR cells, suggesting the existence of partial compensatory mechanisms after HSC commitment. Alternatively, Ikaros may normally function in early hemopoietic progenitors to limit differentiation along the myeloid lineages, a constraint that is removed upon Ikaros inactivation . To determine if the hemopoietic defects manifested in Ikaros mutants are the result of a defect in the production or maintenance of HSCs, we examined the hemopoietic precursor content of embryonic and fetal hemopoietic sites. CFU-S 14 activities were measured in the AGM and the YS on day 10 and the fetal liver on day 14 of Ikaros DN −/− embryos. CFU-S 14 activity was depleted in all three sites in Ikaros DN −/− embryos. The effects of Ikaros mutation on fetal hemopoietic precursors closely match those seen postnatally in the BM and spleen. Depletion of fetal precursors in the Ikaros mutant embryos supports the idea that Ikaros regulates the production of hemopoietic progenitors during ontogeny of the hemopoietic system. Molecular analysis of lin − hemopoietic populations in Ikaros mutant mice revealed a severe reduction in expression of two tyrosine kinase receptors important for HSC development. Surface expression of c-kit is progressively reduced from Ikaros null to Ikaros DN −/− hemopoietic cells, and mRNA expression of flk-2 is missing in both mutant populations. Flk-2 was originally identified as a tyrosine kinase receptor expressed in fetal liver populations enriched for HSCs 33 . It has previously been reported that flk-2 null BM has reduced competitive repopulation activity against wild-type BM, suggesting a potential depletion in HSCs 30 . In addition, the number of B cell precursors in the flk-2 null mice is reduced. Both flk-2 hemopoietic phenotypes correlate with those observed in Ikaros mutants. However, hemopoietic progenitors in Ikaros mutant mice appear to be more severely reduced in number, and B cell precursors are absent. It is possible that lack of the flk-2 tyrosine kinase receptor, compounded with a reduction in levels of cell surface c-kit, and possibly other unidentified factors, which are also required at the early stages of B, T, and HSC differentiation, may account for the more severe HSC and B cell phenotypes manifested in Ikaros mutant mice. Indeed, flk-2 and c-kit double mutants display a far more severe hemopoietic phenotype than that manifested by either mutations alone, resulting in early lethality 30 . It is also significant that the difference in c-kit expression between Ikaros null and DN −/− progenitors directly correlates with the severity in HSC defect manifested in these mutant mice. Expression of the T cell–determining transcription factor, GATA-3, is missing from the Ikaros DN −/− mice but is present among the Ikaros null lin − hemopoietic populations. GATA-3 expression, or lack thereof, correlates with the T cell differentiation potential of Ikaros mutants. Interference from the Ikaros DN isoforms toward the activity of other Ikaros-interacting factors expressed in the early hemopoietic progenitor compartment may impair GATA-3 expression in these cells and block their T cell differentiation potential. Finally, increase in expression of the myeloid-specific GM-CSF receptor within Ikaros mutant early hemopoietic progenitors may underlie the apparent expansion of the myeloid precursors and their progeny despite the dramatic reduction in hemopoietic progenitor numbers. The progressive depletion in long- and short-term hemopoietic progenitors and T cell precursors from the Ikaros null to DN −/− mice supports a role for Ikaros and its interacting factors 21 in the development and differentiation of HSCs. Ikaros proteins are all engaged in a higher order complex with family members and components of two distinct chromatin remodeling complexes 21 . The proper nuclear compartmentalization and gene targeting of these chromatin remodeling complexes manifested in the presence of Ikaros and its family members are likely to be as critical for differentiation and homeostasis of the hemopoietic system as the Ikaros protein itself. The progressive depletion in long- and short-term hemopoietic progenitors and T cell precursors from the Ikaros null to DN −/− mice may reflect progressive mistargeting of these remodeling complexes in the nuclei of HSCs in the absence of Ikaros and through DN interference with the activity of its family members, the expression of which is not affected by the Ikaros mutations (data not shown). Ikaros remodeling complexes may control hemopoietic lineage commitment decisions by potentiating expression of genes that include at least the tyrosine kinase receptors flk-2 and c-kit and possibly the transcription factor GATA-3, required for differentiation along the B and T cell lineages. We have recently shown that Ikaros proteins provide T cell activation thresholds 34 , possibly by regulating changes in chromatin structure 21 . Ikaros may thus control HSC activation by providing thresholds to signaling pathways for c-kit and other receptors. In the absence of Ikaros , the signaling thresholds for these pathways may be lowered, allowing HSCs and their progeny to enter the cell cycle more readily and possibly deplete the most primitive quiescent stem cell pool. Further studies on the cycling status of Ikaros mutant HSC and the effects of cell cycle–promoting factors on the hemopoietic compartment of Ikaros mutant mice will address this central question of self-renewal. | Study | biomedical | en | 0.999999 |
10544194 | Female, 8–10-wk-old mice were used in all experiments. C57BL/6 (B6) mice were obtained from Taconic Farms. (B6 × 129/Sv) F1 (F1) mice were obtained from The Jackson Laboratory. CD1 KO mice were generated in the Transgenic Facility, Harvard Medical School, Boston, MA (Exley, M., manuscript submitted for publication). In brief, the CD1 (both CD1.1 and CD1.2) mutation was created in strain 129/Sv-derived embryonic stem (ES) cells. Mutant ES cell clones were injected into B6 blastocysts to obtain chimeric mice. Heterozygous mutant animals were intracrossed in brother–sister mating to obtain (B6 × 129/Sv)F2 (F2) homozygous mutants. In most cases, control wild-type (WT) mice were F2 mice (The Jackson Laboratory), but F1 cells were used as WT in reconstitution experiments. A confirmatory experiment was performed in B6 CD1 KO mice. During the time the experiments were performed, the CD1 mutation was being backcrossed to the B6 parent for five generations (N5). Progeny that lacked the CD1 gene as determined by DNA analyses were chosen for breeders. The animals were maintained on food and water ad libitum until they reached the desired weight (20–24 g). All animals were treated humanely and in accordance with the Schepens Eye Research Institute–Boston Biomedical Research Institute Animal Care and Use Committee and National Institutes of Health guidelines. ACAID was induced in mice by inoculating OVA (50 μg/2 μl in HBSS; Sigma Chemical Co.) into the ac 10 7 d before sensitizing subcutaneously for DTH. Intravenously induced immune deviation was induced by inoculation of the antigen (OVA, 50 μg/100 μl in HBSS) into the tail vein with a 30-gauge needle 7 d before immunizing for DTH. To induce DTH, mice received a subcutaneous inoculation with OVA (100 μg/ml in HBSS, 50 μl) emulsified in CFA (50 μl) and 1 wk later were tested for the development of DTH by an intradermal inoculation of OVA-pulsed peritoneal exudate cells (PECs; 2 × 10 5 /10 μl HBSS) into the right ear pinnae. Ear swelling was measured 24 and 48 h later with an engineer's micrometer (Mitutoyo/MTI). To test for the efferent-regulatory cell of ACAID, a modified local adoptive transfer (LAT) assay was performed as described elsewhere 14 . In brief, T (effector) cells were generated in B6 mice or F1 3 4 5 by immunizing subcutaneously with OVA in HBSS and CFA. 7 d later the primed T cells were enriched from dissociated spleen cells by removing B cells and macrophages using IMMULAN™ columns . Regulator cells were similarly enriched on IMMUNLAN™ columns from spleen cells of ACAID mice 7 d after ac inoculation of OVA. Stimulator cells were OVA-pulsed PECs as described below. Effector (5 × 10 5 ), stimulator (5 × 10 5 ), and regulator (5 × 10 5 ) cells were mixed and resuspended in 10 μl HBSS for intradermal inoculation into the right ear pinnae of naive mice. Ear swelling was measured with an engineer's micrometer at 24 and 48 h. As a negative control, naive T cells from nonmanipulated mice were used as effector cells and regulator cells. Primed T cells were used as effector cells, and naive T cells from nonmanipulated mice were used as regulator cells for positive control. PECs were obtained from peritoneal washes of mice 3 d after they received an intraperitoneal inoculation of 2.5 ml of 3% aged thioglycolate solution (Sigma Chemical Co.). After counting, PECs were cultured with OVA (5 mg/ml) in a 24-well culture plate in serum-free medium (RPMI 1640 medium, 10 mM Hepes, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin (BioWhittaker), and supplemented with 0.1% bovine serum albumin (Sigma Chemical Co.), ITS + culture supplement (1 μg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na 2 Se, and 0.2 μg/ml Fe(NO 3 ) 3 ; Collaborative Biomedical Products). Nonadherent cells were removed from the cultures after 18 h by three washes, and the remaining adherent cells were collected by vigorous pipetting with cold medium (4°C) before washing (three times in HBSS) to remove free OVA. The Abs used for flow cytometry analysis were as follows: Fc Block™ (anti–mouse FcRγ II/III mAb, 2.4G2), biotin or FITC-conjugated anti-NK1.1 mAb (PK136), biotin-conjugated anti-Ly49C (5E6), biotin-conjugated anti-CD1 mAb (1B1), FITC-conjugated anti-CD3 mAb (145-2C 11 ), CyChrome 5–conjugated anti–TCR-β mAb (H57-597) were all purchased from PharMingen. PE-conjugated anti-B220 mAb (RA3-6B2) and PE-conjugated Mac-1 (M1/70.15) were purchased from CALTAG Laboratories. Rabbit antiasialo GM1 Ab (RαAsGM1) was purchased from Wako Chemicals USA, Inc.; streptavidin-PE was purchased from Jackson ImmunoResearch Labs, Inc. and FITC-conjugated goat anti–rabbit IgG was purchased from Sigma Chemical Co. The Abs used for in vivo treatment were as follows: anti-NK1.1 mAb (PK136, mouse IgG2a) and anti-Ly49C (5E6, mouse IgG2a) were purified from mouse ascites using protein A column chromatography (GIBCO BRL) in our laboratory. Purified mouse IgG was purchased from Sigma Chemical Co. for use as control for anti-NK1.1 mAb and anti-Ly49C. RαAsGM1 was purchased from Wako Chemicals USA, Inc., and purified rabbit IgG was purchased from Sigma Chemical Co. Anti-CD1 mAb (3C11, rat IgM) was also purified from mouse ascites using protein A columns. Purified rat IgM isotype control (R4-22) was purchased from PharMingen and used as control for anti-CD1 mAb. Splenic NK and NKT cells were analyzed by flow cytometry. RBCs were lysed by adding Tris-buffered ammonium chloride to a cell pellet of spleen cells. Staining was performed in the presence of saturating concentration of Fc Block™ (blocks FcRγ II/IIIs). Cells were stained with the following three reagents and colors (using concentrations recommended by the manufacturer): biotin-conjugated anti-NK1.1 mAb counterstained with streptavidin-PE; CyChrome 5–conjugated anti–TCR-β chain mAb; and FITC-conjugated anti-CD3 mAb. In some experiments the cells were stained with RαAsGM1 Ab and counterstained with FITC-conjugated goat anti–rabbit IgG and with CyChrome 5–conjugated anti–TCR β chain mAb. Stained cells were analyzed on an EPICS XL flow cytometer (Coulter). The absolute number of splenic NKT cells detected in flow cytometry was calculated from the percent of NKT cells in the number of viable cells. The total number of viable cells harvested from the spleens before staining was determined by the trypan blue exclusion method. To deplete NK cells in vivo, 100 μl of PBS containing one of the following RαAsGM1 (250 μg), mouse anti-NK1.1 Ab (50 μg), rabbit IgG (250 μg), or mouse IgG (50 μg) was injected into the tail vein of B6 mice. To deplete both NK and NKT cells in vivo, a mixture of anti-NK1.1 mAb and anti-Ly49C (50 μg + 50 μg) or mouse IgG (100 μg) was injected into the tail vein of B6 mice. 24 h later, spleen cells from Ab-treated animals were monitored for the presence of NK or NKT cells by flow cytometry using Abs that detected an NK marker that was different from the target of the Ab used in the depletion treatment. 24 h after the cell depletion treatments, B6 mice were inoculated ac with OVA (50 μg/2 μl). 7 d after ac inoculation, the ability to suppress a primed DTH response was tested in a LAT assay. Before enriching for the regulator T cells from spleens from the ac-inoculated mice, the NK and NKT cells were monitored again to confirm their absence. Purified anti-CD1 mAb (3C11) or control rat IgM mAb (50 μg in 100 μl PBS) was injected into the tail vein of B6 mice to block the interaction of NKT cells with CD1. It is reported that 3C11 blocks the NKT cell–CD1 interaction in vitro 31 . Flow cytometry studies of spleen cells from the 3C11-treated mice confirmed that the CD1 + cells (biotin-conjugated anti-CD1 mAb [1B1] counterstained by streptavidin-PE) were neither depleted nor showed changes in the populations of T cells (FITC-conjugated anti-CD3 mAb), B cells (PE-conjugated anti-B22 mAb), NK and NKT cells (triple staining: FITC-conjugated anti-CD3 mAb, CyChrome 5–conjugated anti–TCR β chain mAb, and biotin-conjugated anti-NK1.1 mAb counterstained by streptavidin-PE), and macrophages (PE-conjugated Mac-1) (data not shown). After RBC lysis, spleen cells were treated with FITC-conjugated anti-NK1.1 mAb, biotin-conjugated anti-Ly49C, and MicroBeads-conjugated anti–mouse pan-NK cells (DX5) (Miltenyi Biotec), and washed twice in PBS (pH 7.2) containing 0.5% BSA and 2 mM EDTA. Ab-labeled cells were treated with anti-FITC MicroBeads and streptavidin MicroBeads (Miltenyi Biotec) for 15 min, and washed twice. To harvest NK and NKT cell–enriched and depleted populations, cells were applied to Type MS + positive selection column with MiniMACS (Miltenyi Biotec). Cells were stained with Cy-Chrome 5–conjugated anti–TCR β chain mAb, and depletion or enrichment was confirmed by flow cytometry. Cell numbers of depleted populations were adjusted to approximate the number used in the control studies. For reconstitution experiments, CD1 + cells were depleted from the spleen cells. Following RBC lysis, column-enriched splenic T cells were incubated with biotin-conjugated anti-CD1 (1B1) and then treated with streptavidin MicroBeads before they were applied to Type MS + positive selection column with MiniMACS. The CD1 + cell–depleted population in the effluent wash was counterstained by streptavidin-PE (Jackson ImmunoResearch Labs, Inc.) and analyzed by flow cytometry to confirm the quality of the depletion technique. CD1 KO mice were γ-irradiated (cesium, 500 rad, Mark 1 irradiator; J.L. Shepherd and Associates) 1 d before receiving 2 × 10 7 /mouse whole spleen cells derived from F1 mice or spleen cells depleted of their NK1.1 + cells by magnetic beads. 7 d later, reconstituted CD1 KO mice were inoculated (ac) with OVA (50 μg/2 μl in HBSS). Spleens were removed 1 wk after the ac inoculation, dissociated cells were pooled, and splenic T cells were enriched as described above. Enriched splenic T cells were transferred to naive F1 mice as regulator cells with effector (derived from F1 mice) and stimulator cells (derived from F1 mice) prepared as described above and tested in a LAT assay. Any host versus graft disease that might have occurred was undetectable and did not interfere with the experimental outcome 15 d after reconstitution. Data were subjected to analysis by analysis of variance and Scheffe's test. A value of P ≤ 0.05 was considered significant. ACAID is a well-established experimental animal model for the study of immune deviation mediated through an immune-privileged site. Modulation of the model and analysis of the subsequent effects on the immune deviation–associated systemic tolerance can be assessed directly in the whole mouse, or in a LAT assay 14 for detection of antigen-specific regulatory T cells associated with ACAID. The postulate that NKT cells are important in ACAID was first assessed by looking for an ACAID-associated increase of NKT cells in the spleen. B6 mice were inoculated (ac) with OVA, and 7 d later the spleens were extirpated, cells dissociated, and the numbers of NK and NKT cells were analyzed by flow cytometry after staining for the TCR β chain and the NK1.1 molecule. Analysis was performed on five individual mice per group. The flow cytometry data showed that the ratio of NKT cells to total gated lymphocytes from spleens (depleted of B cells and macrophages) was increased in all ACAID mice compared with naive mice , subcutaneously or intravenously inoculated mice (data not shown) at 7 d after ac inoculation. In contrast to NKT cells, the number of NK cells did not change in the spleen during ACAID induction. Both the percent and the absolute number of NKT cells in the spleen began to increase as early as day 3 (data not shown), and peaked at day 7 . These data show an association of splenic accumulation of NKT cells, not NK cells, with the induction of ACAID. To directly test the involvement of the NKT cells in the development of ACAID, we examined the potential of CD1-deficient mice to develop immune deviation after ac inoculation of antigen. Since the CD1 molecule is essential for NKT cell development 30 31 , CD1 KO mice do not have NKT cells, but do have NK cells and other lymphocyte subpopulations 32 33 34 . CD1 KO mice and control WT mice were inoculated (ac) with OVA 7 d before subcutaneous sensitization with OVA and CFA, and challenged into the ear pinnae with OVA-pulsed PECs 7 d after subcutaneous sensitization . When ear swelling was measured 24 and 48 h later, it was observed that ac-inoculated CD1 KO mice developed a positive DTH (ear swelling) response, but ac-inoculated WT mice exhibited a suppressed response . While the level of the DTH response in CD1 KO mice was similar to that in WT mice, CD1 KO mice did not develop ACAID. To further assess the NKT cell-dependent ACAID mechanism, spleen cells from ac-inoculated CD1 KO and WT mice were tested in a LAT assay . In brief, CD1 KO mice were inoculated (ac) with OVA 7 d before harvesting, dissociating, and enriching T cells from the spleens for use as regulator cells. Regulator T cells were then cotransferred with OVA-primed T cells (effector cells from F1 mice) and OVA-pulsed PECs (stimulator cells from F1 mice) into the ear of F1 mice. In contrast to regulator cells from WT mice, regulator T cells from CD1 KO mice that received OVA were unable to suppress the DTH response . An experiment was performed in B6 CD1 KO mice (hatched bars) to confirm the role of NKT cells in ACAID in a genetically homogeneous background. In this case, effector and stimulator cells were prepared from B6 mice, and then cells were transferred to the B6 recipient. These results show that NKT cells or other CD1-dependent populations were needed for the development of ACAID in general, and for the generation of the antigen-specific efferent regulatory T cells, in particular. To confirm whether the defect in the CD1 KO mice that led to the failure of ACAID was actually the NKT cell deficiency, we reconstituted CD1 KO mice with whole spleen cells from WT mice (F1) (containing both NKT cells and CD1 + APCs), or spleen cells immunomagnetically depleted of NK1.1 + lymphocytes (that still contained CD1 + APCs). The successful depletion of NKT and NK cells was confirmed by flow cytometry analysis . 7 d after reconstitution, the mice were inoculated (ac) with OVA, and after 8 d spleen cells were harvested for T cell enrichment. A typical profile of enriched splenic T cells from reconstituted CD1 KO mice shows that ∼4.9% of T cells were donor derived (CD1 + ). (Total of CD1 + cells in the non-T splenic cells was 25%; data not shown.) To analyze the regulatory potential of the host CD1 − T cells, the spleen cells were further negatively selected against CD1 expression to yield populations that were CD1 − . These cells were then assessed as regulator cells in the LAT assay. We observed that whole spleen cells were able to reconstitute the ACAID-inducing ability of the CD1 KO mice. In contrast, spleen cells depleted of NKT cells did not restore ACAID-inducing ability . Because CD1 is expressed on all WT bone marrow–derived cells that were transferred, these results also show that antigen-specific-efferent regulatory cells that were generated did not express CD1, indicating that the transferred CD1 + NKT cells were not the effector cell in the regulation of the expression of DTH. The data support the postulate that NKT cells are essential in the induction of ACAID, and function by supporting the generation of antigen-specific efferent-regulatory T cells. To confirm that NKT cells do not have a direct role in efferent regulation of DTH in another way, we depleted cells expressing NK1.1 antigens from the regulator cell population before testing in a LAT assay using specific Ab treatment and magnetic beads selection . Treated cells were then assessed by flow cytometry before cotransferring with primed T cells and OVA-pulsed PECs to the ear pinnae. The results of the LAT assay showed that the NK and NKT cell–depleted populations retained their DTH-regulatory capability. Thus, the efferent-regulatory cell is a conventional NK1.1 − T cell. It is known that activated NKT cells can downregulate their NK1.1 molecules in vitro 35 . Therefore, we were aware that the in vivo–activated NKT cell may not express NK1.1, and the Ab depletion of NK1.1 + cells treatment may not work. However, as also shown in Fig. 1 , after OVA was inoculated in the ac NK1.1 + T cells were clearly present in the spleen. Therefore, together with our observations that CD1 − T cells (from CD1 + NKT cell–reconstituted CD1 KO mice) can function as regulators of DTH , these data confirm that NKT cells are not direct efferent regulators of DTH in ACAID. Another approach to assess the importance of NKT cells in induction of systemic tolerance used Abs specific for NK markers to create CD1 + mice with selective depletion of the NK1.1 + cell populations. B6 mice were depleted of NKT cells and NK cells, or NK cells only, and their ability to generate the efferent regulatory T cell in ACAID was compared to syngeneic mice treated with isotype control Ab. We reported previously that it is difficult to remove the NKT cell population in vivo with Abs to typical NK cell markers 36 . Specifically, the inoculation of mice with anti-NK1.1 mAb or RαAsGM1 Ab depletes NK cells, but not NKT cells, although they both express the target antigens . Because the NKT cells that remained in the spleens after in vivo anti-NK1.1 mAb treatment expressed Ly49C 36 , we mixed the anti-NK1.1 and anti-Ly49C Abs and effectively depleted NKT cells in the mice for these studies . 24 h after the mixed Ab treatment, mice were inoculated (ac) with OVA, and the differently treated groups of mice were tested 1 wk later for their ability to generate efferent-regulatory T cells in a LAT assay. As expected, the NK-only depleted mice, previously treated with either RαAsGM 1 or anti-NK1.1 mAb, developed antigen-specific efferent-regulatory T cells , but the NKT and NK cell–depleted mice did not . Therefore, together with studies in the CD1 KO mice , these data show that the CD1-dependent cell responsible for the development of systemic tolerance and the generation of regulatory T cells in ACAID is the CD1-dependent NKT cell. Clearly CD1 is needed for the development of NKT cells, but it is unknown if CD1 is required for NKT cell function in the generation of regulatory T cells in ACAID. We reasoned that if NKT cell interactions with CD1 were required for ACAID induction, we might be able to block ACAID by blocking the NKT cell interaction with CD1. Previously, anti-CD1 mAbs (3C11) were successfully used in vitro to block NKT cell–CD1 interactions 31 . Therefore, mice were treated with anti-CD1 mAb 1 d before being inoculated (ac) with OVA and 8 d before harvesting the spleens, dissociating the cells, and column-enriching the splenic T cells for use as regulator cells in a LAT assay. 24 h after anti-CD1 treatment (3C11), dissociated spleen cells were stained with a different Ab for CD1 (1B1) to assess the presence of CD1 + cells in the spleen. Flow cytometry analyses showed that the Ab treatment did not alter the ratio or absolute number of CD1 + cells. In addition, anti-CD1 mAb treatment of mice did not alter the ratio or absolute number of NKT and NK cells (stained by anti–TCR-β and anti-NK1.1), T cells (stained by anti-CD3), B cells (stained by anti-B220), and macrophages (anti–Mac-1) in their spleens (data not shown). Groups of mice treated with control Ab developed antigen-specific efferent-regulatory T cells, but mice treated with anti-CD1 mAb did not . Because the Ab treatment did not eliminate CD1 + cells, the most likely explanation is that an interaction between NKT cells and CD1 was blocked by the anti-CD1 mAb. Thus, we postulate that an interaction between the NKT cell and the CD1 molecule is required for the NKT cell to function in ACAID. Since antigen inoculated into the ac is carried to the spleen, an argument could be made that there are few differences between the induction mechanisms of ACAID and the mechanisms involved in intravenously induced systemic tolerance. In fact, it could be argued that the leakage of antigen from the venules in the eye into the blood during ACAID induces intravenous tolerance. However, differences between the mechanisms involved in immune deviation via the ac or intravenous route have been reported 13 . It has also been reported that intravenously induced tolerance generates CD8 + afferent-regulatory T cells in contrast to ACAID, which generates both CD4 + afferentregulatory T cells and CD8 + efferent-regulatory T cells 14 . CD1 KO mice and WT mice were inoculated ac or intravenously with OVA 7 d before subcutaneous sensitization with OVA and CFA and testing for DTH by challenging 14 d later with OVA-pulsed PECs into the ear. As before, ear swelling was measured 24 and 48 h later. As expected, WT mice developed immune deviation regardless of the route of inoculation . Importantly, in contrast to the inability of CD1 KO mice to develop immune deviation when inoculated ac, intravenously treated CD1 KO mice were fully capable of developing immune deviation, and showed reduced ear-swelling responses . Therefore, NKT cells do not participate in intravenously induced immune deviation, and ACAID is indeed a separate entity, with unique and locally maintained mechanisms of regulation. This report shows that NKT cells (a) are required for the development of ACAID; (b) are specifically required for the generation of an efferent-regulatory T cell; (c) may interact with a CD1-expressing cell to mediate regulatory mechanisms for suppressing DTH responses in ACAID; and (d) are not required for intravenous tolerance. In addition, a relationship between ACAID and self-tolerance is suggested by reports that induction of ACAID in mice both prevented the onset of and also suppressed ongoing experimental autoimmune uveitis 37 . Furthermore, while several previously published reports imply a role for NKT cells in preventing certain autoimmune disease in humans 38 39 and in mice 20 40 41 , the mechanisms through which NKT cells might regulate autoimmunity remain unclear. Therefore, these results indicate that the autoimmunity associated with NKT cell defects (such as type 1 diabetes, scleroderma) may be mediated by disruption of organ-specific tolerance mechanisms that are similar to those mediating systemic tolerance to antigens introduced through immune-privileged sites. The systemic tolerance to antigens introduced into the ac (ACAID) involves several steps. In the initial step, antigen introduced into the ac is carried to the spleen by specialized eye-derived F4/80 + APCs 42 . A CD8 + efferent or effector stage regulatory T cell that can suppress a subsequent DTH response to the specific antigen is then generated. This study further shows that CD1-reactive NKT cells are required for the generation of the ACAID regulatory T cell. On the other hand, the LAT studies of CD1 KO mice reconstituted with CD1 + NKT cells show that CD1d expression by ACAID efferent-regulatory cells was not necessary . Anti-CD1 blocking experiments nonetheless indicate a role for CD1, presumably functioning as a ligand for NKT cells in ACAID. Therefore, we suggest that the critical CD1-dependent interaction is between the NKT cell and the specialized ac-derived APC, and that NKT cell interactions with particular APCs may similarly be defective in some autoimmune diseases associated with loss of NKT cell function. The ac contains aqueous humor that is filled with a mixture of immunosuppressive components including TGF-β 3 4 5 6 43 . Moreover, we observed that addition of TGF β2 to thioglycolate-induced PECs in vitro induced an increased expression of their CD1d molecules (Sonoda, K.-H., and J. Stein–Streilein, unpublished observations). Thus, the eye-derived APCs may similarly express increased levels of CD1, and thereby stimulate NKT cells in the spleen. Alternative, and not mutually exclusive, hypotheses are that NKT cells are activated by a CD1-presented endogenous lipid antigen, accessory molecule, or cytokine produced by the APC. A further alternative is that the NKT cells recognize CD1d expressed by another cell type in the spleen. Such a possible CD1 + NKT cell stimulator in the spleen is the marginal zone B cell. Niederkorn and colleagues showed that splenic B cells are needed for ACAID, and suggested that eye-derived APCs “hand over” their antigen and allow the splenic B cells to induce ACAID 44 45 . Consistent with this hypothesis is the observation that the APCs in the spleen that express the highest density of CD1 are the marginal zone B cells 46 . Activated NKT cells may produce large amounts of a variety of cytokines 35 that, in the splenic microenvironment, likely contribute to the development of the efferent-regulatory T cell in ACAID. Cytokines, such as IL-4, may directly modulate activity of the efferent-regulatory cell. While IL-4 is most commonly thought to mediate NKT cell–dependent T cell regulation, we do not propose it to be the critical cytokine in NKT cell–dependent generation of the efferent-regulatory T cell since ACAID occurs in IL-4 KO mice 16 47 48 . Another potentially important cytokine, already commonly associated with the development of ACAID, is TGF-β 43 . A report has been published showing NKT cell production of TGF-β 49 , and another shows that TGF-β may modulate APC function 50 . Thus, the possibility arises that NKT cells respond to the CD1 or other signals by upregulating TGF-β production or its conversion from latent to active form. Strengthening this possibility is a recent report by Kosiewicz et al. showing that both CD4 + CD8 − and CD4 − CD8 − (double negative, DN) T cells from ACAID spleens produced TGF-β 16 . NKT cells are notably either CD4 + or DN 22 . A significant technical observation in this study is that in our hands NKT cells are resistant to in vivo antibody treatments known to remove NK cells. While others report removal of NKT cells by anti-NK1.1 mAb 51 52 , we could only eliminate NK cells and not NKT cells with either anti-NK1.1 mAb or RαAsGM1 Ab alone. However, when we used a mixture of Abs, we effectively eliminated NKT cells as well as NK cells. These results were not surprising, as we previously reported that while activating apoptosis in NK cells, the NK antigen–specific (anti-NK1.1) Ab treatment activated IL-4 synthesis in NKT cells 36 . In our laboratory, when the same Ab was used to label cells for flow cytometry that was used for elimination, we could not find the cells because the antigen was either masked or downregulated. Thus, we consistently used different mAbs for depletion studies and the flow cytometric analyses. In contrast to ACAID, intravenous tolerance could be induced in CD1 KO mice. The relationship between intravenous tolerance and ACAID is unclear, but intravenous tolerance does differ from ACAID in several respects. The intravenous administration of antigen cannot suppress the immune response in previously immunized hosts, whereas presentation of antigen via the ac does downregulate ongoing DTH responses in previously immunized hosts 53 . Moreover, ACAID is mediated by both CD4 + afferent regulatory T cells and CD8 + efferent regulatory T cells 13 , whereas intravenously induced tolerance only required CD8 + afferent-regulatory cells 14 . (In fact, because intravenous tolerance mechanism does not require an efferent-regulatory cell, we could not test the intravenous tolerance capability of CD1 KO mice in a LAT assay.) It is proposed that intravenous tolerance reflects the intrinsic response of T cells to antigen in the absence of costimulatory molecules, and therefore may not require additional cells 54 . In contrast, this report clearly shows that ACAID involves interactions between multiple cells, a process that may be necessary in order to suppress established immune responses. A recent paper by Zeng et al. suggests that NKT cells may be responsible for assisting the generation of antigen-specific regulatory T cells in the bone marrow, perhaps to regulate immune responses to self-antigens displayed on other bone marrow–developing cells 55 . Thus, it seems likely that CD1-reactive NKT cells may not only be unique regulators of self-reactivity in immune privileged sites (ACAID), but also may contribute to self-tolerance through regional specialization in a variety of organs, tissues, and microenvironments in general. | Study | biomedical | en | 0.999994 |
10544195 | The panel of HLA-A2 binding peptides derived from hepatitis B virus (HBV) and hepatitis C virus (HCV) sequences has been described previously 28 29 . Peptide HBV pol 575-83 (FLLSLGIHL) and its variant 573-83, HBV core 18-27 (FLPSDFFPSV) and its variants 17-27 and 18-28, reporter peptides HLTV-I tax 11-19 (LLFGYPVYV), HIV gag 77-85 (SLYNTVATL), R-9-L (RRYNASTEL), R-10-T (RYWANATRST), S-9-R (SRYWAIRTR), S-11-R (SRYWNATIRTR), and F-10-V (FLPSDYFPSV) were all obtained >96% pure from Genosys . HBV epitope variants pol 575-87, 573-87, and core 17-28 were synthesized with >95% purity by the peptide synthesis facility of the Scripps Research Institute. Hybridomas BB7.2 with specificity for HLA-A2 and IVD12 recognizing HLA-DQ were purchased from American Type Culture Collection (ATCC). HLA-A2–expressing B cells used in this study were provided by the Centre pour l'Etude du Polymorphisme Humain (Paris, France) or A. Toubert (Hôpital St. Louis, Paris, France) and included homozygous lines Jesthom and JY and mutant line T2. RMA-S cells transfected with “HHD” HLA-A2/K d molecules were grown with 1 mg/ml gentamycin and 10 −5 M β-ME as described 30 . Peptide binding to human TAP complexes was measured as described 8 using reporter peptide R-9-L at 300 nM. Human B cell lines were washed once in translocation buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl 2 , 2 mM EGTA, 2 mM MgCl 2 , 2 mM dithiothreitol, and 5 mM Hepes, pH 7.3) and permeabilized for 5 min at 37°C with 1 IU/ml streptolysine O before addition of reporter and competitor peptides and ATP (2 mM). After incubation for 20 min at 37°C, cells were lysed by addition of 800 μl cold lysis buffer (500 mM NaCl, 50 mM Tris, 5 mM MgCl 2 , and 1% Triton X-100, pH 7.4) with a cocktail of protease inhibitors. After a 30-min incubation on ice, lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C, and supernatants were recovered for affinity purification of HLA-A2 molecules or glycosylated peptides. These assays were carried out as described by Neefjes et al. 31 using 1.5 × 10 6 cells in a volume of 100 μl and 160 nM iodinated reporter peptide. Reporter peptides R-10-T or S-11-R were used. Glycosylated reporter peptides were recovered by 2-h incubations of cleared supernatants with 100 μl Con A–Sepharose beads at 4°C. Beads were washed four times with assay buffer before counting of bound radioactivity. 100% accumulation corresponded to ∼15,000 cpm. Based on published methods 32 , 5 × 10 6 B cells/sample in 300 μl buffer with 130 nM reporter peptide were used. Reporter peptide S-9-L was used in TAP-proficient cells, and L-9-V was used in TAP-deficient cells. For TAP-independent peptide translocation, no ATP was added. After the incubation period, cells were pelleted to remove excess reporter peptide, washed in 800 μl buffer, and lysed. HLA-A2 molecules were then immunoprecipitated for 20 min (4°C) with 10 μg mAb BB7.2 preadsorbed on 15 μl protein A–Sepharose beads. 100% accumulation corresponded to ∼15,000 cpm. Peptides were iodinated by the chloramin T method using 12 nmol peptide, 0.5 mCi 125 I, and 0.5 mg/ml chloramin T (Merck) for peptides F-10-V, S-9-L, and L-9-V. For R-9-L, R-10-T, and S-11-R peptides, 1 mCi 125 I and 2 mg/ml chloramin T were used. Free iodine was removed on Sephadex G10 columns. For peptides S-9-L and L-9-V, 30% dimethylformamide was added to PBS labeling buffer. Specific activity of iodinated peptides was between 2.5 and 6 × 10 4 cpm/pmol. In competition experiments, cpm corresponding to 50% inhibition of specific peptide binding to TAP or of accumulation of glycosylated or A2-bound peptide in the ER was determined graphically as described 8 . Nonspecific binding, i.e., binding of iodinated peptide to control TAP1 microsomes, or peptide recovered upon incubation of Con A or BB7.2 beads with lysates of nonpermeabilized cells was <2% of specific binding in the absence of competitor unless indicated otherwise. Purified mAb BB7.2 (or glycine at 10 mM) was coupled to cyanobromide-activated Sepharose 4B beads at a ratio of 7.5 mg mAb/ml resin. 3 × 10 9 Jesthom cells were lysed for 1 h at 4°C in 60 ml lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, and a cocktail of protease inhibitors. Clarified lysate was precleared by incubation with 900 μl glycine–Sepharose beads for 3 h and incubated overnight at 4°C with 175 μl BB7.2–Sepharose (corresponding to 1.3 mg mAb). Beads were washed with a series of cold lysis buffers (5 ml each, with 0.1–0.5% Triton X-100) with pH 7.4, pH 8, and pH 9, and, finally, once with 1.8 ml of 250 mM NaCl, 0.1% Triton X-100, and 10 mM Tris, pH 9. HLA-A2 molecules were eluted twice with 500 μl of 50 mM triethanolamine, 150 mM NaCl, and 0.1% SDS, pH 11.5, and immediately neutralized by addition of 150 μl of 1 M Tris, pH 6.8. Eluates were dialyzed against PBS buffer containing 0.05% Triton X-100. HLA-A2 concentration in eluates was quantified in Coomassie-stained SDS-PAGE gels. To measure peptide binding to HLA-A2, we developed an assay derived by modification of published procedures 33 34 . 350 ng of HLA-A2 was incubated in a total volume of 20 μl PBS with 0.05% Triton X-100 and protease inhibitors with 0.5 μg human β 2 m, 3 μl iodinated reporter peptide F-10-V, and competitor peptides. After incubation for 2 d at 25°C, HLA-A2 molecules were separated from free peptide by size exclusion chromatography on Biogel P-30™ spun columns (Bio-Rad Labs.) equilibrated in PBS buffer containing 0.5% Triton X-100. Cpm eluting in the void volume corresponded to HLA-A2–bound peptide and was corrected by subtracting cpm leaking through columns loaded with peptide only. In the absence of competitor peptides, ∼40,000 cpm reporter peptide bound to HLA-A2 molecules. Three approaches were used to generate CTL lines specific for HBV epitopes: human lines primed in vitro, lines from HBV patients, and lines from HLA-A2–transgenic mice. A total of nine independent CTL lines was used for experiments shown and/or referred to in this paper: for HBV pol 575-83, two lines from in vitro immunization, and one from a patient; for HBV core 18-27, one line from in vitro immunization, two from patients, one from conventional and two from single chain–transgenic mice. As most CTL lines lost specificity upon freezing, it was not always possible to use the same line for subsequent experiments concerning one epitope; experiments were therefore generally repeated with different CTL lines. CTL lines were generated by coculture of PBMCs from healthy HLA-A2 + donors with cells from a second donor with a high proliferative response to tetanus toxoid in a transwell system (Costar Corp.) using a modification of published procedures 35 . In brief, PBMCs were seeded into 24-well plates at 4 × 10 6 cells/well in RPMI 1640 with 10% pooled human AB serum in the presence of 10 μg/ml HBV peptide, 1 μg/ml tetanus toxoid, and 10 6 PBMCs of the second donor in the inner well. Unlike in the published protocol, recombinant human IL-7 was added on day 3 at 50 U/ml. Cultures were restimulated weekly with autologous irradiated PBMCs prepulsed with peptide. Starting on day 7, cultures were maintained in 10 U/ml human IL-2 and 50 U/ml IL-7. Once CTL lines exhibited target cell lysis >30%, IL-2 concentration was increased to 50 U/ml. Almost 90% of peptide-specific CTL lines obtained by in vitro immunization did not recognize naturally processed peptide, a phenomenon already documented in previous reports 36 . CTL lines from HBV-infected patients were generated exactly as described previously 37 . D b and mouse β 2 m double-knockout mice expressing an HLA-A2.1/human β 2 m (HHD) single chain molecule 30 and conventional HLA-A2–transgenic mice 38 were injected in the regenerating anterior tibialis muscle of each leg (5 d after injection of cardiotoxin in the same sites) with 50 μg cesium chloride–purified plasmids, corresponding to HBV sequences encoding the pol protein or the core protein cloned into pcDNA3™ (Invitrogen Corp.) with a CMV promoter. 3 wk later, spleens were removed and spleen cells were restimulated with an equal number of irradiated LPS blasts prepulsed with 1 μM peptide; peptide stimulation was repeated weekly until specific cytotoxic activity was detected. Blasts were generated by culture of spleen cells from HHD mice for 3 d at 10 6 cells/ml in RPMI supplemented with 25 μg/ml yeast LPS and 7 μg/ml dextran sulfate. Selected peptides were expressed as minigenes under the control of the p7.5 promoter in vector pSC11ss modified by insertion of a sequence containing BglII, KpnI, NsiI, and NotI sites between the SalI and StuI sites. Complementary oligonucleotides comprising an ATG initiation codon and a stop codon with appropriate cohesive ends were purchased from Genosys, annealed by slow cooling from 70 to 30°C, and ligated overnight at 14°C to modified pSC11ss. To express epitopes preceded by a signal sequence, oligonucleotides corresponding to the signal peptide of adenovirus E3/19K protein 20 were cloned into the KpnI/NotI sites of modified pSC11ss, followed by insertion of minigenes coding for epitopes HBV pol 575-83 or core 18-27 into the NotI/StuI sites downstream. Oligonucleotides corresponding to HBV pol 575-83 and 573-83 were cloned into the SalI/StuI sites; for peptides HBV core 18-27, 17-27, and 17-28, SalI/NsiI sites were used. Insertion of correct minigene sequences was verified by sequencing. Recombinant viruses were generated by cotransfection of CV-1 cells freshly infected by viral strain WR (ATCC) at a multiplicity of infection (MOI) of 0.1–1.0 with plasmids containing minigenes together with 150 ng wild-type vaccinia DNA, followed by selection of recombinant viral plaques in HuTK cells and large-scale production of viruses in HeLa S3 cells according to standard procedures. Recombinant vaccinia viruses coding for the entire HBV polymerase and core proteins have been described previously 37 . Cytolytic activity was measured in 5-h 51 Cr-release assays using a standard protocol with 3,000 targets per well in RPMI 1640 supplemented with 5% FCS. When sensitization by recombinant vaccinia viruses was tested, 1.5 × 10 6 target cells were infected for 1.5 h at an MOI of 10 in medium with 2.5% FCS. Cells were washed once in 0.9% NaCl buffer, incubated for 3.5 h at 37°C in medium with 10% FCS, and then labeled with 100 μCi of Na 2 51 CrO 4 for 1 h at 37°C. Cells were washed in 0.9% NaCl with 2% FCS, incubated for 1 h at 37°C, and washed again. To test sensitization by synthetic or HLA-A2–eluted peptides, target cells were incubated for 1.5 h during or after cell labeling with peptides. In experiments with brefeldin A (BFA), target cells were incubated for 2 h at 37°C with 5 μg/ml BFA; BFA concentration was maintained at 5 μg/ml throughout sensitization by peptide or virus by two-hourly replacement of medium. BFA concentration during the 5-h killing assay was 0.5 μg/ml 39 . Percent specific lysis was calculated according to the formula, (experimental cpm − spontaneous release)/([total release/2] − spontaneous release); only experiments with <20% spontaneous release were considered. HLA-A2–bound peptides were analyzed using a modification of published methods 21 : 1.6 × 10 9 JY cells were infected for 2 h at 10 8 cells/ml and an MOI of 10 with vaccinia virus. After incubation for 6–8 h at 37°C, cells were harvested, washed once in cold PBS with 1 mM PMSF, and lysed as in the protocol for HLA-A2 purification. Clarified detergent lysate was precleared by incubation with 150 μl packed Sepharose beads coupled to mAb IVD12 (corresponding to 750 μg mAb) for 3 h and then incubated overnight at 4°C with 90 μl (i.e., 675 μg) BB7.2–Sepharose beads. IVD12 and BB7.2–Sepharose beads were washed with 10 bed volumes of 50 mM Tris, 150 mM NaCl, pH 8.0, with 0.5% NP-40 (Sigma Chemical Co.), then with 10 volumes of buffer without NP-40, and finally with 10 volumes of 10 mM Tris, pH 8.0. Material bound to beads was eluted by two 5-min incubations in three volumes of 0.1% TFA (Sigma Chemical Co.); eluted peptides were separated from higher molecular weight material by centrifugation through Centricon 10 (Amicon, Inc.) devices and finally dried by vacuum centrifugation. Dried eluate was resuspended in 100 μl 0.1% TFA and fractionated by reversed-phase HPLC using a SMART system (Pharmacia). μRPC C2/C18™ 2.1/10 columns were run in 0.1% TFA (solvent A) and 80% acetonitrile containing 0.081% TFA (solvent B). HPLC conditions were calibrated using synthetic peptides and chosen so as to allow maximal resolution of minimal epitopes and precursors. The following gradients were used: peptides HBV pol 575-83 and 573-83: 0–5 min, linear increase from 0% B to 48% B; 45–57 min, linear increase to 53% B; 57–65 min, linear increase to 90% B. Peptides HBV core 18-27 and 17-27: 0–40 min, linear increase from 0 to 38.8% B; 40–80 min, linear increase to 39.8% B; 80–90 min, linear increase to 90% B; a slightly different gradient was used in the experiment in Fig. 6 E. Flow rate was maintained at 100 μl/min, and fractions of 100 μl (peptides pol 575-83 and 573-83) or 250 μl (peptides core 18-27 and 17-27, HBV core protein) were collected. Fractions were dried in a vacuum centrifuge and resuspended in 50 μl PBS with 5% DMSO. Fractionated peptide elutions were tested using TAP-deficient T2 or RMA-S HHD cells preincubated for 36 h at 27°C to increase surface expression of HLA-A2 30 before pulsing with 5 μl of each fraction; in the experiment shown in Fig. 6 E, 20 μl of each fraction was used for pulsing. To establish a system in which the biological effect of human TAP selectivity and potential ER processing of TAP-translocated peptides could be studied, we sought to identify peptides with low transporter affinities that might enter the ER exclusively in an extended form. Having previously observed that ligands for certain HCI molecules, especially HLA-A2, frequently display low affinities for human TAP 8 14 , we tested a panel of peptides with high or intermediate HLA-A2 binding affinity 6 (50% inhibiting concentration [IC 50 ] of 0.47–400 nM) derived from HBV and HCV proteins for binding to human TAP complexes 40 . Among 34 tested peptides, 8 HBV peptides and 3 HCV peptides have been found to be antigenic in hepatitis patients and can therefore be naturally processed and presented to CTLs 29 37 . Relative affinities measurable in the TAP binding assay range from 0.1 to 3,000. 18/25 HBV peptides , and 5/8 HCV peptides (not shown) derived from HBV proteins had low (IC 50 >300) or unmeasurable TAP affinity. Thus, most peptides derived from HBV and HCV that bind to HLA-A2 and in many cases induce efficient CTL responses in vivo had low TAP affinities, raising the question of how these peptides obtain access to ER-resident HLA-A2 molecules for efficient presentation. Based on the criteria of strong antigenicity in vivo and low TAP affinity, we chose to study two HLA-A2–presented peptides with unmeasurable TAP affinity, HBV pol 575-83 and core 18-27. For each of the two HBV epitopes with very low TAP affinity, we designed precursor peptides by extending epitopes to the closest likely proteasome cleavage site 2 24 flanking their NH 2 and/or COOH termini in the natural protein sequence. Sequences of minimal and extended peptides are shown in Fig. 2 . Peptides extended by an Nt methionine residue were also tested, as such peptides may result from incomplete processing of minigene-expressed epitopes by cytosolic methionine aminopeptidase. We then proceeded to an in vitro evaluation of parameters that should affect efficiency of peptide presentation: HLA-A2 binding affinity, TAP binding and transport efficiency, and efficiency of TAP-independent ER access. To measure HLA-A2 binding affinity, we adopted a published competition binding assay with immunoaffinity-purified HLA-A2 molecules 33 34 . Measurable competitor peptide affinities (IC 50 ) range from 0.1 to 100. Simple extension of epitope pol 575-83 by naturally found residues at either end surprisingly did not change HLA-A2 binding affinity ; however, Nt addition of methionine or extension at both ends decreased affinity. In contrast, all modifications of core 18-27 strongly reduced A2 affinity; this result is in accordance with a previous study that identified this peptide as an optimal synthetic epitope 41 . TAP affinity, the second parameter likely to affect efficiency of presentation, was evaluated with the two available techniques, TAP binding and transport assays. Transport assays measure accumulation of glycosylated peptide in the ER of streptolysin O–permeabilized cells 31 , which is TAP and ATP dependent but also affected by other processes, such as peptide degradation and active depletion from the ER. TAP binding assays evaluate at low temperature the initial step of peptide binding to the transporter in which substrate is selected 9 40 . Largely equivalent results were obtained with the two assays. For both epitopes, Nt extension by one or two residues increased TAP affinity to low to intermediate levels (IC 50 of 57–372 in binding assay). Ct extension of the core epitope but not of pol 575-83 increased affinity, and combination of both extensions resulted in high TAP affinity of the core 17-28 variant (IC 50 of 1.1). Ct-extended variants (pol 573-87 and core 18-28 peptides) competed less efficiently in transport than in binding assays; this may be due to peptide modification by cytosolic carboxypeptidases or peptide degradation in transport assays. Thus, for both epitopes, Nt-extended variants and, for the core epitope, also a Ct-extended variant with higher TAP affinity could be identified. Some peptides can be presented by HCI molecules in a TAP- and signal sequence–independent fashion 42 . Moreover, some epitopes are presented with high efficiency in TAP-deficient T2 cells when expressed as minigenes 43 . To test whether the selected epitopes and their variants could enter the ER in a TAP-independent manner, we adopted a transport assay in which labeled reporter peptides bind to newly synthesized HCI molecules and can be recovered by immunoprecipitation of the latter 32 . To monitor TAP-independent peptide access to the ER, we used a human T cell lymphotrophic virus type 1–derived reporter peptide (L-9-V) with high binding affinity for HLA-A2 that has been shown to be presented efficiently in TAP-deficient cells when expressed as a minigene 43 ; assembly of L-9-V with HLA-A2 was measured in permeabilized T2 cells . The amount of L-9-V entering the ER and assembling with HLA-A2 decreased sevenfold in the absence of functional TAP complexes (not shown); addition of ATP did not increase recovery of HLA-A2–bound peptide, suggesting that TAP- and signal peptide–independent ER access is a relatively inefficient process that does not require ATP. As shown in Fig. 4 A, the two minimal epitopes and most single-extended precursor peptides competed for assembly of L-9-V with HLA-A2 in T2 cells and were therefore able to enter the ER in TAP-deficient cells; differences in IC 50 values were relatively small. Double-extended precursors did not compete at all. High amounts of three unrelated 9-mer peptides binding to HLA-B40 and the HLA-B27–binding peptide S-9-R, a peptide reported to enter the ER when expressed as a minigene in TAP-deficient cells 43 , also did not compete (not shown). This suggests that TAP-independent ER entry does not involve interaction with a saturable substrate binding site of a transporter or channel. As reporter and competitor peptides competed for TAP-independent ER access as well as binding to HLA-A2, it is possible that the observed differences were due to distinct HLA-A2 binding affinities. However, measured A2 affinities did not fully explain competition efficiencies in TAP-independent transport assays, so that distinct efficiencies of ER access are likely to contribute to experimental results. Thus, TAP-independent peptide entry into the ER of permeabilized cells displayed relatively low efficiency as well as selectivity and appeared not to correspond to an active process, as indicated by lack of ATP requirement and absence of sensitivity to competition. We finally tested peptides in an in vitro assay that is likely to reflect the combined effect of the parameters evaluated above. In this assay, peptides competed for TAP-dependent and -independent ER entry and binding to HLA-A2 molecules of reporter peptide S-9-L with high TAP affinity (IC 50 = 2.3; reference 8) and an intermediate HLA-A2 binding affinity . The two epitopes showed a distinct pattern of relative competition efficiencies of minimal and extended epitope forms . Isolated Nt extension by one or two residues (including a single methionine) significantly increased efficiency of competition; however, Ct or double extension slightly increased competition efficiency of the pol peptide, whereas it dramatically decreased that of the core peptide. In view of the evaluation of isolated parameters described above, the distinct efficiencies of accumulation can be interpreted as follows. Increased efficiency of single Nt-extended variants could only be due to higher TAP affinities, as TAP-independent ER access of these peptides was unchanged and HLA-A2 affinities were equal (pol 573-83) or significantly decreased (methionine–pol 575-83, both core variants). Importantly, the low HLA-A2 affinities of three Nt-extended precursors did not appear to affect efficiency of competition; this suggests that either the negative effect on HLA-A2 affinity of Nt extension observed in equilibrium binding assays is not present during initial peptide assembly with HLA-A2 measured in the assay described here or that Nt extensions were removed by an ER-resident trimming activity during the assay, thereby increasing peptide affinity for HLA-A2. Although Ct- or double-extended variants of HBV core 18-27 were very similar to Nt-extended variants with respect to reduced HLA-A2 affinities and increased TAP affinities, they competed for assembly with HLA-A2 in permeabilized cells with greatly reduced efficiency and thus differed strikingly from Nt-extended variants. In view of previous reports demonstrating the absence of a Ct trimming activity in the ER 10 19 , this discrepancy suggests that Nt but not Ct peptide trimming in the ER occurs during transport experiments as described here and that Ct-extended variants do not compete efficiently in these experiments because of their low affinity for HLA-A2. Alternatively, only the negative effect of Ct extensions on HLA-A2 affinity, but not that of Nt extensions observed in equilibrium binding assays, may act during initial peptide assembly with HLA-A2. Taken together, HCI and TAP affinities both contributed to selection of epitope forms that can enter the ER and assemble with HCI molecules in our experimental system in vitro. Extended epitope forms with increased TAP affinities entered into the ER with greatly increased efficiency; subsequent efficient assembly with HLA-A2 was restricted to epitope forms with high HLA-A2 affinities and epitope precursors with lower HLA-A2 affinities due to Nt but not Ct extensions. However, a TAP-independent pathway provided access to the ER even to epitope forms with very low TAP affinities, albeit with reduced efficiency. As very small quantities of antigenic peptides can be sufficient for T cell recognition, and because some factors influencing peptide presentation by intact cells may not be readily measurable by the applied techniques in vitro, we decided to examine the importance of the described in vitro findings for presentation of the various epitope forms to specific CTL lines. We expressed minimal epitopes, Nt-extended precursors, and minimal epitopes preceded by the E3/19K signal sequence as minigenes in recombinant vaccinia viruses in order to obtain high peptide levels in the cytosol or ER, respectively. In parallel, HLA-A2–restricted specific CTL lines were generated using three different approaches: in vitro priming and expansion of PBMCs from healthy donors by successive restimulation with autologous irradiated PBMCs pulsed with synthetic minimal epitopes 35 , short-term expansion of secondary CTL lines by peptide restimulation of PBMCs from patients with acute HBV infections 37 , DNA immunization with plasmids encoding full-length HBV proteins of two transgenic mouse lines expressing HLA-A2/K b heavy chain and human β 2 m encoded by two transgenes 38 , or a single chain construct 30 . All used CTL lines met the following criteria: (a) specific recognition of target cells pulsed with synthetic minimal and Nt-extended epitope variants and (b) recognition of naturally processed epitope presented by HLA-A2 + target cells. Most CTL lines were tested for relative efficiencies of recognition of short and extended epitope forms. All tested lines specific for pol 575-83 and core 18-27 recognized both short and Nt-extended epitope forms; however, recognition of epitope precursors generally required 10–50-fold higher peptide concentrations than that of minimal epitopes (not shown). In the case of the core epitope, CTL recognition of Nt-extended longer peptides is also in accordance with a previous study 41 . CTL recognition of synthetic precursor peptides was not due to contamination by truncated peptides 44 ; high resolution HPLC analysis of synthetic precursor peptides combined with mass spectrometric analysis demonstrated absence of contaminating minimal epitopes (not shown). CTL lines generated by immunization in vivo (patients/transgenic mice) generally lost recognition of short synthetic peptides at 10 −12 M, whereas lines derived by in vitro immunization lost recognition at 10 −10 M. To ascertain that experiments with vaccinia viruses reflected intracellular assembly of peptides with HLA-A2 molecules and not binding to cell surface HLA-A2 molecules of peptides released from lysed infected cells, we also performed control experiments in which export of newly ER-assembled HLA-A2 molecules to the cell surface was inhibited by BFA 39 . As expected, this treatment completely inhibited presentation of minigene-expressed epitopes but did not affect presentation of synthetic peptides (not shown). As shown in Fig. 5 , CTL lines recognizing epitope pol 575-83 killed target cells much more efficiently when these expressed the Nt-extended precursor 573-85 than cells expressing the minimal peptide 575-83. Lysis of cells expressing the precursor was similar to lysis of cells pulsed with short synthetic peptides or expressing full-length pol protein, whereas cells expressing the minimal epitope were hardly recognized at all in an experiment with a CTL line from a patient . A similar although less dramatic difference in recognition was obtained with two CTL lines obtained by in vitro immunization . In contrast, one of these lines recognized TAP-deficient T2 cells expressing the two epitope forms with equal, intermediate efficiency, demonstrating that more efficient sensitization by expression of the precursor form was due to TAP-mediated ER access of this epitope variant . An even more dramatic effect of Nt epitope extension was observed for epitope core 18-27 and its precursor, 17-27 . In this case, the HBV patient–derived line did not kill cells expressing the minimal epitope but recognized precursor peptide–expressing cells and cells expressing the core protein or the signal peptide–coupled epitope with equally high efficiency. Equivalent results were obtained with a CTL line derived from another patient and a line generated by in vitro immunization (not shown). In conclusion, investigation of presentation of minigene-expressed epitopes and Nt precursors confirmed results obtained in vitro and underlined the dominant role of TAP in selecting variants of two HLA-A2–presented epitopes for presentation, as well as the low efficiency of TAP-independent ER access. Concordant results of in vitro and ex vivo experiments suggested that all major parameters controlling presentation of the two epitopes had been analyzed in vitro. However, it was not clear whether the TAP-selected precursors were presented in an unmodified or shortened form. To find out whether precursor peptides could be processed in the ER, we analyzed HLA-A2–presented peptides from minigene-expressing cells. HLA-A2 molecules were purified from JY cells expressing minimal or Nt-extended forms of epitopes pol 575-83 and core 18-27, and peptides associated with purified HLA-A2 molecules were analyzed according to published methods 21 . Acid-eluted peptides were fractionated under HPLC conditions that allowed maximal separation of synthetic peptides corresponding to minimal epitopes and the two Nt-extended precursors for each of the epitopes; peptides extended by an Nt methionine were included in calibration experiments to allow detection of vaccinia-expressed minimal epitopes having undergone incomplete processing by cytosolic methionine aminopeptidase. Each fraction was tested for its capacity to sensitize HLA-A2 + cells for lysis by specific CTL lines. Unspecific lysis was determined as the mean lysis obtained by sensitization with fractions from control IVD12-coated beads and was always <15%. In the case of epitope pol 575-83, only the epitope forms encoded by the minigenes could be detected in fractions eluted from HLA-A2 . Specifically, elutions from cells expressing precursor 573-85 did not contain sensitizing activity with the retention time corresponding to the minimal epitope; in addition, elutions from cells expressing the short epitope form did not contain sensitizing material in the fraction corresponding to the methionine-extended epitope (not shown). The same result was obtained with a second CTL line obtained from an HLA-A2– transgenic HHD mouse (not shown). Thus, there was no evidence for cleavage of the pol 573-83 precursor peptide in the ER. Significant amounts of the short epitope were detected in HLA-A2–eluted material from cells expressing the minimal epitope pol 575-83 . We have so far not been able to quantify the peptide contained in these fractions, although indirect evidence allows us to conclude that elutions from precursor peptide–expressing cells are likely to contain significantly more sensitizing peptide than those from cells expressing the short epitope. Due to its generation by in vitro immunization with pol 575-83, the CTL used for analysis of fractions recognized peptide 575-83 with 40–50-fold higher efficiency than peptide 573-83 . As CTL recognition of sensitizing fractions from cells expressing pol 575-83 and 573-83 was equally efficient, it is likely that fractions from cells expressing and presenting peptide 573-83 contain substantially more peptide than those from cells expressing peptide 575-83. Detection of a low amount of peptide 575-83 in elutions from pol 575-83/vaccinia–infected cells is consistent with low-level killing of such cells by the same CTL line . Results for elutions from cells expressing the core epitope or its precursor were strikingly different from those for the pol epitope . Both cells synthesizing the short and the extended epitope form exclusively presented the minimal epitope; in addition, HLA-A2 molecules from cells expressing the precursor peptide contained substantially larger quantities of this peptide than A2 molecules from cells expressing the short form . The epitope variant extended by an Nt methionine could not be detected in cells expressing core 18-27 preceded by a methionine initiation codon (not shown). Results shown in Fig. 6 C were obtained with a patient-derived CTL line; equivalent results were obtained with three other CTL lines raised in conventional or HHD-transgenic mice (not shown). Thus, in cells expressing precursor peptide HBV core 17-27, apparently complete Nt processing in the ER was observed. When peptides eluted from cells expressing complete HBV core protein were analyzed, sensitizing activity also comigrated exclusively with the fraction corresponding to peptide core 18-27 , demonstrating that this peptide is also the dominant epitope form generated from the source protein. Detection of peptide HBV core 18-27 but not 17-27 might be due to more efficient CTL recognition of the shorter peptide, a standard feature of all CTL lines described in this paper, including the two lines generated from HHD-transgenic mice used for analysis of eluted material . To address this issue, dilutions of synthetic peptides and of sensitizing fractions of eluted material were analyzed for CTL recognition. Both CTL lines recognized peptide 18-27 with 10-fold higher efficiency than 17-27 (not shown). CTL lines recognized a 500-fold dilution of the fraction from cells expressing the core 17-27 minigene and a 40-fold dilution of the fraction from cells expressing the HBV core protein . Consequently, cells expressing minigene core 17-27 contained at least 50 times more peptide core 18-27 than 17-27, whereas cells expressing HBV core protein contained at least four times more of the short peptide. Titration experiments with synthetic peptide standards and elution fractions also allowed us to calculate the number of HLA-A2–extracted HBV core 18-27 molecules per cell. This number was 3,610 for cells expressing the core 17-27 minigene, 438 for the core 18-27 minigene, and 20 for core protein. Two principal novel findings emerge from this study: (a) the selectivity of human TAP can have a substantial biological impact on peptide entry into the ER and (b) similar to signal peptide or Jaw 1–coupled epitopes, TAP-transported peptides can be modified by a highly efficient aminopeptidase activity in the ER. To our knowledge, this is the first conclusive demonstration that the human peptide transporter can participate in peptide selection for presentation by HCI molecules. Thus, previously established rules governing peptide affinity for human TAP 8 14 are biologically relevant. In a comparison of the data reported here with earlier demonstrations of the biological impact of murine and rat TAP-B transporters 10 12 , an important difference needs to be emphasized. Significant biological effects of peptide selection by restrictive rodent transporters have been demonstrated in settings where TAP and MHC preferences for Ct peptide residues were incompatible. Due to the absence of Ct peptide processing in the ER 10 , these incompatibilities cannot be compensated for after TAP transport and impair the efficiency of class I antigen processing so severely that incompatible TAP/MHC combinations have not survived in rat evolution 11 . This study addresses the biological impact of TAP selection in a setting where Ct preferences of TAP and MHC class I molecules are adapted overall (as is the case in humans), and some peptides display low TAP affinities due to Nt as well as Ct sequence elements. In this setting, we find a dramatic effect of TAP affinity similar to previous studies of rodent transporters. However, in the absence of an overwhelming conflict between Ct preferences of human TAP and MHC class I molecules, transport of Nt-extended peptides followed (when required) by Nt processing in the ER allows highly efficient presentation of epitopes with very low TAP affinities. This suggests that, in humans, high permissiveness of the class I processing system downstream from proteolytic peptide generation is not due to absence of selectivity of TAP transporters but to a combination of TAP transport of epitope variants with Nt peptide processing in the ER. Our analysis of a panel of HBV- and HCV-derived peptides , with many of them representing in vivo immunodominant epitopes 37 , suggests that precursor peptide selection by human TAP is a relatively common occurrence for HLA-A2–presented peptides. The frequent requirement for ER processing of ligand precursors for this molecule is likely to reflect the poor compatibility of HLA-A2 and TAP preferences. In a recent analysis of the compatibility of human TAP preferences with those of nine HCI molecules, HLA-A2 ligands showed the poorest and HLA-B27 ligands the best adaptation to TAP selectivity 14 . However, close analysis of the TAP affinities of in vivo antigenic HBV peptides suggests that antigen processing may favor peptides that do not require ER processing. Whereas only 2/12 peptides with very low TAP affinities (IC 50 >1,000) are known to be antigenic in vivo, this is the case for 3/6 peptides with intermediate to high TAP affinities (IC 50 <100). This may indicate limited efficiency of the ER aminopeptidase or a requirement of specific flanking sequences for its activity. As it is not possible to quantitate antigenic peptides in the cytosol, we do not know the expression levels of the minigene-expressed epitope forms. In a study by Anton et al., vaccinia virus–expressed, MHC class I–bound epitopes were detected at at least 50,000 copies per cell 45 . We cannot entirely rule out that 9- or 10-mer epitope forms are produced at lower levels than 11-mer precursors, although there is no experimental evidence supporting this hypothesis. However, in complete accordance with results obtained with minigene-infected cells, synthetic precursor peptides assembled much more efficiently with HLA-A2 in permeabilized cells than shorter minimal epitopes. This suggests strongly that the dramatic difference between the efficiencies of sensitizations by minigene-expressed precursor and minimal peptides was due to more efficient TAP transport of precursors and not to lower expression of shorter epitope forms. We therefore conclude that, in the studied cases, high cytosolic concentrations of minimal epitopes could not compensate completely for low TAP affinities. It is likely that ER entry of minimal or extended epitope forms produced at physiological, i.e., lower concentrations generally requires higher TAP affinities than in the case of the two HBV-derived epitopes and their variants. For the core 18-27 epitope, we were able to determine the nature and number of peptide molecules eluted from HLA-A2 molecules of cells expressing different epitope forms. This analysis revealed that peptide core 18-27 is the dominant HLA-A2–bound epitope form in cells expressing its precursor 17-27 and also in cells expressing the complete HBV core protein. Thus, in the case of this immunodominant HBV epitope, the naturally processed epitope form is excluded from the ER by low TAP affinity, demonstrating that TAP-imposed ER import of epitope precursors for luminal processing plays a role in natural antigen processing. Whatever the nature of precursor peptides of core 18-27 generated by cytosolic degradation of the HBV core protein, such precursor peptides must undergo ER processing similar to peptide core 17-27. Quantitative analysis of eluted fractions also demonstrated almost 10-fold higher amounts of core 18-27 peptide in elutions from cells expressing the precursor peptide, as compared with cells expressing the minimal peptide. The relatively low number of 20 peptide molecules per cell expressing HBV core is compatible with immunodominance of this epitope in HBV-infected patients. As few as three peptide molecules per cell can give rise to efficient CTL lysis 46 . Anton et al. found a low number of 30 molecules per cell for an immunodominant viral epitope generated from the vaccinia-encoded source protein; as in this study, peptide copy number was dramatically increased upon expression of the epitope as minigene 45 . Results of Pamer et al. also suggest that immunodominance does not require high peptide copy numbers 47 . The relatively high peptide copy number in cells expressing the core 18-27 minigene clearly conflicts with CTL recognition of vaccinia-infected targets ; three independent CTL lines from different individuals recognized targets expressing whole core protein with high efficiency but targets expressing core 18-27 barely or not at all. We propose that the number of 438 peptide molecules per cell may be due to binding of cytosolic peptides to HLA-A2 molecules during overnight incubation of cell lysates for A2 immunoprecipitation. Although current consensus holds that antigenic peptides can only be recovered from cells expressing a restricting MHC class I molecule 1 48 , to our knowledge there is no experimental evidence to rule out that peptide–MHC complexes can be formed after cell lysis. Based on reports that as many as 50,000 peptides per cell can be presented by MHC class I molecules in cells expressing vaccinia-encoded minigenes 45 , it can conservatively be assumed that at least 100,000 peptide molecules per cell are synthesized in such cells. Provided that these peptides are not (or not entirely) degraded and at least partly set free in detergent lysates, this would correspond to a maximum peptide concentration of 8 nM during immunoprecipitation of HLA-A2 under the conditions employed in this study. This concentration is not very far from the K D of 40 nM of peptide core 18-27 (Lauvau, G., and P.M. van Endert, unpublished data). Corroboration of the hypothesis of cytosolic peptide association with HLA-A2 after cell lysis will require lysate mixing experiments followed by peptide elution and analysis. Based on the identification of epitopes that cannot enter the ER in their minimal form, we have been able to study ER processing of TAP-translocated peptides. We find that precursor peptide pol 573-83 is presented without modification, presumably due to its high-affinity binding to HLA-A2; efficient binding of up to 12-mer peptides to HLA-A2 has been reported previously 49 . Apparently complete processing of precursor peptide core 17-27 with low HLA-A2 affinity suggests that ER processing of precursor peptides may be controlled by HCI binding affinity but unaffected by peptide length; both precursor peptides are 11-mers. Although the reported data establish clearly that TAP-transported precursor peptides can be processed in the ER, it remains unclear whether the involved mechanism is identical to that implicated in release of epitopes coupled to signal sequences 19 . We have so far not been able to investigate in our system the effect of protease inhibitors that have been reported to inhibit assembly of signal sequence–derived peptides with HLA-A2 50 . Although the observation of an unusual length of signal sequence–derived peptides in TAP-deficient cells 27 may indicate distinct peptide maturation mechanisms in the two cases, involvement of an identical or similar mechanism is suggested by the apparent lack of Ct processing in both systems 19 21 . It is also possible that several proteolytic events are involved in epitope generation from signal sequences, only one of which also acts on peptides delivered by TAP. We can now only speculate on the mechanism of Nt peptide processing. Our analysis of HLA-A2–eluted peptides may provide clues in this regard. Nt peptide processing may be carried out by a fluid phase or directly HCI-associated aminopeptidase. Although both scenarios could explain processing of precursor core 17-27, the complete absence of processing of precursor pol 573-83, as detected in elutions, is difficult to reconcile with random fluid phase processing. We therefore favor the alternative scenario, in which unstable HCI association of Nt-extended epitope precursors would be detected by a sensor mechanism integrated into or coupled to the putative peptidase. Sensor and processing mechanisms may preferentially associate with HCI molecules interacting with TAP–tapasin complexes. This hypothesis could also account for the frequent finding of long peptides bound to HLA-A2 in TAP-deficient cells and to HLA-B27 in normal cells; a recent report suggests that HLA-B27 may frequently assemble with peptides in a tapasin-independent fashion 26 . Several proteins involved in peptide loading on HCI molecules are candidates for the putative role of aminopeptidase. These include HCI molecules themselves 51 , the ER-resident chaperone gp96 52 , and the putative chaperone tapasin 4 . | Study | biomedical | en | 0.999994 |
10544196 | Human peripheral blood was collected in 10% (vol/vol) 0.1 M EDTA, layered onto 1-Step Polymorphs gradient (Nycomed Pharma) and centrifuged at 400 g for 30 min at room temperature. Neutrophil and mononuclear cell layers were collected, resuspended in Dulbecco's PBS (DPBS) without calcium and magnesium (GIBCO BRL), and centrifuged for 15 min at ∼750 g . Red blood cells were lysed in the neutrophil fraction by resuspending the pellet in E-Lyse (Cardinal Associates) for 5 min on ice. Both cell fractions were washed two times with ice-cold DPBS. The mononuclear cells were allowed to adhere to protein-coated plastic for 2–3 h, and nonadherent cells were gently washed off the plate. After an additional 12 h, the nonadherent dendritic cells were washed off the plate and depleted of B lymphocytes and T lymphocytes with anti-CD19 and anti-CD2 Dynabeads (5 beads per cell; Dynal). The remaining cells were cultured in 50 ng/ml GM-CSF, 40 ng/ml IL-4 DMEM, and 10% FCS plus additives for 7 d to generate immature DCs, and in some cases 24 h additional culture in 10 ng/ml LPS was used to mature the dendritic cells. CD4, CD8, CD14, CD56, and CD19 populations were purified from mononuclear cells with the relevant microbeads (Miltenyi Biotec) using 20 μl of beads for 10 7 mononuclear cells in PBS, 1% BSA, 5 mM EDTA at 5 × 10 7 cells/ml for 30 min at 4°C. They were then pelleted, resuspended in PBS with 1% BSA and 5 mM EDTA at 5 × 10 7 cells/ml, and passed over a VS column (Miltenyi Biotec) in a magnetic field to remove nontagged cells. Cells were removed by forcing 20 ml of PBS with 1% BSA and 5 mM EDTA over the VS column, outside the magnetic field. Human small intestine LPLs and IELs were isolated from patients undergoing gastric bypass surgery for morbid obesity as described previously 31 . IEL preparations were on average 80% CD3 + , 70% CD8 + , 5% CD4 + , whereas LPL preparations were 85% CD3 + , 40% CD8 + , 45% CD4 + , consistent with previous analysis 31 . All human subject protocols were approved by the Human Resources Committee at the Robert Wood Johnson Medical School, Brigham and Women's Hospital, or Massachusetts General Hospital. Anti-CD4, -CD8, -CD14, -CD19, -CD49d, -CD56, -CD62L, -CLA, -CD45RA, and -CD45RO dye-linked mAbs for immunofluorescence studies were all obtained from PharMingen, while anti-αE–FITC was obtained from Coulter Pharmaceuticals. Anti-CCR4 mAb (2B10) was generated in the laboratory to CCR4/L1.2 transfectants. OKT3, an anti–human CD3 mAb, was obtained from American Type Culture Collection, and anti–human CD28 mAb was purchased from Becton Dickinson. IP-10, eotaxin-1, B cell–attracting chemokine 1 (BCL-1), IL-8, IFN-inducible T cell α chemoattractant (I-TAC), regulated on activation, normal T cell expressed and secreted protein (RANTES), fractalkine, leukotactin, liver-expressed chemokine (LEC), eotaxin-3, CKα2, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, MIP-4, I-309, and MDC were synthesized at LeukoSite, Inc., by f-moc chemistry (433A automated peptide synthesizer; Perkin-Elmer Biosystems). These chemokines were purified and folded as described previously 32 . For biotinylated thymus-expressed cytokine (TECK), the chemokine domain of TECK was synthesized (TECK 24–99). Lys 99 was derivitized during the synthesis with an aminocapropyl-biotin moiety. A COOH-terminal glycine was added for synthesis convenience. The TECK chemokine domain had the same potency as full-length TECK for chemotaxis of GPR-9-6/L1.2 transfectants (data not shown). All other recombinant chemokines were obtained from either PeproTech or R&D Systems. The human endothelial cell line ECV304 was purchased from American Type Culture Collection. All cytokines were obtained from R&D Systems. A peptide consisting of the NH 2 terminus of GPR-9-6 was generated by American Peptide Company having the sequence NH 2 -MADDYGSESTSSMEDYVNFNFTDFYC. BALB/c mice were immunized intraperitoneally with 10 μg each of GPR-9-6 peptide/KLH conjugate prepared in complete Freund's adjuvant (CFA) at day 1, IFA at day 20, and PBS at day 40. At day 60, the mice were boosted with 10 μg of GPR-9-6 peptide/KLH in PBS, and after 4 d the spleens were removed and fused to SP2/0 myeloma cells (American Type Culture Collection). Fusions were screened by ELISA with plates coated with GPR-9-6 peptide. Hybridomas producing anti–GPR-9-6 mAbs were checked for reactivity with GPR-9-6 transfectants and subcloned for further characterization. As described previously 33 , 6-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 and 2 μg/ml OKT3, then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems) were cultured at 10 5 –10 6 cells/ml in DMEM with 10% FCS and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti–IL-4 (1 μg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and anti–IFN-γ (1 μg/ml) were used to direct to Th2. After 4–5 d, the activated Th1 and Th2 lymphocytes were washed once in DMEM and cultured for 4–7 d in DMEM with 10% FCS and IL-2 (1 ng/ml). After this, the activated Th1 and Th2 lymphocytes were restimulated for 5 d with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 μg/ml) to prevent apoptosis. After 4–5 d, the Th1 and Th2 lymphocytes were washed and then cultured again with IL-2 for 4 d. Activated Th1 and Th2 lymphocytes were maintained in this way for a maximum of three cycles. Direction of the Th1 and Th2 lines was confirmed by intracellular cytokine staining and ELISAs for IL-4 and IFN-γ. 3-μM pore diameter Transwell inserts were generally used, with the exception of the bulk migration experiments for the phenotyping of migrated cells, in which 5-μM inserts were used. The cells under study were washed once in RPMI and resuspended at 4 × 10 6 cells/ml for Th1/Th2 lymphocytes, cell lines, and transfectants, and at 10 7 cells/ml for resting CD4 lymphocytes, in RPMI with 0.5% heat-shock antigen (HSA) and 10 mM Hepes. An aliquot of 200 μl of cell suspension (input of 8 × 10 5 and 2 × 10 6 cells, respectively) was added to each insert. Chemokine in 500–600 μl of RPMI with 0.5% HSA and 10 mM Hepes was added to the lower well. After 2–4 h, the inserts were removed and the number of cells which had migrated through the insert to the lower well was counted for 30–60 s on a Becton Dickinson FACScan™ with the gates set to acquire the cells of interest. Using this technique, 100% migration would be 25,000 cells for Th1/Th2 cells and 75,000 cells for resting CD4 lymphocytes, where this number represents the cells in the lower well counted on the FACScan™ over 30–60 s. In all cases, the data points were the result of duplicate wells, with the mean value shown and the error bars representing the sample standard deviation. To determine the migration of lymphocyte subsets, the number of cells in each subtype was determined by multi-color FACScan™ analysis for the starting population of each chemotactic assay 34 . The number of cells belonging to the same subtype was determined in the migrated population, and the percentage of migration was determined from these two numbers. Eight wells were used for each treatment and pooled for FACScan™ analysis. As it has been demonstrated previously that most chemokine receptors couple to the G i class of G proteins, we wanted to determine if GPR-9-6 also couples to the G i isoform. Pertussis toxin was used to block G i -mediated cellular responses. 2 × 10 6 cells/ml in prewarmed RPMI plus 10% FCS were incubated with pertussis toxin (GIBCO BRL) at a final concentration of 100 ng/ml for 2 h at 37°C, washed twice with medium, and resuspended in chemotaxis medium. Migration was assayed as described above. Cells were resuspended at 10 7 cells/ml in FACS ® buffer (PBS with 5% FCS, 0.1% azide, and 10% human serum to block Fc receptors), and primary mAb was added. After 20 min, the cells were washed twice in FACS ® buffer, and F(ab) 2 anti–mouse IgG–PE (absorbed against human Ig) was added. After 20 min, the cells were washed twice in FACS ® buffer and then blocked in 10% mouse serum before the second mAb directly linked to a fluorochrome was added. After 20 min, the cells were washed three times in FACS ® buffer and analyzed. Throughout the staining, the cells were kept on ice. We used an IgG 2b isotype control followed by F(ab) 2 anti–mouse IgG–PE as a negative control, and isotype control staining always fell below 10 FL1 or FL2 units. 4 × 10 5 cells in 25 μl of PBS with 10% rabbit serum, 1% FCS, and 0.1% sodium azide (assay buffer) were preincubated with either assay buffer (no treatment), 1,000 nM TECK/MCP-1, or 50 μg/ml 3C3/IgG 2b for 30 min. 25 μl of biotinylated TECK at a final concentration ranging up to 100 nM was then added, and after 1 h, the cells were washed twice with 200 μl of wash buffer (PBS with 1% FCS and 0.1% azide) and resuspended in 25 μl of Wallac Eμ-labeled streptavidin diluted 1:400 in assay buffer. After 30 min at room temperature, the cells were washed in wash buffer and transferred to a 96-well solid white nonspecific binding plate and, after spinning to remove supernatant, 200 μl of Wallac Enhancement Solution was added. The fluorescence was counted on a multilabel counter at 340/613 nm and reported in relative fluorescence units (RFUs). 10 7 cells/ml in DPBS were labeled for 30 min with Fura-2 AM (Molecular Probes) at 2 μM, washed three times in DPBS, and resuspended at 10 6 cells/ml in DPBS containing 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10 mM Hepes (pH 7.2), and 5.5 mM glucose. Chemokine was then added as indicated in the figure legend, and the calcium flux was measured on a fluorimeter using 10% NP-40 and 10 mM EGTA to establish the maximum and minimum Ca 2+ mobilized. Plasmid DNA was isolated using Genomic tips as recommended by the manufacturer (Qiagen). DNA ligations, restriction endonuclease digestions, and gel electrophoresis were performed as described previously 35 . DNA purification through agarose gel extraction was performed using the QIAEXII Gel Extraction kit as recommended by the manufacturer (Qiagen). Plasmid DNA was introduced into Escherichia coli by chemical transformation (GIBCO BRL). Enzymes were purchased from New England Biolabs, GIBCO BRL, or Boehringer Mannheim. RNA was isolated from frozen tissues or cells using either the standard guanidinium isothiocyanate method 35 or the RNeasy kit as recommended (Qiagen). DNA sequencing was performed by MacroMolecular Resources (Colorado State University, Fort Collins, CO) using an ABI DyeRhodamine Terminator cycle sequencing kit and an ABI Prism DNA Sequencer (model 377; Perkin-Elmer Applied Biosystems) according to the manufacturer's specifications. Sequences were analyzed using SeqMan (DNASTAR). Primers were synthesized by MacroMolecular Resources or by Operon Technologies, Inc. Primers were designed for use in the PCR to amplify the coding region of GPR-9-6 based on the nucleotide sequence deposited in EMBL/GenBank/DDBJ . BamHI and XbaI sites were incorporated into primer pair BAZ201 5′-TCGAAG GGATC C CTAAC ATGGCTGATGACTATGGC -3′ and BAZ202 5′-AAGAAG TCTAGA ACCCC TCAGAGGGAGAGTGCTCC -3′ for directional cloning ( bold , coding sequence; italic , enzyme site). 5 μg of total human genomic DNA (Clontech) was used as the template in the Pfu PCR cycles, with 60 mM Tris-HCl (pH 9.5), 1.5 mM MgCl 2 , 100 pmol primers, 200 μM dNTP, and 5 U PfuI polymerase (Invitrogen) in 100 μl volume. The cycle parameters were an initial melt at 95°C for 2 min, then 35 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 2 min 15 s, followed by a final extension of 72°C, 7 min in a DNA thermal cycler (Perkin-Elmer Corp.). Primers were designed to amplify the complete coding region of TECK based on the published nucleotide sequence. HindIII and XbaI sites were incorporated into the primer pairs for directional cloning. BAZ203 5′-TCGAAG AAG CTT ATGAACCTGTGGCTCCTG -3′ and BAZ204 5′-AAGAAG TCTAGA TCACAGTCCTGAATTAGC -3′ were used to amplify TECK ( bold , coding sequence; italic , enzyme site). 5 μg of human thymus RNA was reverse transcribed with oligo(dT) in 20 μl volume. The cDNA was mixed with 200 μM dNTP, 100 pmol primers, 60 mM Tris-HCl (pH 9.5), 1.5 mM MgCl 2 , and 10 U AmpliTaq in 50 μl volume. The cycle parameters were an initial melt at 95°C for 2 min, then 35 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min, followed by a final extension of 72°C, 7 min. The human thymus was obtained from Boston Children's Hospital (Boston, MA). Reverse transcription (RT)-PCR amplifying TECK (BAZ203, BAZ204) and GPR-9-6 (BAZ201, BAZ202) was performed using equal amounts (0.5 ng) of cDNA template from thymus, small intestine, colon, and brain, as well as 5 μg genomic DNA (Clontech). The RNA used to prepare the cDNA template was triple oligo(dT) column purified. The same conditions and PCR profile were used as the AmpliTaq PCR cycle described above, except that 30 cycles were performed. Amplification with glyceraldehyde 3-phosphate dehydrogenase (G3PDH) primers (Clontech) was used to demonstrate equivalence of template. Amplification with G3PDH intron B specific primers (BAZ205 5′-TCCCCTGCCAGCCTAGCGTTGACC-3′ and BAZ206 5′-CCCCACTATGCCACCCCAGGAATG-3′) was used to confirm the absence of contaminating genomic DNA in the Clontech cDNAs. After agarose gel electrophoresis, the PCR products were visualized in the presence of ethidium bromide with an ultraviolet light source. DNA fragments of predicted size (∼450 bp for TECK and ∼1 kb for GPR-9-6) were isolated and cloned into pBluescript II KS+ (Stratagene) and pcDNA3 (Invitrogen) for sequence analysis and further manipulation. The coding region of GPR-9-6 was amplified by PCR and directionally cloned into the BamHI/XbaI sites of pcDNA3. Transfectants were generated in the murine pre-B lymphoma cell line L1.2 as described previously 36 , and maintained in RPMI 1640 supplemented with 10% FCS, 2 mM l -glutamine, 50 U/ml penicillin/streptomycin, 0.55 mM β-mercaptoethanol, 10 mM Hepes, 1 mM sodium pyruvate, and 1 mg/ml G418 (Geneticin) for selection. The transfectants were then stained by mAbs with reactivity against the GPR-9-6 peptide (see below) and analyzed by FACScan™ to confirm surface expression of GPR-9-6, then cloned by limiting dilution. Transfected cells were treated with 5 mM n -butyric acid for 24 h before experimentation. Northern blots were either purchased from Clontech or prepared as follows: total RNA was separated by electrophoresis on 1.2% formaldehyde agarose gels and transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech) by the capillary method as described previously 34 and cross-linked using a Stratalinker (Stratagene). Full-length gel-purified TECK, GPR-9-6, and β-actin (Clontech) were radiolabeled using High Prime reagents (Boehringer Mannheim) according to the manufacturer's specifications. Hybridizations were performed in ExpressHyb Solution (Clontech) using the manufacturer's suggested protocol. Length of autoradiography exposure is described in appropriate figure legends. Due to its close phylogenetic association with other known leukocyte chemokine receptors such as CCR6 and CCR7, we cloned GPR-9-6 by PCR, using primers designed from the deposited EMBL/GenBank/DDBJ sequence. GPR-9-6/L1.2 transfectants were prepared and stained with mAbs raised against the first 30 amino acids of the NH 2 terminus of GPR-9-6 coupled to KLH. One mAb, designated 3C3, reacted with GPR-9-6/L1.2 transfectants but not with parental L1.2 cells. 3C3 was found to have an IgG 2b isotype. In cross-reactivity studies, 3C3 did not cross-react with CCR1–7 or CXCR1–4 transfectants. We show the data for CCR6 here, as it is one of the more closely related chemokine receptors to GPR-9-6 . Also, the NH 2 -terminal peptide of GPR-9-6 blocked the binding of mAb 3C3 to GPR-9-6 transfectants (data not shown), further validating the specificity of this mAb. mAb 3C3 was used to identify surface expression of GPR-9-6 on various cell lines and primary cells. Out of a panel of cell lines, only Molt-4 and Molt-13 T cell lines expressed GPR-9-6 ( Table ). In addition, GPR-9-6 was expressed on a small subset of CD4 lymphocytes (2–4%) as well as on a small subset of CD8 lymphocytes . A subset of B lymphocytes also expressed GPR-9-6. Monocytes, basophils, eosinophils, neutrophils, immature and mature dendritic cells, and NK cells did not express GPR-9-6 . GPR-9-6 was expressed on >90% of thymocytes that expressed all levels of TCR, although a small subset of TCR high GPR-9-6 − thymocytes was evident. In three-color experiments, GPR-9-6 was found on the majority of CD4 + , CD8 + , and CD4 + CD8 + thymocytes and on ∼50% of immature CD4 − CD8 − thymocytes (data not shown). To determine which subset of CD4 and CD8 lymphocytes expressed GPR-9-6, three-color flow cytometry was performed . The CD4 lymphocytes that expressed GPR-9-6 were mainly of memory phenotype, and those cells that expressed the highest levels of GPR-9-6 were all of memory phenotype. Interestingly, almost all of the GPR-9-6 + CD4 lymphocytes expressed high levels of the α4β7 integrin, which is expressed on gut-trafficking T lymphocytes, and lacked expression of CLA, which mediates T lymphocyte trafficking to inflamed skin. The subset of memory CD4 lymphocytes defined by expression of αE and CD62L was also subdivided into GPR-9-6 positive and negative subpopulations. Upon analysis over multiple donors, a subset of CD4 lymphocytes that expressed low levels of GPR-9-6 was evident. This subset was more apparent when we used four-color analysis and gated on memory and naive CD4 lymphocytes, as defined by CD45RO expression . Expression of GPR-9-6 on memory CD4 lymphocytes was more than threefold higher than that on naive CD4 lymphocytes, as shown in Fig. 4 where the ratio of the mean fluorescent intensities of GPR-9-6 on naive and memory CD4 lymphocytes approaches 3.5 when an anti–mouse IgG–PE second stage was used. GPR-9-6 low CD4 lymphocytes expressed CD62L, a homing receptor involved in lymphocyte trafficking to organized lymphoid tissues via high endothelial venules (HEVs), and low levels of β7. In contrast, the majority of the GPR-9-6 high memory CD4 lymphocyte subset did not express CD62L, but expressed high levels of β7. Like GPR-9-6 + CD4 lymphocytes, GPR-9-6 + CD8 lymphocytes also expressed high levels of β7 and were CLA − . CD45RO and CD45RA are not as extensively used as memory markers for CD8 lymphocytes. However, GPR-9-6 was expressed on CD45RA low CD8 lymphocytes. Also, as observed for CD4 lymphocytes, a subset of GPR-9-6 low cells was evident that was CD45RA high CD62L + , whereas GPR-9-6 high CD8 lymphocytes were CD62L − . Finally, the majority of αE + CD8 lymphocytes expressed GPR-9-6, whereas GPR-9-6 low CD8 lymphocytes were mainly αE − . Further, αE + CD8 lymphocytes that did not express GPR-9-6 expressed lower levels of αE than αE + GPR-9-6 + CD8 lymphocytes. The selective expression of GPR-9-6 on a subset of intestinal trafficking CD4 and CD8 blood lymphocytes suggests that the receptor may be involved in lymphocyte homing to the intestine. Therefore, we examined the expression of GPR-9-6 on T lymphocytes isolated from the small intestine. Flow cytometric analysis revealed that GPR-9-6 was expressed on all intestinal IELs and LPLs . Compared with GPR-9-6 expression on PBL lymphocytes, the chemokine receptor was found to be significantly enriched on T lymphocytes in intestinal tissue. As GPR-9-6 was expressed on a subset of memory T lymphocytes, we attempted to induce expression of GPR-9-6 on umbilical CD4 lymphocytes by activation in the presence of directing cytokines to generate Th1 and Th2 lymphocytes. Although the Th1 and Th2 lymphocyte lines showed the appropriate differential production of IL-4, IL-13, and IFN-γ upon activation (data not shown), chronic activation of these cells in the presence of IL-12 or IL-4 to generate Th1 or Th2 lymphocytes failed to induce the expression of GPR-9-6 . Further, chronic activation of umbilical CD4 lymphocytes in the presence of IL-1–13, IL-15, IL-17, IL-18, and TGF-β did not induce expression of GPR-9-6 (data not shown). As expected, CXCR3 was upregulated selectively on Th1 lymphocytes . Interestingly, activation of T lymphocytes through cross-linking of TCR resulted in transient downregulation of GPR-9-6 expression , with reexpression of GPR-9-6 observed upon removal of the TCR stimulation and culture in IL-2. This effect was not specific to the chemokine receptor GPR-9-6, as the other chemokine receptors expressed by T lymphocytes that we examined (CCR5 and CCR6) were also transiently downregulated upon TCR cross-linking . Expression by T lymphocytes of other CD molecules such as CD29, CD44, and CD49d was unaffected by T lymphocyte activation (data not shown). From a panel of chemokines including MCP-1–4, MIP-1αβ, MIP-3αβ, eotaxin-1–3, RANTES, I-309, thymus and activation-regulated chemokine (TARC), MDC, liver-expressed chemokine (LEC), CKα2, secondary lymphoid-tissue chemokine (SLC), human CC chemokine 1, fractalkine, lymphotactin, monokine induced by IFN-γ (MIG), IP-10, I-TAC, B cell–attracting chemokine 1 (BCL-1), IL-8, gro-αβψ, leukotactin, stromal cell–derived factor (SDF)-1α/β, MIP-3, and MIP-4, only TECK induced chemotaxis of GPR-9-6/L1.2 transfectants . TECK-mediated chemotaxis of L1.2/GPR-9-6 transfectants was inhibited by anti–GPR-9-6 mAb 3C3 , while pretreatment of the transfectants with pertussis toxin also inhibited migration to TECK . The observation that pertussis toxin abolished TECK-mediated transfectant chemotaxis indicates that chemokine receptor GPR-9-6 couples to the G i class of G proteins. TECK did not act on any of the other transfectants tested (CCR1,2,4–7 and CXCR1–4), with the exception of CCR3/L1.2 transfectants, for which high concentrations of TECK were weakly chemotactic (data not shown). Also in examining our panel of cell lines, we found that only GPR-9-6 + Molt-4 and Molt-13 cell lines migrated to TECK ( Table ). Migration of Molt-4 and Molt-13 cells to TECK was blocked by anti–GPR-9-6 mAb 3C3 (data not shown). Primary thymocytes were also chemotaxic for TECK in a dose-dependent manner , and this migration was blocked by anti–GPR-9-6 mAb 3C3, but not by an IgG 2b control . In contrast, anti–GPR-9-6 did not block thymocyte migration to SDF-1α, which binds to CXCR4 on thymocytes . We also found that a small subset of CD4 lymphocytes (<0.5% of input) migrated to TECK and that this chemotaxis was blocked by anti–GPR-9-6 mAb 3C3, but not by an anti-CCR4 mAb, 2B10. In contrast, 3C3 mAb had no effect on TARC-induced CD4 lymphocyte chemotaxis . In our experiments, TECK was not chemotactic for other total cell populations in peripheral blood, including B lymphocytes, CD8 lymphocytes, neutrophils, monocytes, and eosinophils (data not shown). In our preliminary chemotaxis studies, we were able to demonstrate that TECK induced significant but very low levels of migration of peripheral blood CD4 lymphocytes. This reflects the low percentage of CD4 lymphocytes that express GPR-9-6 and the background migration of the total population of CD4 lymphocytes. As GPR-9-6 was expressed at high levels mainly on small subsets of memory β7 high CD4 and CD8 lymphocytes, we examined the phenotype of the memory CD4 and CD8 lymphocytes that migrate to TECK using multi-color FACScan™ analysis with anti-CD4, anti-CD8, anti-CD45RA, anti-CLA, anti-α4β7 mAb Act1, and anti-αE . Act1 is an mAb that recognizes a combinatorial epitope on α4β7 and therefore can be used to specifically examine expression of this integrin on leukocytes. The gut-homing α4β7 + CLA − memory CD4 lymphocytes were enriched in the migrated population, whereas skin-homing α4β7 − CLA + memory CD4 lymphocytes and α4β7 − CLA − memory CD4 lymphocytes were not . Therefore, the memory CD4 lymphocytes that migrate to TECK are contained in the gut-homing α4β7 + CLA − memory CD4 lymphocyte subpopulation. For the CD8 lymphocytes, the anti-CD45RA mAb was used to define the memory CD8 lymphocytes as CD45RA low/− . As GPR-9-6 was expressed at high levels on CD8 lymphocytes that express high levels of αE or β7, we analyzed the expression of αE and α4β7 on the migrated CD8 lymphocytes. Higher random migration of CD45RA low/− CD8 lymphocytes was evident, but the cells responsive to TECK were greatly enriched in the αE + α4β7 + CD45RA low/− CD8 lymphocyte subset . In addition to its ability to induce cell migration, TECK also induced a more robust Ca 2+ flux in Molt-4 cells than was observed with SDF-1α, whose receptor (CXCR4) was also expressed by Molt-4 cells . MDC did not induce Ca 2+ flux in Molt-4 cells, which do not express CCR4, the receptor for MDC. As TECK was chemotactic for GPR-9-6–expressing cells, we evaluated whether TECK binds to GPR-9-6. Although tyrosine-iodinated TECK did not bind to GPR-9-6 + Molt-4 cells, biotinylated TECK bound in a dose-dependent and saturable manner. In contrast, biotinylated TECK did not bind to GPR-9-6 − CEM cells . Binding of biotinylated TECK to Molt-4 cells was inhibited by either 500 nM TECK or by the anti–GPR-9-6 mAb 3C3, but not by either 500 nM MCP-1 or an IgG 2b control mAb . Preincubation of GPR-9-6 transfectants, CD4 cells, and B lymphocytes with TECK inhibited the staining with anti–GPR-9-6 mAb 3C3, whereas MDC had no effect . Although these experiments were performed at 4°C and with azide, in the absence of a nonblocking anti–GPR-9-6 mAb, we cannot be certain of whether these results reflect either blocking or downmodulation of the chemokine receptor. However, these data do demonstrate that the staining pattern we observe with mAb 3C3 is not due to nonspecific binding of mAb 3C3 to lymphocytes. Due to the expression of GPR-9-6 on mucosal homing lymphocytes, we examined the distribution of TECK and GPR-9-6 transcripts in lymphoid and mucosal tissues . TECK was selectively expressed in thymus and small intestine . GPR-9-6 was expressed at high levels in thymus and weakly in spleen and PBLs . Although we could not detect GPR-9-6 transcripts by Northern blot analysis in small intestine, we were able to detect GPR-9-6 message in small intestine and thymus using the more sensitive technique of RT-PCR . Message for either gene was not detected in brain or colon. Also as predicted from our expression data on cell lines with mAb 3C3, GPR-9-6 was expressed by Molt-13 cells (and Molt-4 cells, data not shown) but not in GPR-9-6 − JY, KU812, and EOL cells. In additional Northern blot analysis, TECK and GPR-9-6 were not detected in Th1, Th2, or T regulatory type 1 lymphocytes, LAK (lymphokine-activated NK) cells, monocytes, CD34-derived dendritic cells, monocyte-derived dendritic cells, astrocytes, high umbilical vein endothelial cells, or pulmonary vein endothelial cells (data not shown). Selective expression of adhesion molecules on T lymphocyte subsets mediates trafficking to distinct physiologic locations, such as peripheral LNs 37 , intestinal sites 15 38 , and different tissue inflammatory sites 13 39 40 41 . It is thought that specific chemokine receptors expressed on these lymphocyte subsets interact with chemokines expressed in these areas to mediate leukocyte activation, arrest, and transendothelial migration. Therefore, it is possible that lymphocyte subsets defined by their expression of certain adhesion molecules may also express known, orphan, or as yet undiscovered chemokine receptors that will be important in trafficking of the lymphocytes into these sites. Our work describes one such chemokine receptor that is selectively expressed at high levels on thymocytes, gut-homing memory CD4 and CD8 lymphocytes, and intestinal mucosal lymphocytes. GPR-9-6 was originally chosen as a potentially interesting orphan chemokine receptor due to its strong phylogenetic linkage with other known chemokine receptors including CCR6 and CCR7. To evaluate its function, we generated GPR-9-6 transfectants and performed studies to determine which chemokines induced chemotaxis of GPR-9-6 + cells. Out of all the chemokines tested, only TECK 42 acted as a chemoattractant for GPR-9-6/L1.2 transfectants. In contrast, TECK did not induce chemotaxis of L1.2 cells transfected with CCR1,2,4–7 or 9 or CXCR1–4 or 5. Although TECK also induced chemotaxis of CCR3/L1.2 transfectants, this was a relatively weak effect, inducing only 20% of the chemotactic mobility observed with eotaxin-1. Thus, TECK appears to have a restricted effect. In addition, in examining tumor cell lines for chemotaxis to TECK, GPR-9-6 expression directly correlated with chemotaxis. TECK-mediated chemotaxis of GPR-9-6 + transfectants, tumor cell lines, thymocytes, and CD4 lymphocytes was inhibited by anti–GPR-9-6 mAb 3C3. Thus, GPR-9-6 appears to represent the main chemokine receptor through which TECK acts on these cell populations, and to be a selective receptor for TECK. Like other chemokine receptors, the chemotaxis of GPR-9-6/L1.2 transfectants to TECK appears to be mediated exclusively through G i signaling, as treatment with pertussis toxin abolishes transfectant migration to TECK. In dose–response curves, 150 nM TECK resulted in optimal chemotaxis of GPR-9-6–expressing transfectants. This falls into the range of 1 nM to 1 μM at which other leukocyte chemokines are active. However, as a recombinant TECK produced by E . coli (PeproTech) was used for these studies, it remains possible that proper posttranslational modifications of the chemokine may not have occurred and that the recombinant TECK has a somewhat different activity than naturally produced TECK. In addition, further cleavage of TECK by factors outside the cell in vivo could generate more active fragments, as is the case for CKβ8 43 . Thus, the actual dose–response curve in vivo may differ from that determined using the reagents currently available. In published reports and in our own analysis, TECK is prominently expressed in the thymus. Therefore, we evaluated whether GPR-9-6 is expressed on thymocytes. Indeed, GPR-9-6 mRNA was detected prominently in thymic tissue based on Northern blot analysis. In addition, based on two- and three-color flow cytometry, GPR-9-6 polypeptide was expressed on the cell surface of the majority of thymocytes bearing all levels of TCR. Thus, GPR-9-6 is apparently expressed at all stages of T lymphocyte development. However, there was a small subpopulation of TCR high thymocytes that lacked GPR-9-6 expression. Further, most naive peripheral blood T lymphocytes derived from adults were either GPR-9-6 − or expressed low levels of GPR-9-6 based on FACS ® analysis. Thus, it appears likely that GPR-9-6 is downregulated near the time of thymocyte exit to the periphery. The expression of both GPR-9-6 and its ligand in the thymus demonstrates that TECK and GPR-9-6 probably interact in vivo as well as in vitro. This chemokine receptor–ligand pair is likely to play a role in thymocyte development, perhaps through effects on thymocyte localization or activation. TECK was originally cloned from fetal intestine 42 , and our Northern blot analysis revealed abundant TECK mRNA expressed in small intestine tissue derived from adults. Thus, it seemed possible that TECK might also function to recruit circulating lymphocytes to the intestine. To evaluate this possibility, we determined the expression of GPR-9-6 on PBLs. Although most PBLs lacked cell surface GPR-9-6, there was a small subset of peripheral T lymphocytes that was GPR-9-6 + . In three-color experiments, GPR-9-6 was found predominantly on memory CD4 and CD8 lymphocytes that coexpressed high levels of the gut-homing receptor, α4β7. However, in the case of CD8 lymphocytes, we observed a greater correlation of GPR-9-6 and αE expression. GPR-9-6 + T lymphocytes also lack expression of CLA, which is consistent with a possible role of TECK in gut T lymphocyte localization. Furthermore, TECK induced chemotaxis of a small subset of CD4 lymphocytes that were enriched in the β7 + memory CD4/CD8 subset that expresses GPR-9-6. In addition, although we did not detect chemotaxis of total CD8 lymphocytes to TECK, we were able to identify a subset of αE + α4β7 + CD45RA low/− CD8 lymphocytes that was enriched in the population of cells that migrated in response to TECK, suggesting that TECK induced migration of this CD8 lymphocyte subset. Thus, the GPR-9-6 expressed on memory CD4 and CD8 lymphocytes is functional to induce chemotaxis. In contrast to GPR-9-6 expression, CCR4 is expressed on CLA + or CLA − α4β7 − circulating memory CD4 lymphocytes 13 38 44 , but not on α4β7 high cells. Thus, CCR4 and GPR-9-6 may define nonoverlapping subsets of memory CD4 lymphocytes that traffic to different sites. GPR-9-6 was also expressed on a substantial proportion of B lymphocytes in peripheral blood, both with 3C3 and also with a second anti–GPR-9-6 mAb (data not shown), and staining of B lymphocytes with 3C3 was blocked by TECK. However, B lymphocytes were not chemotaxic for TECK. This may reflect either the reduced motility of these cells or the failure of GPR-9-6 to mediate chemotaxis of B lymphocytes, as observed for CCR1 on neutrophils. Although the evidence suggests that a subset of B lymphocytes that express GPR-9-6 does exist, the function of GPR-9-6 on these cells is not clear. If GPR-9-6 plays a role in the localization of T lymphocytes to the intestine, its expression might be enhanced on T lymphocytes in this tissue site. Indeed, while we did not detect GPR-9-6 transcripts in small intestine using relatively less sensitive Northern blot analysis, GPR-9-6 transcripts were detected in small intestinal tissue via RT-PCR. Further, flow cytometry revealed GPR-9-6 expression on almost all intestinal IELs and lamina propria T lymphocytes isolated from normal individuals undergoing gastric bypass surgery. Thus, expression of GPR-9-6 was greatly enriched on CD4 and CD8 lymphocytes present in intestinal tissue compared with its expression on peripheral T lymphocytes. The expression of TECK in small intestine and of GPR-9-6 on a subset of intestinal trafficking T lymphocytes indicates that TECK may play a role in the trafficking of GPR-9-6 + CD4 and CD8 lymphocytes to intestinal sites. It is possible that expression of TECK by postcapillary venules in the lamina propria or by Peyer's patch HEVs may activate GPR-9-6 + T lymphocytes and lead to leukocyte arrest via α4β7–mucosal addressin cell adhesion molecule 1 (MAdCAM-1) interactions, and thus in the selective extravasation of memory intestinal T lymphocytes within the intestine. Additionally, as the majority of resident intestinal IELs and LPLs express GPR-9-6, TECK may play a role in their localization within intestinal tissue or effector action at these sites. For example, it is possible that selective expression of TECK by epithelial or other cells in the small intestine could direct GPR-9-6 + T lymphocyte migration after the cells have crossed the endothelium and entered the intestinal tissue. Finally, although the majority of CD4 lymphocytes that express GPR-9-6 are memory cells, a small subset of naive phenotype CD4 lymphocytes also expresses GPR-9-6, albeit at lower levels. These may represent T lymphocytes that have recently emigrated from the thymus. Alternatively, a subpopulation of naive T lymphocytes may traffic to sites of TECK expression in the small intestine. It is of interest that while most GPR-9-6 high memory CD4 lymphocytes do not express CD62L, GPR-9-6 low naive CD4 lymphocytes do express CD62L. High levels of expression of α4β7 (such as those observed on memory CD4 lymphocytes) are thought to be sufficient to mediate lymphocyte tethering and rolling via MAdCAM-1 on lamina propria as well as HEVs in Peyer's patches. However, low levels of expression of this integrin (such as those observed on naive CD4 lymphocytes) are not thought to be sufficient 16 17 . It is possible that the coexpression of CD62L and low levels of α4β7 on GPR-9-6 low naive CD4 lymphocyte could allow initiation of rolling on Peyer's patch HEVs, with resulting arrest after lymphocyte activation and LFA-1 and α4β7 avidity upregulation. We postulate that factors present in the mucosal environment lead to the induction of GPR-9-6 on T lymphocytes as well as TECK expression. Cytokines present in Th1/Th2 environments, at least in vitro, induce expression of certain chemokine receptors, such as CCR4 on Th2 and CXCR3 on Th1 lymphocytes, as well as the production of the chemokines that bind these receptors 25 26 27 28 29 30 . However, these conditions did not upregulate GPR-9-6 expression on T lymphocytes. Also, our attempts to induce expression of GPR-9-6 on activated umbilical CD4 lymphocytes with cytokines IL-1–13, IL-15, IL-17, IL-18, and TGF-β, previously shown to induce αE on T lymphocytes 45 , failed to identify a cytokine that upregulates GPR-9-6 expression. Therefore, there must be an as yet undefined mechanism by which GPR-9-6 expression is controlled on T lymphocytes. In addition to studying the effect of cytokines on chemokine receptor expression, we wanted to determine if GPR-9-6 was modulated after T lymphocyte stimulation via antigen receptor cross-linking. Upon activation via TCR cross-linking, expression of GPR-9-6 is downregulated, as are other chemokine receptors such as CCR5, CCR6, and CXCR4 46 . When the TCR is disengaged, GPR-9-6 is reexpressed by the T lymphocyte. As TCR cross-linking mimics antigen presentation, we conclude that upon TCR cross-linking via appropriate MHC class II–peptide complexes expressed by APCs, T lymphocytes will downregulate chemokine receptors such as GPR-9-6. By this method, T lymphocytes will be held in association with either dendritic cells or B lymphocytes until the relevant signals involved in antigen presentation or T–B cognate interactions have occurred. In summary, we have shown that orphan chemokine receptor GPR-9-6 is expressed on the majority of thymocytes, intestinal IELs, LPLs, and on discrete subsets of T lymphocytes that traffic to intestinal sites. The CC chemokine TECK interacts with GPR-9-6 and mediates the chemotaxis of cells bearing this receptor. Based on the demonstration that GPR-9-6 induces chemotaxis of cell populations in a dose-dependent fashion in response to a chemokine, we propose to name this orphan chemokine receptor CCR-9. These findings, together with the selective expression of TECK and GPR-9-6 in thymus and small intestine, imply a dual role for GPR-9-6, both in T cell development and in the intestinal immune response. While this manuscript was in submission, Zaballos et al. 47 reported that TECK acts as a chemoattractant for HEK293/human GPR-9-6 transfectants as well as for the Molt-4 cell line. | Study | biomedical | en | 0.999996 |
10544197 | CB.17 scid/scid and 129 × C57BL pfp/rag-2 double-knockout (RAG/NK −/− ) mice (Taconic) were maintained in microisolator cages in our animal facility at Baylor College of Medicine. Age-matched BALB/c mice were used as controls. Mice were irradiated (2 Gy) by exposure to a 137 Cs source. Intrathymic injections were performed as described 17 . In brief, mice were anesthetized by methoxyflurane (Metofane; Mallinckrodt Veterinary, Inc.), and a midline incision was made in the skin overlying the lower cervical and upper thoracic region. The upper third of the sternum was bisected longitudinally with fine scissors to expose the thymus. Suspensions (5–10 × 10 6 cells) of bone marrow cells in PBS were injected into the anterior superior portion of either thymus lobe (10–40 μl/site) using a 1-ml syringe equipped with a 28-gauge needle. The incision was then closed with sutures, and animals were allowed to recover in a warm enclosure. DNA was prepared as described previously 4 . In brief, thymic cell suspensions were subjected to lysis buffer containing SDS and proteinase K, followed by phenol extraction and ethanol precipitation. PCR assays were performed by using 100 ng genomic DNA in a total volume of 50 μl with 1 U of Taq polymerase (Perkin-Elmer Corp.) in a buffer containing 2 mM MgCl 2 . 30 cycles of amplification were carried out in a PE 9600 thermal cycler (Perkin-Elmer Corp.). Each cycle consisted of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR products were separated on a 6% polyacrylamide gel, blotted onto GeneScreen Plus nylon membrane (DuPont), and hybridized to 32 P end-labeled internal oligonucleotide probes. Probes: Vβ8 (DR154), 5′-GGGCTGAGGCTGATCCATTA-3′ 7 ; Jα49 (DR183), 5′-GGACTCACTGTGAGCTTTGC-3′ 8 ; and Vγ2 (DR147), 5′-ACCATACACTGGTACCGGCA-3′ 18 . PCR primers: Vβ8.1,2,3 (DR144), 5′-GAGGAAAGGTGACATTGAGC-3′ 7 19 ; Jβ2.6 (DR155), 5′-GCCTGGTGCCGGGACCGAAGTA-3′ 7 20 ; Vα8 (DR197), 5′-cgccactctccataagagcagcagc-3′; Jα49 (DR184), 5′-CATGCCC-ATCAGTTGGTGTGAAAG-3′(8); Vγ2(DR148), 5′-AAGGAA-TTCATCGAAAGCTTTAGGAG-3′ 18 ; and Jγ1 (DR127), 5′-CCCTCGAGCTTTGTTCCTTCTGCAAATAC-3′ 21 . Thymi were homogenized, and cells were washed and counted. Cells were stained with anti-CD4 (RM4-4), anti-CD3 (145-2C 11 ), and anti-CD8 (53-6.7) mAbs that were conjugated with Cy-Chrome, PE, and FITC (PharMingen). Thymocytes were analyzed on an EPICS XL-MCL cytometer (Coulter Immunology). Thymocytes were gated by forward and side scatter properties; three-parameter histograms are shown. We employed an adoptive transfer approach using young adult (4–6-wk-old) pfp −/− / RAG-2 −/− mice (hereafter termed RAG/NK −/− ) as recipients. These animals, generated by crossing RAG-2–deficient mice with perforin-deficient mice, were used as hosts because they lack mature T and B cells and are incapable of rescuing V(D)J recombination after irradiation 22 . Therefore, any cells bearing TCR rearrangements could be attributed to the SCID donor. Because NK cells are the major mediators of graft rejection in this system, loss of the perforin cytolytic function ensures the survival of the donor cells. We first attempted to test the possibility that irradiation rescue might be mediated by intrathymic precursor cells. Although thymocytes from BALB/c mice were capable of generating DP thymocytes when introduced into RAG/NK −/− recipients by intrathymic injection, in >40 intrathymic transfers of thymocytes from irradiated SCID mice, we failed to observe consistent development of DP thymocytes (data not shown). Furthermore, sensitive PCR analyses revealed no rescue of TCR rearrangements (data not shown). These data could indicate that intrathymic precursors are not the major mediators of the irradiation rescue effect; however, we cannot rule out the possibility that technical factors limited our ability to detect rescue. We next asked whether cellular elements residing in the bone marrow, such as primitive lymphocyte precursors, might be capable of mediating the irradiation response. As a control, we transferred unirradiated wild-type (BALB/c) bone marrow cells (of the same MHC haplotype as SCID mice) by intrathymic injection and analyzed recipient thymi after 16–23 d. RAG/NK −/− host thymi were reconstituted by injections of wild-type bone marrow, giving near normal to normal cellularity with appreciable proportions of both DP and SP thymocytes . This effect was not observed after transfer of unirradiated SCID bone marrow cells . In four out of five experiments, intrathymic injection of adult SCID bone marrow cells harvested immediately after whole body irradiation resulted in increased thymic cellularity and appearance of a distinct population of DP thymocytes in host animals after 16–23 d . Representative unirradiated and irradiated SCID thymocyte profiles are shown for comparison . In three out of five animals, distinct populations of CD4 SP cells (4–7% of total thymocytes) were also apparent. Further analysis of this CD4 + population revealed high levels of surface CD3 similar to those in the wild-type controls . Consistent with this finding, CD3 levels on the surfaces of the DP cells arising in transfers from irradiated SCID bone marrow were higher than those seen in irradiated SCID thymocytes . These data suggest that the transfer of irradiated bone marrow to RAG/NK −/− hosts results in even more effective rescue of thymocyte differentiation than simple irradiation of SCID mice. However, it should be noted that populations of SP thymocytes and stimulation of intermediate levels of CD3 expression have been observed previously in irradiated adult 6 and newborn 5 SCID mice. To determine whether V(D)J recombination was rescued in RAG/NK −/− hosts reconstituted with irradiated SCID bone marrow cells, we employed semiquantitative PCR assays for detection of TCR-β, -γ, and -α rearrangements. As expected, TCR-β, -γ, and -α coding joints were detected in RAG/NK −/− hosts reconstituted with wild-type bone marrow cells . TCR-β and -γ coding joints were detected in thymocytes of RAG/NK −/− hosts injected with irradiated SCID bone marrow at levels comparable to those seen in irradiated SCID thymocytes . Sequence analysis of cloned TCR Vβ8-Jβ2 rearrangements revealed that 7/15 junctions recovered from a single animal were unique, and all showed features of Vβ8 rearrangements isolated from irradiated SCID mice 5 . Only trace amounts of TCR Vα8-Jα49 coding joints were detected in recipients of irradiated SCID bone marrow or in irradiated SCID thymocytes , consistent with previous analyses of TCR-α rearrangements in irradiated SCID mice 8 9 . Taken together, these results show that irradiated SCID bone marrow cells can promote rescue of both thymocyte differentiation and TCR rearrangements when transferred to unirradiated host animals. These data suggest the possibility that effects on the bone marrow may play an important role in the thymocyte responses observed in irradiated SCID mice. The three cardinal effects of irradiation treatment on differentiation of SCID thymocytes are progression to the DP stage, proliferation, and rescue of V(D)J rearrangements. As these effects are limited to thymocytes, we and others have suggested that these phenomena result from effects of irradiation on intrathymic precursor cells, perhaps aided by important contributions from irradiated thymic stromal cells 5 6 7 8 9 . Our data demonstrate that transfer of irradiated bone marrow cells reconstituted all three features. These results show that irradiation of thymic stromal elements is not required for irradiation rescue and indicate that the irradiation effect can be mediated by lymphoid precursor cells at an earlier stage of differentiation than was previously thought. The failure of irradiated SCID thymocytes to promote rescue of rearrangements or differentiation when transferred to unirradiated host animals could mean that thymocytes are not the targets of the irradiation effect. However, technical factors could have prevented us from detecting an irradiation rescue response in this system. This interpretation is supported by studies of fetal thymic organ cultures. Although rescue of TCR rearrangements was not assessed, differentiation to the DP stage was observed in day 17 fetal thymi placed in organ culture immediately after irradiation of the SCID fetuses (Williams, C., J. Danska, and C. Guidos, personal communication). The current model for explaining the irradiation rescue effect postulates that irradiation transiently affects double strand break repair pathways in DN thymocytes that are (a) committed to the T cell lineage and (b) actively undergoing rearrangement at the TCR-γ, -δ, and -β loci 5 6 7 8 9 . Both of these postulates are undermined by our results, which show that irradiation of SCID mice affects lymphocyte progenitors before they leave the bone marrow, a much earlier stage of T cell differentiation than was previously thought. In fact, lymphocyte precursors capable of repopulating the thymus isolated either from bone marrow or indeed from the thymus are not T lineage restricted and can repopulate the B cell compartment if allowed to enter the bone marrow of a lethally irradiated host 23 24 25 . Although the presence of TCR-β and -γ germline transcripts in bone marrow cells 26 27 suggests that these loci may be accessible to the recombinase machinery, only incomplete (D–J) TCR-β rearrangements have been detected in bone marrow cells by PCR 27 . Furthermore, we could not detect V(D)J rearrangements in SCID bone marrow (with or without irradiation) using PCR assays (Wang, C., M. Bogue, and D. Roth, unpublished data). Analysis of DNA from highly purified T lineage precursors isolated from murine bone marrow also failed to reveal evidence for TCR-γ or -β rearrangements 1 2 3 . Finally, as the bone marrow lymphoid precursors are not restricted to the T lymphocyte lineage, these cells should not be committed to rearrangement of their TCR loci at this stage. The considerations described above indicate that irradiation of cells before the onset of V(D)J recombination can rescue rearrangements. This possibility led us to consider a new model for the irradiation rescue effect. We suggest that irradiation induces a persistent response in bone marrow lymphoid progenitor cells that facilitates the joining of V(D)J recombination intermediates created at a later time during lymphocyte differentiation. There are several examples of such an effect in the literature. Increased radioresistance after exposure to low doses of X irradiation has been observed in mammalian cell lines, mouse tissues, and human lymphocytes (for review see reference 28 ). Based on these and other observations, it has been suggested that low doses (as little as 0.05–1 Gy) of irradiation induce persistent DNA repair activities (for review see reference 28 ). Induction of these activities in SCID cells might allow coding ends to bypass the SCID defect via an alternative joining mechanism. These activities would have to persist until the bone marrow precursor cells commit to the T lineage and initiate TCR rearrangements. The length of time required for these events is not known, nor do we know how long radiation-induced radioresistance might persist in this system. In cycling fibroblasts, induced radioresistance can persist for three generations 29 . Furthermore, other studies have shown that exposure of cells (including lymphocytes) to a single, low dose of ionizing radiation produces genomic instability that continues for many generations 30 31 . This phenomenon is correlated with an increased incidence of neoplastic transformation 31 and could reflect induction of aberrant recombination or repair activities 30 . These observations raise the possibility that radiation-induced alterations in DNA repair activities in bone marrow precursor cells might persist for many cell divisions. Irradiation-induced genomic instability could also be involved in generating the lymphomas that inevitably develop in irradiated SCID mice. It is, therefore, of great interest to determine if irradiated bone marrow precursors are capable of transferring lymphoma to unirradiated host animals. Why are TCR-α rearrangements not efficiently rescued? We have considered two possible explanations. First, as TCR-α recombines later in differentiation than the other TCR loci, perhaps the irradiation effect does not persist long enough. Many rounds of cell division are initiated at the DN–DP transition, and the irradiation-induced change in DNA repair potential may not be maintained through many cell cycles. This is consistent with the observation that induced radioresistance persists in fibroblasts for only three cell cycles 29 . A second, nonexclusive possibility is based on the observation that successful rearrangements at the other TCR loci, but not TCR-α, can undergo substantial amplification during the proliferation that accompanies the DN–DP transition. Thus, rescue of all loci may actually be rather inefficient, yet proliferation of cells that contain rare successful rearrangements amplifies the rescued rearrangements at the other loci but not at TCR-α. This hypothesis is supported by two observations: (a) >90% of rescued TCR-β rearrangements are in frame 5 and (b) rare TCR-α rearrangements have been detected in irradiated SCID thymocyte RNA by reverse transcriptase–PCR 5 , and we have repeatedly detected very low levels of TCR-α rearrangements in irradiated SCID thymocytes 8 . These could result from irradiation-mediated rescue of TCR-α recombination without the benefit of selective amplification provided by the proliferation that accompanies the DN–DP transition. A second question raised by our results is the failure of rescue to occur in the B cell lineage, an issue that is especially perplexing if irradiation is indeed inducing DNA repair activities in a lymphoid progenitor cell capable of giving rise to either B or T lymphocytes. We have considered several possible explanations. First, the SCID or RAG/NK hosts may lack appropriate niches in the bone marrow stroma, so that precursor cells cannot proceed with their differentiative program. Another possibility is that the cells in the B cell lineage may fail to proliferate sufficiently after irradiation to allow detection of rescued rearrangements. Studies in RAG −/− mice, which are incapable of V(D)J rearrangement, have shown that irradiation provides a signal for proliferation and differentiation that appears to be thymocyte specific 22 . This signal may be required for expansion of precursor lymphocytes containing rescued rearrangements. Further experiments will be required to answer these interesting questions. | Study | biomedical | en | 0.999997 |
10544198 | Mononuclear cells (MNCs) were obtained from whole blood from disease-free volunteers by density gradient centrifugation using Ficoll metrizoate (Lymphoprep; GIBCO BRL). MNCs were depleted of CD56 + NK cells using the MACS separation system (Miltenyi Biotec) and CD56-specific, paramagnetic beads. In pilot experiments, CD8 + or CD4 + cells were also depleted, and in all cases depletion was verified using flow cytometry (see below). In experiments with superantigens, cells were activated with SEA (Sigma Chemical Co.) in complete media (CM) containing RPMI (GIBCO BRL) supplemented with 10% fetal bovine serum (Hyclone) and 2 mM l -glutamine for 3 d, and SEA-reactive T cells were expanded in IL-2 and added to adherence-purified monocytes. Otherwise, purified OKT3 antibody (Coulter) was added to NK cell–depleted MNCs at a 1:1,000 dilution that strongly activated T cells in initial titration experiments, and cells were harvested after 3 d, washed, and used for experiments or expanded in IL-2 as noted in the figure legends. An aliquot was saved for flow cytometric analysis of cell surface phenotype, and remaining cells (>90% CD3 + ) were plated in wells that had been coated with 10 μg/ml of rabbit anti–mouse Ig and 3 μg/ml of either anti-CD3 (clone UCHT-1; PharMingen) or an isotype-matched irrelevant control antibody. Initial dose–response experiments demonstrated that the effects of anti-CD3 saturated at 1–2 μg/ml. Additional anti-CD3 antibodies that were used with similar results were 4B5 (Boehringer Mannheim) and HIT3a (Immunotech), and anti-CD4 antibodies (RPA-T4; PharMingen) were also used as an additional control. Cells were analyzed using flow cytometry as described previously 31 using the following antibodies: T cell and activation markers, UCHT-1 and 4B5 (anti-CD3), RPA-T4 (anti-CD4), HIT8a (anti-CD8), M-A251 (anti-CD25), CF1 and Mik-β3 (anti-CD122), AG184 (anti-γ c ); NK cell marker, 3G8 (anti-CD16); monocyte marker, IV.3 (anti-CD32 monocyte-specific epitope); and 48607 (anti–CC chemokine receptor 2 [CCR2]). AG184, M-A251, HIT8a, and Mik-β3 were obtained from PharMingen, 4B5 and 3G8 from Boehringer Mannheim, CF1 from Immunotech, IV.3 from Medarex, and 48607 from R&D Systems. Analyses were done using a FACScan™ flow cytometer with CELLQuest software™ (Becton Dickinson). Cell extracts were obtained as described previously 29 . Extracts corresponding to 3.3 × 10 5 cells (∼12 μg of protein) were incubated for 15 min at room temperature with 0.5 ng of 32 P-labeled double stranded oligonucleotide containing the gamma-activated sequence (GAS) STAT-binding site from the IFN regulatory factor (IRF) promoter in a 15-μl binding reaction containing 40 mM NaCl and 2 μg of poly-dI-dC (Amersham Pharmacia Biotech), as described previously 29 , and complexes were resolved on nondenaturing 4.5% polyacrylamide gels. For immunoblotting, cell lysates were fractionated on 7.5 or 10% polyacrylamide gels using SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore), and incubated with specific antibodies, and enhanced chemiluminescence was used for detection. mAbs against Janus kinase (Jak)1 (clone 73) and STAT5 (clone 89) were obtained from Transduction Laboratories, anti-cyclin D3 (D-7) from Santa Cruz Biotechnology, anti-underphosphorylated retinoblastoma protein (Rb, G99-549) from PharMingen, and antiphosphotyrosine (4G10) and anti-tyrosine phosphorylated STAT5 from Upstate Biotechnology. Polyclonal antibodies against Jak1 (Q-19), STAT5 (C-17), and cyclin D3 (C-16) were from Santa Cruz Biotechnology, and anti-tyrosine phosphorylated Jak1 was from Quality Control Biochemicals. In most cases, results were confirmed using two different antibodies. Immunoprecipitations were performed by adjusting cell extracts obtained from 20 × 10 6 cells to a volume of 0.5 ml and incubating with 2–4 μg of specific antibody for 4 h at 4°C. Immunoprecipitates were collected using protein A and protein G agarose beads (Pierce Chemical Co.), washed twice in lysis buffer and once in PBS, resolved by SDS-PAGE, and analyzed by immunoblotting. Jak1 and Jak3 immunoprecipitates were obtained from 40 × 10 6 cells that had been lysed in buffer containing 0.5% NP-40, 10% glycerol, 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM Na 3 VO 4 , and protease inhibitors, as described previously 29 , using Jak1 (Q-19) or Jak3 antibodies (provided by J. O'Shea, National Institutes of Health, Bethesda, MD). After washes in lysis buffer and kinase buffer containing 20 mM Hepes (pH 7.4), 50 mM NaCl, 5 mM MgCl 2 , 5 mM MnCl 2 , and 0.1 mM Na 3 VO 4 , 25% of immunoprecipitates were saved for immunoblotting, and the remainder was incubated for 30 min at room temperature in 40 μl of kinase buffer with 1 μCi of [γ- 32 P]ATP. 1 μM unlabeled ATP was added to Jak3 kinase reactions. 32 P-labeled proteins were washed in kinase buffer and PBS, and analyzed using SDS-PAGE and autoradiography. Kit225 cells, a human IL-2–dependent cell line 32 , were maintained in CM containing 20 U/ml of IL-2, and deprived of IL-2 for 24 h before transfection. 10 7 cells were transfected using electroporation and the Bio-Rad Gene Pulser (320 V, 960 μF) with 20 μg of total DNA (10 μg of 5× gamma response region [GRR] GAS-luciferase reporter ), 2 μg of lacZ encoding plasmid, and 8 μg of empty control vector or vector encoding constitutively active MEK1 (containing the S218E, S222D mutations and an NH 2 -terminal deletion of residues 30–49) or kinase-inactive MEK1 29 . Cells were stimulated with 100 U/ml of IL-2 for 6 h, and luciferase activity was measured as described previously 29 , and normalized for β-galactosidase activity encoded by the cotransfected control plasmid. Total cellular RNA was isolated using Trizol (GIBCO BRL) according to the instructions of the manufacturer. For reverse transcription (RT)-PCR, RNA was treated with RNase-free DNase, and cDNA was obtained using Moloney murine leukemia virus reverse transcriptase (GIBCO BRL). 2.5% of each cDNA was subjected to 25 cycles of PCR using conditions that result in a single specific amplification product of the correct size as described previously 33 : 30 s denaturation at 94°C, 1 min annealing at 55°C, and 30 s extension at 72°C in a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp.). dNTPs were used at 100 μM, and 1 μCi of [α- 32 P]dATP was added to each reaction. No amplification products were obtained when reverse transcriptase was omitted, indicating the absence of contaminating genomic DNA. Amplification was empirically determined to be in the linear range. Oligonucleotide primers for the cytokine-inducible SH2 protein (CIS) and PGE2R genes were provided by C. Beadling and K. Smith (Weill Medical College of Cornell University ), and additional primer sequences are Fos: CCG AGA TTG CCA ACC TGC TGA A and CAC TGG GCC TGG ATG ATG C; suppressor of cytokine signaling (SOCS)1 (three sets): GGA CGC CTG CGG ATT CTA CTG G and TCG CGG AGG ACG GGG TTG AG, CGA CGG CGG CCA GAA CCT TC and GTG AAA GCG GCC GGC CTG AAA GT, and CTC CGG CTG GCC CCT TCT GTA G and TGA AAG CGG CCG GCC TGA AAG TG; SOCS3: CAC TAC ATG CCG CCC CCT GGA G and TCG CCC CCG GAG TAG ATG TAA T; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): GTG AAG GTC GGA GTC AAC and TGG AAT TTG CCA TGG GTG. 10 5 cells in 100 μl of medium were seeded in triplicate in 96-well tissue culture plates that had been coated with 10 μg/ml of rabbit anti–mouse Ig and 3 μg/ml of anti-CD3 or control antibody. Cells were pulsed for 4 h with 10 μCi/ml of [ 3 H]thymidine and harvested using an automated cell harvester (Harvester 96; Tomtec), and [ 3 H]thymidine incorporation was quantitated using a Microbeta Trilux Scintillation Counter (Wallac). Cell counts were performed in duplicate, and cell viability was determined using trypan blue and propidium iodide exclusion. IL-2 is a major growth factor for activated T cells and plays an important role in lymphocyte expansion and differentiation during an immune response (for reviews, see references 35 36 37 38 ). Activation of STAT5 is an important component of IL-2 signaling 38 , and the effect of TCR cross-linking on IL-2 induction of STAT5 DNA binding activity was analyzed. Since staphylococcal superantigens are high-affinity naturally occurring TCR ligands that initially lead to vigorous immune responses, which subsequently are strongly limited 1 , the effect of SEA on IL-2 signaling was determined. Freshly isolated MNCs from normal donors were depleted of NK cells, activated with SEA, and SEA-reactive T cells were expanded in IL-2. T cells were then added to tissue culture wells containing APCs in the presence or absence of SEA for 1 h, stimulated with IL-2 for 10 min, and cell extracts were assayed for STAT DNA-binding activity by electrophoretic mobility shift assays . Treatment with IL-2 resulted in the rapid induction of a DNA–protein complex (lane 3) that bound the IRF GAS oligonucleotide specifically and corresponded to STAT5 ( 33 ; and data not shown), and treatment with SEA resulted in significant inhibition of IL-2 activation of STAT5 DNA binding activity (lane 4); similar results were obtained with SEB (data not shown). Immunoblotting demonstrated comparable levels of STAT5 in all extracts , indicating that SEA treatment suppressed activation of DNA binding and did not decrease STAT5 protein levels. To determine whether APCs or APC products were required for inhibition, TCRs were cross-linked using plate-bound anti-CD3 antibodies in the absence of APCs. Activation of STAT5 DNA binding was suppressed by TCR ligation . Suppression of STAT5 activation by TCR ligation (40–90% inhibition relative to control) was consistently detected in >40 independent experiments using different blood donors, and was observed with a broad range of IL-2 doses (between 2 and 100 U/ml), with cross-linking times of 0.5–4 h, with purified CD4 + or CD8 + cells, and with several antibodies that ligate the TCR, but not with irrelevant or anti-CD4 antibodies (data not shown). Consistent with previous reports that TCR signaling is less dependent on costimulation in activated T cells 39 , co-cross-linking of CD28 had no additional effect, but pharmacological mimicking of maximal TCR stimulation using PMA plus ionomycin more strongly inhibited STAT5 activation (data not shown). Tyrosine phosphorylation of STAT5 is required for DNA binding 38 , and the effect of SEA or TCR cross-linking on IL-2–induced tyrosine phosphorylation was examined. IL-2–induced tyrosine phosphorylation of STAT5 was suppressed by SEA treatment or TCR cross-linking . Immunoblotting demonstrated comparable levels of STAT5 in all extracts . Rapid and transient activation of STAT5 tyrosine phosphorylation, which has been reported by one group after TCR cross-linking (detected after 7 min, and absent after 15 min ), was not detected in the time frame of these experiments. The results showing inhibition of STAT5 tyrosine phosphorylation confirm the DNA binding results , and suggest that TCR ligation blocks STAT5 activation. The mechanism of inhibition of IL-2 signaling by TCR cross-linking was further explored using the more simple system with plate-bound antibodies, which eliminated potential contributions from APCs or ligation of additional T cell surface proteins. Activation of STAT5 by IL-2 depends on membrane-proximal events including the phosphorylation and activation of Jak1 and Jak3 kinases 38 . IL-2–induced tyrosine phosphorylation of Jak1 and Jak3 was suppressed by TCR cross-linking , and Jak kinase activity was suppressed in parallel . Similar results were obtained using an antibody specific for tyrosine-phosphorylated Jak1 (data not shown). These results indicate that TCR cross-linking suppressed the activation of IL-2 signaling upstream of STAT5 activation, and suggest that additional IL-2 signaling pathways downstream of the Jaks may be affected. Activation of the Akt kinase (also termed protein kinase B) by IL-2 is important for proliferation and suppression of apoptosis 32 41 , and the effect of TCR cross-linking on activation of Akt was investigated using immunoblotting with antibodies specific for the activated, phosphorylated form of Akt . TCR cross-linking effectively suppressed IL-2 activation of Akt (lane 4). These results indicate that TCR cross-linking in activated T cells blocks IL-2 signal transduction at a proximal step, and suppresses the activation of several signaling pathways important for the proliferative response to IL-2. TCR ligation could block STAT5 activation by inhibiting the expression or the function of components of the IL-2R–Jak-STAT signaling pathway. Expression levels of the IL-2R and the associated Jak1 and Jak3 kinases were investigated . Flow cytometry experiments demonstrated that, as expected, TCR ligation resulted in decreased cell surface expression of CD3 , thus confirming the efficacy of TCR engagement 39 . In contrast, TCR cross-linking did not have a significant effect on cell surface expression of the IL-2R α (CD25), β (CD122), or γ c chains . TCR cross-linking also had no effect on cellular levels of Jak1 or Jak3 . The results demonstrating no changes in IL-2R, Jak1, Jak3, and STAT5 levels after TCR cross-linking indicate that cross-linking does not modulate the expression of the components of the IL-2R–Jak-STAT pathway that are required for STAT5 activation, and suggest instead that TCR cross-linking modulates their function. We have previously demonstrated that triggering of the kinase cascade leading to MEK and ERK activation blocks IL-6 activation of Jaks upstream of STAT3 activation without altering expression of IL-6Rs or Jaks 28 29 , and others have shown that activation of the MEK-ERK pathway can lead to suppression of proliferation 26 27 . Since cross-linking of the TCR activates a kinase cascade involving PKC→Raf→MEK1, MEK2→ERK1, ERK2, we investigated whether these kinases play a role in inhibition of IL-2 signaling . The specific PKC inhibitor GF109203X, but not the calcineurin inhibitor cyclosporin A, reversed TCR inhibition of STAT5 activation , and inhibition was also reversed by the MEK inhibitor PD98059 . These results suggest that the PKC-dependent activation of the MEK-ERK pathway plays an important role in inhibition of IL-2 signaling. The role of this pathway was further investigated using PMA, a direct activator of PKC and a strong activator of downstream MEKs and ERKs. Preincubation for 15 min with PMA resulted in a strong suppression of STAT5 activation , consistent with the rapid inhibition of IL-6 signaling by this pathway 29 . PMA treatment did not alter IL-2R expression (data not shown), and cytokine signaling was not globally inhibited by PMA, since IFN-α signaling was not inhibited in T cells or in myeloid cells 29 . The role of MEKs and the functional significance of inhibition of IL-2 signaling mediated by these kinases was investigated using transient transfections of the IL-2–responsive Kit225 cell line and reporter gene assays . IL-2 treatment induced a sevenfold increase in activity of the 5× GRR GAS-luciferase reporter gene, which contains five copies of the GAS site from the FcγRI promoter, and was previously shown to be induced in these cells in an IL-2– and STAT5-dependent manner 32 . This induction was nearly completely blocked when cells were cotransfected with a plasmid encoding constitutively active MEK1, but not kinase-inactive MEK1 . These results, taken together, indicate that activation of the PKC→ Raf→MEK→ERK pathway plays an important role in inhibition of IL-2 signaling by TCR ligation in activated T cells, consistent with previously reported MEK-dependent inhibition of proliferation in PC12 cells and primary fibroblasts 26 27 . We have previously shown that rapid MEK-ERK–dependent inhibition of IL-6 signaling occurs independently of new protein or RNA synthesis 29 , presumably through phosphorylation of preexisting signaling components. Inhibition of IL-2 signaling by PMA occurred within 15 min ; inhibition by TCR ligation occurred reproducibly within 30 min, and was less consistent with shorter incubation periods, possibly secondary to the time required for cells to settle from suspension onto the bottom of antibody-coated wells (data not shown). The requirement for new protein and RNA synthesis for inhibition of IL-2 signaling by TCR ligation was investigated using cycloheximide and actinomycin D under conditions where these agents effectively blocked new protein and RNA synthesis, respectively ( 28 29 31 ; and data not shown). TCR ligation blocked IL-2 activation of STAT5 in the presence of cycloheximide and actinomycin D , indicating that inhibition can occur in the absence of new synthesis of inhibitory molecules. These results do not exclude the possibility that TCR ligation may induce expression of inhibitory molecules that contribute to inhibition of IL-2 signaling, especially at later time points. SOCS1 and SOCS3 are the two members of the SOCS family of proteins that are currently known to inhibit Jak-STAT signal transduction, and therefore the expression of these genes after TCR ligation was examined . Semiquantitative RT-PCR that had been validated using Northern hybridization was used as described previously 33 . Positive control panels show that Fos and IFN-γ mRNA levels were dramatically induced by TCR ligation with the expected kinetics (third and fourth panels). SOCS3 mRNA was modestly and transiently increased after TCR ligation (top panel), suggesting that SOCS3 may contribute to inhibition of IL-2 signaling. Activation of the MEK-ERK pathway by PMA did not increase SOCS3 mRNA levels (top panel, lane 6), indicating that inhibition of IL-2 signaling by the MEK-ERK pathway is not mediated by SOCS3. SOCS1 mRNA levels were regulated in a complex fashion by TCR ligation (second panel); this result was obtained in multiple independent experiments using three different sets of SOCS1 primers. Although we have not yet resolved whether the regulation of SOCS1 may involve opposing effects on transcription and RNA stability, it is clear that neither TCR ligation nor PMA treatment resulted in increased SOCS1 levels. Taken together, the results indicate that one rapid, MEK-dependent component of inhibition of IL-2 signaling is independent of the synthesis of new inhibitory proteins, and suggest that inhibition of IL-2 signaling by the MEK-ERK pathway is not dependent on induction of SOCS1 or SOCS3 proteins. We have not excluded the possibility that induction of inhibitory proteins, such as SOCS3, may contribute to inhibition of IL-2 signaling, possibly at later time points. The functional consequences of TCR-induced inhibition of IL-2 signaling were investigated by analyzing expression of CIS, PGE2R, and CCR2, which are induced by IL-2, but not by the TCR 34 42 43 . Ligation of the TCR before addition of IL-2 effectively blocked the induction of CIS and PGE2R mRNA , and suppressed expression of CIS and PGE2R genes even when cells were exposed to high doses of IL-2 before TCR ligation . PMA plus ionomycin more strongly inhibited expression of IL-2–dependent genes , which correlated with stronger inhibition of STAT5 (data not shown). CIS transcription is STAT5 dependent 43 , and the CIS and PGE2R genes are regulated by IL-2 at the transcriptional level, indicating that TCR ligation likely inhibited gene expression by blocking IL-2–induced transcription. TCR-mediated suppression of IL-2 induction of CCR2 42 was confirmed in our system using flow cytometry . These results, together with the suppression of IL-2 activation of reporter gene activity , demonstrate that inhibition of IL-2 signaling by TCR ligation in activated T cells has important functional consequences for modulation of gene expression. Important events for IL-2–induced progression into S phase of the cell cycle include upregulation of cyclin D3 and cdk2 levels 32 44 45 , leading to increased cdk activity and phosphorylation of Rb. TCR ligation in rapidly proliferating, IL-2–dependent T cells resulted in downregulation of cyclin D3 and cdk2 levels . Downregulation of cyclin D3 and cdk2 levels would be predicted to result in increased levels of underphosphorylated Rb, and suppression of proliferation. As predicted, TCR cross-linking resulted in increased levels of underphosphorylated Rb . The effects of PMA or PMA plus ionomycin were comparable to, or greater than, the effects of TCR ligation , and PMA plus ionomycin increased underphosphorylated Rb levels more dramatically in other experiments (data not shown). These results show that TCR ligation, or pharmacologic mimicking of TCR ligation, blocks IL-2 signals that are important for cell cycle progression. Proliferation of activated T cells in these cultures was highly dependent on exogenous IL-2 (data not shown), consistent with previous reports 22 . TCR ligation in rapidly proliferating, IL-2–dependent T cells led to a 65% decrease in T cell proliferation as assessed by [ 3 H]thymidine incorporation . Cell counts performed on parallel wells at the same time point (after 4 h of TCR ligation) demonstrated comparable numbers of viable cells (data not shown), indicating that decreased [ 3 H]thymidine incorporation reflected inhibition of proliferation rather than cell death. PMA inhibited proliferation comparably to TCR cross-linking, and PMA plus ionomycin was more effective . The effect of TCR ligation on T cell expansion was determined. 24 h after plating of proliferating IL-2–dependent T cells, control cultures demonstrated a 90% increase in the number of viable cells, whereas cultures subjected to TCR ligation demonstrated only a 10% increase in viable cells . The decreased cell numbers in TCR-stimulated cultures were primarily secondary to suppression of growth, although a small increase in cell death was detected 24 h after TCR ligation . Direct cross-linking of Fas receptors using agonist antibodies at this early point in culture (after 2 d of culture in exogenous high dose IL-2) had minimal effect on cell numbers, indicating that the decrease in T cell numbers detected was not secondary to a Fas-mediated mechanism. As predicted, TCR signaling affected the distribution of T cells in different phases of the cell cycle, and Fig. 8 C shows a decrease in actively cycling T cells in S and G2/M phases of the cell cycle after SEA treatment. SEA treatment induced an increase in apoptosis, as assessed by numbers of cells containing subdiploid levels of DNA, from 4 to 12% of cells , and the possible role of a Fas-mediated mechanism in apoptosis has not yet been investigated. TCR ligation resulted in decreased cell numbers throughout the subsequent 5 d of culture, with a resulting fourfold lower yield of viable cells in TCR-stimulated as opposed to control cultures . The effect of PMA was comparable to, and the effect of PMA plus ionomycin greater than, the effect of TCR ligation . Increased cell death contributed more to the lower cell recovery 5 d after TCR cross-linking than 1 d after TCR ligation . Cells became sensitive to apoptosis triggered by cross-linking cell surface Fas receptors during the final 2–3 d of these cultures (data not shown), and the relative contributions of cytokine withdrawal and Fas-dependent mechanisms to this increased cell death have not yet been resolved. These results demonstrate that TCR-induced inhibition of IL-2 signaling leads to growth arrest, and suggest that inhibition is linked to subsequent cell death. In T cell activation, initial encounter with antigen and TCR ligation induces quiescent cells to enter the G1 phase of the cell cycle and to express cytokine receptors, and renders them competent to progress through the cell cycle in response to cytokines and growth factors such as IL-2 22 . In this initial phase, the cells are not cytokine dependent, and MEK-ERK signaling downstream of TCR ligation plays a role in activation and priming for expansion 25 . Upon subsequent exposure to cytokines, T cells enter a proliferative phase during which they are cytokine dependent for growth and survival 22 . At this point, persistence of antigen leads to negative feedback and limitation of clonal expansion. The best-characterized mechanism of negative feedback is TCR-triggered apoptosis mediated by death receptors such as Fas and TNF receptor 1 14 , and defects in apoptosis have been implicated in autoimmunity in mice and human patients 14 46 47 48 . The results described herein identify TCR-induced, rapidly acting, and MEK-dependent inhibition of IL-2 signaling as a novel physiological mechanism that underlies growth arrest of primary proliferating T cells. Given the recent evidence that TCR-induced apoptosis is dependent on prior growth arrest 15 , it is possible that TCR-induced blockade of IL-2 signaling and subsequent growth arrest may represent an important first step that, along with additional factors, makes cells susceptible to undergo apoptosis at a later time point. Apoptosis may be secondary to decreased survival signals or may occur by a Fas-dependent mechanism, and the relationship between inhibition of IL-2 signaling and apoptosis will be further investigated in future work. Growth arrest was observed before the onset of significant levels of apoptosis , and may correspond to an additional level of limiting T cell responses that functions in addition to apoptosis. This growth arrest may contribute to the limitation in T cell expansion in the absence of cell death that has been described under certain circumstances, such as high dose paralysis occurring in the presence of high doses of antigens, and in persistent infections 2 9 11 12 23 . IL-2–dependent T cell expansion was limited but not blocked, suggesting that this mechanism does not completely prevent the normal progression of an immune response, but that interplay between TCR and cytokine signals of varying intensities and at different time points in T cell activation determines the magnitude of the response. At first glance, inhibition of IL-2 signaling and proliferation by an MEK-dependent mechanism may appear paradoxical, since activation of the MEK-ERK pathway by the TCR in resting T cells primes the cells for proliferation 22 25 49 , and activation of the MEK-ERK pathway by Ras has been linked to proliferation in transformed cells 50 . However, it is becoming increasingly clear that the effects of MEK-ERK signaling on cell growth depend on the physiological state of the cell, and either proliferation or growth arrest and senescence may occur 25 26 27 . During activation, T cells progress from a quiescent, cytokine-independent state to a proliferative state where growth and survival are exquisitely cytokine dependent. It is only during this cytokine-dependent state that the antiproliferative aspects of TCR-triggered MEK-ERK signaling become apparent, through inhibition of cytokine signal transduction. TCR ligation and MEK-ERK signaling blocked IL-2 induction of STAT5, activation of a STAT5-dependent reporter gene , and activation of expression of the IL-2–inducible CIS, PGE2R, and CCR2 genes . The CIS gene is activated primarily by STAT5 43 , and thus TCR-mediated inhibition of CIS expression provides a functional correlate to inhibition of STAT5 activation. It is not yet clear if inhibition of PGE2R and CCR2 expression is secondary to blocking STAT5 or other IL-2 signaling pathways, but inhibition of these genes clearly demonstrates that TCR inhibition of IL-2 signaling has important functional consequences for T cell phenotype. Inhibition of IL-2 signaling also resulted in inhibition of IL-2–dependent proliferation. Suppression of proliferation may be mediated, at least in part, through inhibition of STAT5, which has been shown to play an important role in proliferation of primary T cells 51 52 53 . Another important mechanism by which IL-2 stimulates T cell proliferation is through induction of cdk2 54 and cyclin D3 expression 32 45 , with concomitant phosphorylation of Rb and progression from G1 into S phase of the cell cycle. IL-2 regulation of cyclin D3 and Rb has been shown to be mediated by Akt 32 , and activation of Akt was blocked by TCR cross-linking . As predicted, this resulted in decreased levels of IL-2–induced cyclin D3 and cdk2 expression, and decreased phosphorylation of Rb . Thus, our results demonstrate that TCR ligation blocked IL-2–dependent proliferation of T cells by blocking at least two signaling pathways important for proliferation. A recent report by Welte et al. 40 described rapid activation of STAT5 tyrosine phosphorylation that was detected 7 min after TCR ligation in the murine D10 Th2 clone, and was very transient, such that tyrosine phosphorylation was not detected 15 min after TCR ligation. TCR activation of STAT5 tyrosine phosphorylation was significantly weaker than activation by IL-2, and activation of STAT5 DNA binding after TCR ligation was detected only in transfected 293 cells that overexpressed the relevant signaling components 40 . In our system, STAT5 activity was examined 0.5–4 h after TCR ligation, and thus we would not have detected very rapid and transient activation of STAT5. Our results are consistent with a previous report that TCR ligation did not induce STAT5 activation 55 . Welte et al. suggested that TCR-mediated STAT5 activation played a role in TCR-induced proliferation based on results obtained using overexpression of a dominant negative STAT5-Y694F, which contains a mutation in the STAT5 tyrosine residue that becomes phosphorylated. In contrast to Welte et al. 40 , we show that TCR ligation blocks subsequent activation of STAT5 by IL-2, and blocks IL-2–dependent proliferation . The most likely explanation for these different findings is that they represent differences between quiescent T cells, where TCR ligation induces proliferation 40 , and actively cycling T cells, where TCR ligation induces growth arrest . Alternatively, although D10 cells do not produce IL-2, they produce proliferative cytokines such as IL-4, and high levels of overexpressed STAT5-Y694 may have resulted in loss of specificity in receptor docking and blocked proliferation 40 by blocking STAT signaling by cytokines such as IL-4. Inhibition of cytokine signaling is a rapidly emerging area, and several constitutive mechanisms of downmodulating STAT activity have been described, including dephosphorylation, proteolytic degradation, or association with inhibitory molecules 56 57 58 59 60 61 . We have previously described a rapidly inducible mechanism of inhibition of IL-6 signaling 28 29 . Similarities between the mechanisms of inhibition of IL-6 signaling in myeloid cells and of IL-2 signaling in T cells include rapid induction within 15–60 min of addition of inhibitory factors, independence of new protein or RNA synthesis (at early time points), blockade of signaling at a proximal step in signal transduction without any changes in expression of receptors, Jaks, or STATs, and dependence on the MEK-ERK kinase cascade. These results suggest that inhibition of IL-2 and IL-6 signaling is achieved through MEK-dependent modification of IL-2 and IL-6 signaling components. Another previously described inducible mechanism for inhibiting the Jak-STAT pathway is cytokine-mediated induction of expression of SOCS/Jak binding (JAB)/STAT-inducible STAT inhibitor (SSI) proteins, which interact with and inhibit Jaks 62 63 64 . Induction of SOCS3 may contribute to the inhibition of IL-2 signaling , and inhibition of IL-6 signaling by GM-CSF at later time points also depended on new protein synthesis, likely of SOCS proteins 28 . Thus, two distinct inhibitory mechanisms, mediated by MAPKs and SOCS proteins, may function in tandem to achieve effective inhibition of signaling at both early and later time points after addition of an inhibitory factor. Inhibition of IL-2 and IL-6 signaling in T cells, myeloid cells, and several cell lines by the MEK-ERK pathway indicates that this inhibitory mechanism functions in a number of cell types and physiological settings. Interestingly, additional important immune receptors that activate the MEK-ERK pathway, FcRs and CR3, have also been shown to inhibit cytokine and Jak-STAT signaling, although the mechanisms of inhibition and the role of the MEK-ERK pathway were not resolved 65 66 . Thus, since the major receptors that mediate interactions of immune cells with antigenic stimuli, antigen, Fc, and complement receptors, activate the MEK-ERK pathway, cross-talk between the MEK-ERK and Jak-STAT pathways may play a particularly important role in modulation of cytokine signaling in immune cells. | Study | biomedical | en | 0.999996 |
10544199 | C57BL/6J (Ly5.1), C57BL/6 (B6)–IL-12β tm1Jm 27 , and B6-Tnfsf5 tm1Imx (CD40L −/− ; reference 28 ) mice were purchased from The Jackson Laboratory. C57BL/6TacfBr-[KO]Aβ b mice 29 were purchased from Taconic Farms Inc. B6-Ly5.2 mice were obtained from Charles River Laboratories through the National Cancer Institute animal program. The OT-I mouse line was provided by W.R. Heath (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) and F. Carbone (Monash Medical School, Prahran, Victoria, Australia) 30 and was maintained as a B6-Ly5.2 or B6-Ly5.1 line on a recombination-activating gene (RAG) −/− background. B6-CD40 −/− mice 14 were provided by Dr. Hitoshi Kikutani (Osaka University, Osaka, Japan) via Dr. Nancy Philips (University of Massachusetts Medical Center, Worcester, MA). This method was adopted from Kearney et al. 3 . 2.5–4 × 10 6 pooled CD8 LN cells from OT-I–RAG −/− (Ly5.1 or Ly5.2) mice were injected intravenously into B6 (Ly5.1 or Ly5.2) mice. 2 d later 5 mg of OVA (Grade VI; Sigma Chemical Co.) was administered by intraperitoneal injection. Lymphocytes were isolated at the indicated times and analyzed for the presence of transferred cells by flow cytometric detection of Ly5 differences. Antibody treatments were performed by intraperitoneal injection of 200 μg of MR1 (anti-CD40L) or hamster Ig as control 11 . MR1 was provided by Dr. R. Noelle, Dartmouth Medical School, Hanover, NH. Injections were given daily starting 1 d before immunization. Cells were labeled with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Inc.) at a concentration of 5 μm for 10 min at 37°C 31 32 . Each experiment was performed a minimum of three times. Mice were infected by intravenous injection of 1 × 10 6 PFU of vesicular stomatitis virus (VSV), Indiana serotype. 6 d later lymphocytes were isolated, and VSV nucleoprotein (N)-specific CD8 T cells were detected using H-2K b tetramers containing the N protein–derived peptide RGYVYQGL, or as a control the OVA-derived peptide SIINFEKL (Research Genetics) 33 . MHC tetramers were produced essentially as described previously 34 35 . Briefly, H-2K b containing the BirA-dependent biotinylation substrate sequence was folded in the presence of human β2-microglobulin and the N peptide. Biotinylation was performed with biotin–protein ligase (Avidity). Tetramers were then produced from biotinylated HPLC–purified monomers by addition of streptavidin–allophycocyanin (Molecular Probes, Inc.). IELs and LP cells were isolated as described previously 36 37 . For cytotoxicity assays, panning of Percoll-fractionated IELs on anti-CD8 mAb–coated plates was performed to remove contaminating epithelial cells. Without panning, lytic activity of IELs is less consistent between replicates, but overall activity is similar to that of panned cells. Lytic activity of LPLs is measured without prior panning and is similar to that of IELs, indicating that the anti-CD8 panning step does not effect lytic function (data not shown). LNs and spleens were removed, and single cell suspensions were prepared using a tissue homogenizer. Peripheral LNs included brachial, axillary, and superficial inguinal nodes. The resulting preparation was filtered through Nitex, and the filtrate was centrifuged to pellet the cells. Lymphocytes were resuspended in PBS, 0.2% BSA, 0.1% NaN 3 (PBS/BSA/NaN 3 ) at a concentration of 10 6 –10 7 cells/ml followed by incubation at 4°C for 30 min with 100 μl of properly diluted mAb. The mAbs were either directly labeled with FITC, PE, Cy5, allophycocyanin, or were biotinylated. For the latter, avidin-PE-Cy7 (Caltag Laboratories) was used as a secondary reagent for detection. After staining, the cells were washed twice with PBS/BSA/NaN 3 and fixed in 3% paraformaldehyde in PBS. Relative fluorescence intensities were then measured with a FACSCalibur™ (Becton Dickinson). Data were analyzed using WinMDI software (Joseph Trotter, Scripps Clinic, La Jolla, CA). Cytolytic activity was measured using ( 51 Cr)sodium chromate–labeled EL4 cells (an H-2 b thymoma), with or without the addition of 10 μg/ml of the OVA-derived peptide SIINFEKL. Serial dilutions of effector cells were incubated in 96-well round-bottomed microtiter plates with 2.5 × 10 3 target cells for 6 h at 37°C. Percent specific lysis was calculated as: 100 × [(cpm released with effectors) − (cpm released alone)]/[(cpm released by detergent) − (cpm released alone)]. Our previous results indicate that adoptively transferred OVA-specific T cells migrating into the intestinal mucosa become potent CTLs after immunization with OVA. To test whether CD40–CD40L interactions were involved in this activation, we treated mice during immunization with a blocking mAb specific for CD40L 11 . Naive OT-I cells do not enter the intestinal mucosa 26 38 but a small population of naive cells (∼0.5%) can be detected in the peripheral LNs (PLNs) and mesenteric LNs (MLNs) by virtue of differences in Ly5.1/Ly5.2 expression between the transferred and host lymphocytes . 3 d after intraperitoneal immunization with sOVA, a large increase in OT-I cells in PLNs and MLNs was observed. Although the magnitude of this response is dependent on antigen dose, immunization with 0.5 mg sOVA resulted in detectable T cell activation and migration to the mucosa (data not shown). Responses in MLNs were partially inhibited (∼30%), whereas PLN OT-I responses were not affected . Although the inhibition of the MLN response is not impressive, it was consistently observed, as was the lack of inhibition of the PLN response. However, treatment with anti-CD40L mAb resulted in ∼70–80% inhibition of appearance of OT-I cells in the IELs and LP compartments. This inhibition was not merely a kinetic delay in the response, as the effect of early anti-CD40L blockade was maintained throughout the response (8 d and longer after immunization; data not shown). Thus, there was an apparent requirement for CD40–CD40L interactions to generate an optimal mucosal CD8 T cell response. The effect of anti-CD40L mAb on mucosal OT-I accumulation could have been due to inhibition of migration or of proliferation. In addition, inhibition of proliferation outside of the mucosa could result in fewer cells migrating to the mucosa. To determine the point at which CD40L interactions were involved in activation, we transferred CFSE-labeled OT-I cells and then immunized with OVA in the presence or absence of anti-CD40L mAb. 3 d after immunization, lymphoid tissues were analyzed for the presence of donor cells and their CFSE fluorescence was measured. CFSE is distributed equally between daughter cells upon division and can be detected over nine divisions (i.e., no fluorescence after nine divisions ). In all sites, OT-I cells from control mice had divided at least nine times and thus retained no fluorescence above background . This was true even at time points earlier than 72 h when fewer cells had appeared in the intestinal mucosa, indicating that substantial proliferation of OT-I cells had occurred elsewhere before entry into the mucosa (data not shown). Transferred cells in PLNs from MR1-treated mice also exhibited little fluorescence, indicating that the cells had divided at least nine times, and that CD40L blockade had not greatly affected the proliferation of cells in the PLNs. In MLNs, ∼40% of the cells from anti-CD40L–treated mice had undergone eight divisions, at least one less than control OT-I cells. In the LP from MR1-treated mice, ∼50% of the OT-I cells had divided less than nine times, with 28% having divided seven times, and 7% having undergone six divisions. In IELs from MR1-treated mice, ∼60% of the cells had undergone less than nine divisions, and 22% had divided seven times. This inhibition of proliferation correlated well with the decreased percentage and total cell number (data not shown) of OT-I cells in LP and IELs. These results suggested that CD40L-mediated proliferative signals, whether delivered within or outside of the mucosa, were important for generation of the mucosal CD8 response. The results thus far demonstrated a role for CD40L in CD8 T cell activation, but did not indicate whether this effect was direct or indirect. To test this, we transferred OT-I–RAG −/− cells into CD40L-deficient mice and attempted to block activation with MR1. In this experiment, only the transferred CD8 T cells were capable of expressing CD40L, so that any inhibitory effect must be mediated at the level of the OT-I cells. After OT-I cell transfer, immunization of CD40L −/− mice with sOVA resulted in substantial proliferation in the periphery and appearance of OT-I cells in the LP . Treatment with anti-CD40L mAb resulted in partial inhibition of OT-I accumulation in MLNs and much greater inhibition of OT-I accumulation in the LP . Little inhibition of OT-I expansion in PLNs was observed (data not shown). These results were similar to those obtained in CD40L-competent mice , and indicated that CD40L expressed by CD8 T cells was responsible for activation of OT-I cells destined for the mucosa. Although anti-CD40L mAb treatments have been widely employed to test in vivo function, it was possible that mAb binding to CD40L directed untoward effects on the T cells. Therefore, we performed adoptive transfer and immunization in mice lacking CD40 . 4 d after antigen administration, substantial populations of OT-I cells were detected in PLNs, MLNs, LP, and IELs of normal mice. However, in the absence of CD40, OT-I cells were reduced by ∼80% in IELs and LP, just as they were with MR1 treatment. Interestingly, the inhibition of OT-I cell expansion in both PLNs and MLNs was greater than that observed with MR1 treatment. However, a difference remained between PLNs (∼40% reduction) versus MLNs (∼75% reduction). To further examine the role of CD40 in mucosal CD8 T cell responses, we visualized the response of endogenous antiviral CD8 T cells using MHC tetramers. B6 or CD40 −/− mice were infected with VSV, and 6 d later spleen cells, LPLs, and IELs were isolated. N-specific CD8 T cells were identified using H-2K b –N peptide MHC tetramers. In normal mice, N-specific cells consistently comprised ∼10% of the splenic CD8 T cells, whereas in the CD40 −/− mouse shown the response was partially inhibited, with 4.5% of the CD8 cells reactive with N-peptide . However, the difference observed in the splenic response between control (10.4 ± 1.8, n = 6) versus CD40 −/− (7.6 ± 2; n = 6) mice was not statistically significant. In contrast, the anti-VSV CD8 response in LP was greatly inhibited in the absence of CD40. In the experiment shown, 21% of LP CD8 cells from control mice were N-specific, while only 2.5% were N-specific in CD40 −/− mice . The difference between the response in control mice (19.7 ± 0.8, n = 6) versus CD40 −/− mice (4 ± 0.6; n = 6) was highly significant ( P = 0.001) and was independent of total CD8 cell numbers (data not shown). Although we do not yet know whether CD40L expressed by antiviral CD8 cells is involved in activation, the results nevertheless demonstrated the importance of CD40–CD40L interactions in the mucosal antiviral CD8 response. The adoptive transfer system allows a comparison of lytic activity on a per cell basis, and so whether CD40L triggering results in enhanced lytic activity was tested. We analyzed CTL activity of transferred OT-I cells from IELs and spleen from immunized, normal, or CD40 −/− mice . sOVA immunization induced poor lytic activity in OT-I cells in spleen despite their activated state, as determined by phenotype and proliferation (data not shown, and reference 26). In contrast, sOVA immunization resulted in appearance of potent OT-I IEL effectors . In the absence of CD40, OT-I proliferation was severely inhibited in the IEL compartment, as shown in Fig. 4 . Nevertheless, when the lytic activity of OT-I effectors was compared on a per cell basis, OT-I IELs in CD40 −/− mice were equally effective at inducing antigen-specific lysis as IELs from control mice. This result demonstrated that CD40–CD40L interactions were essential for optimal induction of proliferation of CD8 T cells, but that differentiation to CTLs in the intestinal mucosa was CD40 and CD40L independent. The MR1 blocking studies predicted that CD4 cells were not likely to be involved in the sOVA activation of mucosal OT-I cells. To test this directly, the lytic activity of OT-I IELs was measured after adoptive transfer and immunization of MHC class II–deficient mice. The absence of MHC class II had no effect on generation of CTLs in the mucosa . Proliferation of OT-I cells in MHC class II–deficient mice was also no different from that observed in normal mice (data not shown). Since IL-12 is produced by activated APCs and has been suggested to be involved in potentiation of CTL activity 39 40 41 42 , we also performed transfers into mice lacking IL-12. The absence of IL-12 did not affect the induction of lytic activity or the proliferation of OT-I cells in the intestinal mucosa . The results presented here identified for the first time a critical role for CD40–CD40L interactions in mucosal CD8 T cell responses. Blockade or removal of CD40L interactions demonstrated a more stringent requirement for CD40–CD40L engagement to generate a CD8 response in mucosal effector sites (LP, IELs) compared with responses in peripheral secondary lymphoid tissue. This was true for the response of adoptively transferred OT-I cells , as well as for an endogenous antiviral CD8 T cell response . Recently, it was reported that the splenic anti-VSV CTL response was CD40 independent as determined by measuring lytic activity 43 . Our present results using MHC tetramer staining corroborate these findings . Perhaps more importantly, our data showed that despite the fact that the splenic anti-VSV CD8 response was CD40 independent, the LP CD8 response had a stringent requirement for CD40. These results demonstrated a role for tissue-specific regulation of the CD8 immune response via CD40. The results also suggested, but did not prove, that CD8 cells migrating to the mucosa encountered APCs in the LP, which induced further proliferation and differentiation. There are few, if any, professional APCs in the intestinal epithelium, so the inhibition of the IEL response by CD40–CD40L blockade or absence was likely the result of a decrease in the LPL response and/or the peripheral response, thereby resulting in fewer cells available for migration to the epithelium. Indeed, the inhibition of the LPL response always paralleled the decrease in the IEL response. If the mucosal response is in fact modulated by interaction of migrating CD8 cells with LP APCs, then the resulting effects could be linked to the status of APCs in the LP. That is, LP APCs may be at a heightened activated state compared with those in nonmucosal areas. This possibility was supported by the induction of CTL activity in mucosal effector sites but not in peripheral tissues. DCs from the gut migrate to MLNs 44 45 , and this could form the basis of the observed role for CD40L in that site. However, signals in addition to CD40L triggering must be involved in CTL induction, since activated OT-I cells in MLNs exhibited poor lytic activity (data not shown). Overall, the results supported a generalized role for CD40–CD40L interactions in amplification of intestinal CD8 T cell responses, whether or not the relevant CD8 T cell–APC interaction occurred in the mucosa. Our finding that CD40L expressed by activated OT-I cells was interacting with CD40 expressed by APCs made sense, considering that MHC class II–restricted CD4 T cells were not required for the OT-I response. The importance of CD40L expressed by OT-I cells was unequivocally established by showing that inhibition of OT-I expansion by CD40L blockade occurred when OT-I cells were the only cells capable of expressing CD40L . However, this finding does not preclude an indirect role for CD4 T cells, when they are present, in CTL expansion via an effect on APCs. Thus, CD4 cells could induce upregulation of CD40 on APCs, which would subsequently interact with CD40L expressed by CD8 T cells. Nevertheless, the recent demonstration that CD8 T cells can directly activate dendritic cells 43 supports the concept that some CD8 responses do not require CD4 T cells to activate APCs. In all other in vivo responses examined thus far, CD40L expressed by CD4 T cells was critical for CD8 T cell activation 19 20 21 46 47 . However, in contrast to the system in which CD40 triggering is required to induce CTLs to OVA-loaded spleen cells 20 , induction of mucosal CTLs to soluble antigen was CD40 independent. That is, despite the fact that the increase in OT-I cells in LP and IELs was greatly inhibited by CD40L blockade, the lytic activity of the remaining cells was not affected. Thus, although CD40 triggering in some situations can drive CTL differentiation 21 , this is apparently not a requisite step for mucosal CTL induction. It should be noted that in previous studies it was not possible to determine the effect of CD40 on proliferation versus CTL differentiation, since antigen-specific T cells were not quantified. The importance of CD40/CD40L in the mucosal CD8 T cell response indicates that the nature of the CD40 expressed by intestinal APCs may be distinct from that of APCs in nonmucosal sites, perhaps in terms of level of expression as well as activation status of the cell. The significant numbers of activated B cells in the LP could participate in the response as APCs. Further, the density and/or anatomy of the APC network in the LP may regulate T cell interactions differently from those occurring in secondary lymphoid tissue. Although we do not yet know the factors required for mucosal induction of CTLs, we showed here that IL-12 was not essential. Exogenous IL-12 augments alloreactive CTL activity in vitro 39 and in vivo 41 . Yet, it is not clear from these studies whether IL-12 acts directly on CD8 T cells or affects CD4 T cells which then influence the CTL response. Moreover, the primary alloreactive CTL response in vivo was unaffected in IL-12–deficient mice 27 . In another report, low doses of IL-12 increased the number of CD8 cells in lymphocytic choriomeningitis virus–infected mice, whereas high doses of IL-12 inhibited the CTL response 42 . Thus, although splenic CD8 cells may respond to exogenous IL-12, it remains unclear whether IL-12 is involved in systemic CD8 T cell immunity. In the case of mucosal T cell responses, IL-12 appears to play a significant role. In a model of colitis in which IFN-γ–producing Th1 cells are involved, inhibition of IL-12 results in abrogation of the disease 48 . Furthermore, inhibition of CD40L–CD40 interaction inhibited the induction of the disease by blocking priming of Th1 cells by IL-12 49 . IL-12 may also play a role in some mucosal CD8 responses. Mucosal immunization of mice with a multideterminant HIV peptide results in the appearance of antigen-specific CTLs in spleen, Peyer's patches (PPs), and LP. However, CTL priming was completely abrogated when IL-12 was blocked or IFN-γ was absent 50 . In our system, primary mucosal CTL development was IL-12 independent. This finding may be related to the demonstration that this response was also CD4 T cell independent, while the colitis model and the response to the HIV peptide require CD4 T cells. Therefore, as discussed above, the requirement for IL-12 in CD8 responses may be indirect and related to the action of this cytokine on CD4 T cells. Our findings support a model for mucosal CD8 T cell activation in which sequential encounters with APCs may occur. As shown here for sOVA immunization and VSV infection, CD40–CD40L interaction allowed a significant amplification of the mucosal CD8 response. This system could provide a powerful mechanism for potentiation of mucosal immune responses. The goal of the immune system is to send functional CTLs to sites of potential pathogen entry, the intestine being foremost on this list. When the intestinal mucosa is infected, antigens are most likely presented to T cells in the draining organized secondary lymphoid tissue such as PPs and MLNs, followed by migration of activated T cells to the LP. Our hypothesis suggests that upon entry into the LP, CD8 T cells may reencounter antigen presented by APCs and receive further proliferative and differentiative signals. This theory predicts a second level of regulation for CD8 T cells patrolling the mucosa: i.e., if antigen is not encountered in the mucosa, further activation of CD8 T cells does not occur, thereby conserving resources and decreasing the opportunity for destructive autoimmune reactions. Deciphering the specialized signals for T cell activation in the intestinal mucosa will provide tools for manipulating inflammatory bowel disease and for potentiation of mucosal vaccination and immune barrier function. | Study | biomedical | en | 0.999996 |
10544200 | Mouse NIH 3T3 cells were grown in DME supplemented with 10% newborn calf serum. Primary mouse embryonic fibroblasts (MEFs) prepared from BALB/cJ mice and B12 cells 24 were grown in MEM with 10% FCS. The Smith strain of MCMV (VR-194; ATCC) and the recombinant viruses were propagated on third-passage MEFs and purified by sucrose gradient centrifugation. Tissue culture–grown virus preparations were used throughout. Plasmid constructions were performed by standard methods 25 . Plasmid p152KO used for generating m152 − recombinant viruses was constructed by ligation of a 5-kb NotI–BamHI fragment comprising a loxP -flanked lacZ cassette 26 into the XhoI/NheI-digested plasmid pEcoOΔMB (all sites were blunt-ended by treatment with Klenow DNA polymerase). Plasmid pEcoOΔMB contains a 5.0-kb EcoRI–MluI fragment of the MCMV genome (MCMV nucleotides 209,756–214,714) encompassing the m152 gene 27 . To generate recombination plasmid pm152gpt, the Escherichia coli gpt gene was flanked with loxP sites and inserted into an XhoI site of plasmid pEcoOΔMB at the 3′ end of the m152 gene. Recombinant viruses were generated by homologous recombination in NIH 3T3 as described previously 26 . LacZ + recombinants were identified by 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal) staining and isolated by at least five rounds of limiting dilution passage. Recombinant viruses carrying the gpt marker were first enriched by selection in medium that was supplemented with 12.5 μg/ml mycophenolic acid (GIBCO BRL) and 100 μg/ml xanthine (Sigma Chemical Co.) 28 and further purified by limiting dilution. LacZ − and gpt − mutants were generated by a single passage through the recombinase Cre + cell line, N2 26 . LacZ − recombinants were identified as white plaques after X-gal color screening and purified by limiting dilution. Gpt − mutants were selected on STO cells in medium containing 20 μg/ml 6-thioguanine (Sigma Chemical Co.) as described previously 29 . To characterize the recombinant virus genomes, viral DNA was isolated from infected cells and analyzed by Southern blot analysis 26 . B12 cells were infected with wild-type MCMV or m152 recombinant viruses. Cells were pulse labeled at 37°C for 60 min with 500 μCi/ml [ 35 S]methionine (1,200 Ci/mmol; Amersham) in methionine-free MEM supplemented with 5% dialysed FCS and chased in the presence of 10 mM nonlabeled methionine for 2 h. Labeled cells were washed in ice cold PBS and disrupted in lysis buffer (140 mM NaCl, 20 mM Tris/HCl, pH 7.6, 5 mM MgCl 2 , 1% NP-40, and 1 mM PMSF). Cytoplasmic extracts were precleared by incubation with normal mouse serum, antiactin mAb (Boehringer Mannheim), and protein A–coupled Sepharose (Pharmacia). Immunoprecipitations were performed with anti-K d mAb MA-215 ascitic fluid, and immune complexes were retrieved using protein A–coupled Sepharose. Endoglycosidase H (Endo H; Boehringer Mannheim) digestion and SDS-PAGE were performed as described previously 23 . Target cells were labeled for 90 min with Na 2 51 CrO 4 , and a 4-h standard release assay was performed with 10 3 target cells and a graded number of effector cells in fivefold dilution steps as described 21 30 . In short, for selective and enhanced expression of immediate-early (IE) genes, MEFs were infected with 0.5 PFU of recombinant viruses or wild-type MCMV per cell by centrifugation (800 g , 30 min). Infection was performed in the presence of cycloheximide (50 μg/ml), which was removed 3 h later by washing with medium containing actinomycin D (5 μg/ml). Limited E gene expression after CH treatment was achieved by removal of cycloheximide using inhibitor-free medium and by adding actinomycin D to the final concentration of 5 μg/ml after 1.5 h. To generate pp89-specific polyclonal CTLs, MCMV-primed spleen cells were restimulated with pp89-derived antigenic peptide 21 , and recombinant IL-2 (100 U/ml) was added 5 d later. Cultures were restimulated with gamma-irradiated syngeneic MEFs pulsed with antigenic peptide at a concentration of 10 −8 M. Data represent the mean percentage of specific lysis from three replicate cultures . BALB/c (H-2 d haplotype) and C57BL/6 mice (H-2 b haplotype) were bred at the Central Animal Facilities at the Medical Faculty, University of Rijeka. Mice homozygous for the μ chain mutation (C57BL/6 background; reference 31 ) were provided by Dr. Klaus Rajewsky (Institute for Genetics, Cologne, Germany) and were backcrossed on the BALB/c background for 10 generations. Mice heterozygous (μMT −/+ ) and homozygous (μMT −/− ) for the μ chain mutation were distinguished by ELISA for the presence or absence of IgM in mouse sera, as described previously 32 . Mice homozygous for the β2 microglobulin mutation (β 2 m −/− ; supplied by Dr. Rudolf Jaehnisch, Whitehead Institute of Biomedical Research, Cambridge, MA) fail to express ternary MHC class I complexes and are devoid of CD8 + T lymphocytes 33 . Mice homozygous for the deletion of the gene encoding the CD8 molecule (CD8 −/− ) were obtained from the Centre de Developpement des Techniques Avancées pour l'Expérimentation Animale, Institut de Transgenose, Orleans, France. The absence of CD8 + T lymphocytes in β 2 m −/− and CD8 −/− mice was verified by flow cytometry as described previously 34 . Neonatal mice, 24 h and 4 d postpartum, were injected intraperitoneally with recombinant viruses or wild-type MCMV. 6–8-wk-old mice were injected either in the posterior footpad or i.p. with 2 × 10 5 PFU of virus in a volume of 50 and 500 μl of diluent, respectively, as described 35 . Plaque assays were performed in MEF as described previously 36 37 . Statistical significance of differences between the experimental groups was determined by the Mann-Whitney exact rank sum test. Virus titers (x and y) were considered significantly different for P (x versus y) < alpha = 0.05 (one sided), where P is the observed probability value and alpha is a selected significance level. In vivo depletion of CD4 + and CD8 + T lymphocyte subsets was performed by intraperitoneal injection of mAbs (rat anti–mouse) to CD4 (YTS 191.1) and/or CD8 (YTS 169.4) molecules 38 . Adult and newborn mice received 1 mg and 250 μg of antilymphocyte antibodies, respectively, at the time of injection and every fifth day throughout the experiment. The efficacy of T lymphocyte depletion was >95%, as assessed by cytofluorometric analysis of spleen cells using FITC- or PE-conjugated antibodies directed against mouse CD4 and CD8 molecules . Donor T lymphocytes were harvested from spleens of uninfected (nonprimed) or latently infected (MCMV-primed) mice. Recipient mice were injected with 2 × 10 5 PFU of virus in a rear footpad 12 h after gamma irradiation (6.5 Gy). Immediately after infection, 2 × 10 5 nylon wool–purified cells were injected intravenously into recipient mice. Mice that did not receive cell transfer were used as negative controls. Mice were killed on day 14 after infection, and tissues were harvested for virus titer determinations. To investigate the significance of the m152 gene product in the course of infection, a targeted deletion of the m152 gene and subsequent reintroduction of this gene into the MCMV genome was performed . The recombinant virus ΔMC95.21 was generated by homologous recombination between the wild-type MCMV genome and the recombination plasmid p152KO. In this plasmid, a 1.2-kb XhoI–NheI fragment containing the m152 gene was replaced by a lox P-flanked E . coli lacZ gene. The lacZ marker was excised by passaging the ΔMC95.21 recombinant through the recombinase Cre + cell line N2 26 to create the m152 − lacZ − deletion mutant ΔMC95.24. To generate a revertant virus, the m152 gene, together with the loxP -flanked gpt gene, was reinserted by homologous recombination into the ΔMC95.21 genome. After positive selection 28 of the m152 + gpt + virus rMC95.26, the gpt marker gene was again removed by passaging the virus through the recombinase Cre + cell line N2 to generate the m152 + gpt − revertant virus (designated rMC96.27). Southern blot analysis of the recombinant virus genomes confirmed the recombination events at the expected positions . In the mutant virus genomes, the original 23.3-kb HindIII E fragment and the 5.9-kb EcoRI O fragment were replaced by expected new HindIII and EcoRI fragments. HindIII fragments of 20.0 and 6.5 kb and EcoRI fragments of 4.8 and 3.5 kb are evident in ΔMC95.21, whereas in the m152 − lacZ − deletion mutant, ΔMC95.24, a 22-kb HindIII fragment and a 4.7-kb Eco RI fragment were found. In the genomes of the rMC95.26 and rMC96.27 revertant viruses, HindIII fragments of 21.2 and 2.9, and 23.2 kb, and EcoRI fragments of 6.4 and 0.4 kb, and 5.5 and 0.5 kb, were observed, respectively. Note that the size of the HindIII E fragment in the rMC96.27 genome is identical to that in the wild-type MCMV genome, whereas a new EcoRI site purposely introduced outside of the m152 open reading frame enabled us to discriminate between the constructed revertant and wild-type MCMV. Comparison of the HindIII, EcoRI, and XbaI digestion patterns of the recombinant genomes with those of the wild-type MCMV genome confirmed that the recombinant viruses were free of detectable deletions or insertions in any other region of the viral genome (data not shown). The altered glycosylation pattern of newly synthesized molecules can be used to locate the export block of nascent MHC class I molecules in MCMV-infected cells 1 22 23 . Correctly assembled MHC class I complexes retained by the m152 gene product in the ERGIC/cis-Golgi compartment of MCMV-infected cells are not processed by medial-Golgi enzymes to complex glycans. Therefore, the majority of MHC class I molecules from cells infected with wild-type MCMV exhibit high mannose N-linked glycans typical for this compartment that are sensitive to Endo H and migrate faster in gels after digestion with Endo H . In contrast, MHC class I complexes in cells infected with the m152 deletion mutant ΔMC95.21 as well as in uninfected cells acquire Endo H–resistant glycans, indicating the normal egress from the ERGIC/cis-Golgi compartment. As expected, the MHC class I transport was affected again in cells infected with the revertant virus rMC96.27, demonstrated by the reappearance of molecules sensitive to Endo H digestion. The transport arrest of MHC class I molecules by the MCMV m152 gene product at early (E) times of virus replication prevents surface expression of these molecules and thus the recognition and lysis of infected cells by specific CTLs 1 . A deletion of this gene should restore the recognition of infected cells by CTLs under the experimental conditions. To test this, MEFs were infected with the m152 deletion mutant ΔMC95.21, the revertant virus rMC96.27, or wild-type MCMV. Infected cells were arrested in the IE or E phase of the MCMV replication cycle and used in a CTL assay with MHC class I–restricted CTLs specific for the MCMV antigen pp89 21 30 36 . As expected, recognition and cytolysis were equivalent for cells infected with all three viruses during the IE phase of the viral replication cycle, a time at which the m152 gene product is not yet expressed . However, recognition was impaired during the E phase when cells infected with wild-type or revertant virus were used as targets. In contrast, efficient recognition of ΔMC95.21-infected cells was seen, confirming that retention of MHC class I molecules and the associated block in antigen presentation is mediated under these conditions exclusively by the m152 gene. Multistep growth curves of recombinant and wild-type viruses served to assess whether the deletion of the m152 gene affects virus growth in cell culture. After infection of NIH 3T3 fibroblasts at a multiplicity of infection of 0.1 PFU per cell, replication of the m152 deletion mutant and revertant were indistinguishable from that of MCMV wild-type virus . Identical results were obtained by comparing the replication capacity of the m152 deletion mutants, the revertant virus, and MCMV wild type on primary MEFs (not shown), indicating that the m152 gene product is completely dispensable for virus growth in fibroblasts. Considering the fact that three different MCMV genes affect nascent MHC molecules and that m152 merely represents the gene that is expressed first, it was not clear whether or not the deletion of this gene would have any detectable impact on the susceptibility of the virus to immune control in vivo. Whereas adult mice control the infection with tissue culture–derived wild-type MCMV effectively, young mice allow virus replication to high titers 39 40 . To detect even minor differences in virulence due to deletion of the single m152 gene, we assayed virus replication in neonatal mice. To avoid the potential influence of marker gene products on the biological properties of mutant viruses, the in vivo experiments were performed mainly with the m152 deletion mutant ΔMC95.24 and the revertant virus rMC96.27, although the other mutants gave comparable results (data not shown). Neonatal mice were injected with 100 PFU of the m152 deletion mutant, the revertant virus, or wild-type MCMV and monitored for 30 d. After infection with wild-type MCMV or revertant virus, 53 and 75%, respectively, of animals succumbed to infection . In contrast, infection with the m152 deletion mutant was survived by the majority of mice (25% mortality). With respect to clinical signs, all three groups of mice exhibited during the first week of infection significant runting and a general failure to thrive compared with mock-infected controls. By 14–20 d after infection, however, most animals that survived the infection with the m152 deletion mutant had recovered. In contrast, clinical signs persisted throughout the course of observation for wild-type MCMV and revertant virus–infected mice. The different disease courses correlated with the body weights of infected mice. On day 26 after infection, the average body weight of mice that survived infection with ΔMC95.24 was comparable to that of the control group (9.79 ± 1.86 and 10.9 ± 1.16 g, respectively), whereas mice infected with the revertant virus still appeared runted (7.04 ± 1.70 g; data not shown). To assess whether the differences were due to an altered tissue tropism associated with the m152 deletion, virus titers were determined for lungs, spleen , and salivary glands (data not shown). The mutant ΔMC95.24 yielded lower titers in the spleen and lungs as compared with wild-type MCMV and the revertant virus. Although the differences in virus titers in tissues of neonatal mice did not exceed 1–2 log 10 steps, this finding was reproducible both in MCMV-sensitive (BALB/c) as well as MCMV-resistant (C57BL/6) mouse strains. In the salivary glands, this observation could not be made. In this organ, the virus titer yielded by the m152 deletion mutant was indistinguishable from that of the wild-type and revertant virus. In this context, it is of interest to note that we have demonstrated earlier that the salivary gland represents the only organ in which MCMV replication is exempt from CD8 + T cell control 41 . Altogether, the lack of the m152 gene results in an attenuated course of infection and in restricted virus growth. Immunodeficient mice were used to assess whether the attenuated phenotype of the m152 deletion mutant indeed reflected an enhanced sensitivity to T cell control. BALB/c mice were immunodepleted by gamma irradiation and by injection with cytolytic antibodies to T lymphocytes and NK cells. In immunodepleted animals, all three viruses replicated to high titers without significant titer differences (data not shown). This demonstrated already that the attenuated phenotype of the m152 deletion mutant is caused by an increased sensitivity to immune control mechanisms. The m152 deletion mutant replicates to lower virus titers than the revertant virus in undepleted BALB/c as well as C57BL/6 mice . This growth restriction was abrogated after depletion of T lymphocyte subsets , indicating that the attenuated phenotype of the deletion mutant is caused by an enhanced sensitivity to T cell control. B cell–deficient (μMT −/− ) mice were employed to identify the relative role of T cell subsets . Due to the lack of specific antibodies, MCMV spreads rapidly in μMT −/− mice, and detection of infectious virus is facilitated 35 . 8-wk-old μMT −/− mice were depleted of only CD8 + T lymphocytes, depleted of both CD8 + and CD4 + T cell subsets, or left undepleted. Virus titers were determined 10 d after infection. The growth restriction of the m152 deletion mutant was notable in particular in the lungs of nondepleted mice, resulting in titer differences ranging from 2 to 3 log 10 . After depletion of CD8 + T lymphocytes or of both T cell subsets, mutant and revertant virus reached comparable virus titers . These data demonstrate that CD8 + T cells are the relevant cell subset responsible for the replication inhibition associated with the m152 gene deficiency. Furthermore, the attenuating effect is also seen in adult mice. Although the differences are not significant, in BALB/c μMT −/− mice, the m152 deletion mutant reached slightly lower titers than the revertant, even after depletion of T cell subsets. The m152 gene function affects antigen presentation in the MHC class I pathway. Therefore, in mice in which this presentation pathway is defective, the specific defect of the virus should be phenotypically complemented. To test this, we used MHC class I–deficient C57BL/6 β 2 m −/− mice 33 and mice deficient for CD8 + T lymphocytes due to the deletion of the CD8 gene (C57BL/6 CD8 −/− mice). 4-d-old mice were infected with 1,000 PFU of either the m152 deletion mutant or the revertant virus. In contrast to the situation in immunocompetent mice, no difference in the titers between the two viruses was found in three replicate experiments performed in β 2 m −/− and CD8 −/− mice. One representative experiment is shown in Fig. 6 . Essentially, the same message was obtained in adult CD8 −/− mice infected with the m152 deletion mutant or the revertant virus. However, adult mice of the C57BL/6 strain cleared both viruses so efficiently that the titers in tissues were below the threshold levels when assayed 10 d after infection. Therefore, to enhance the virus replication and to get a measurable virus load in tissues, we had to deplete NK cells in vivo (data not shown). Altogether, these experiments show that attenuation of the m152 deletion mutant is directly linked to functions required for antigen presentation and recognition in the MHC class I pathway. Adoptive cell transfer into immunodepleted recipients was used to determine the sensitivity of MCMV N2 virus-primed as well as to naive lymphocytes. Lethal MCMV infection in gamma-irradiated BALB/c mice is therapeutically prevented by adoptive transfer of as few as 10 5 MCMV-primed CD8 + T cells, whereas the same number of naive lymphocytes or primed CD4 + T cells is ineffective 37 41 42 . As the product of the m152 gene downregulates presentation of viral antigens in the MHC class I pathway, we expected to see an increased sensitivity of the mutant to primed T cells and perhaps also a more effective priming of T lymphocytes. To test this, 2 × 10 5 lymphocytes derived from BALB/c mice, either MCMV primed or naive, were intravenously transferred into syngeneic gamma-irradiated recipients 12 h after infection with wild-type MCMV, the m152 deletion mutant, or the revertant virus strain. Adoptive T cell control of MCMV is a selective function of CD8 T cells but not of CD4 T cells and is more effective in spleen and liver than in the lungs 37 42 . Accordingly, the replication of the m152 deletion mutant is more efficiently controlled in these organs than the revertant virus. Small numbers (∼10 5 ) of naive T lymphocytes fail to protect mice against MCMV infection 41 . This was reproduced for mice infected with the revertant virus; however, the number of 2 × 10 5 naive lymphocytes already decreased the titers of the m152 deletion mutant . This is a function of T lymphocytes, as depletion of the CD8 T cell subset eliminated this activity (data not shown). Transfer of graded numbers of naive cells into gamma-irradiated mice showed that the number of naive T cells had to be increased by 100-fold to achieve an effect on wild-type MCMV comparable to the effect on revertant MCMV (data not shown). We therefore concluded that deletion of the m152 gene increases the antigenicity of the virus. Herpesvirus genomes contain several genes coding for potential immunomodulatory functions. Shared between viruses of the α- and the β-herpesvirus family is the expression of gene functions that interfere with peptide presentation in the MHC class I pathway in vitro. Herpesviruses are highly species specific, and so are the functions of the genes that affect this pathway. No cellular homologue has been detected for any of these genes so far 4 . For a better understanding of the contribution of each individual gene to the biology of the virus infection, experiments in the natural host are required. Here, we report on the in vivo function of the immunomodulatory protein encoded by gene m152 of MCMV during infection of its natural host. To prove that a gene has a predicted immunoregulatory function in vivo, three aspects must be addressed. First, the deletion of the gene from the genome should not affect virus growth in cells in the absence of immune control. Second, a phenotype seen in vivo should be lifted by a targeted revertant of the virus. Third, the attenuation due to lack of the immunomodulatory function of the virus should be phenotypically complemented in a host that is genetically or functionally disabled to exert the control that is specifically affected by the deleted viral gene product. Only the fulfillment of all three requirements confirms the prediction of the in vitro studies. Not all virus genes that have an effect on specific immune effector mechanisms in vitro show this effect in vivo as their main function. One such example is the Fc receptor function encoded by gene m138 of MCMV. The Fc receptor is expressed at the cell surface and selectively binds mouse IgG in vitro. The deletion of m138 results in strong attenuation of the mutant virus in vivo that is lifted by the specific revertant. However, in Ig-deficient mice, the attenuation is still present, proving that attenuation of the virus due to the deletion of the Fc receptor is not linked to Ig control 26 43 . The m152 gene encodes the glycoprotein gp40, which arrests the export of nascent mouse but not human MHC class I molecules 16 . If this was the major function of the protein, then the deletion of the gene should be dispensable for virus growth in fibroblasts but should restrict replication in immunocompetent animals. This prediction was fulfilled by the m152 deletion mutant viruses. Virus growth in vivo but not in fibroblasts was affected by the mutation. Furthermore, the MHC class I complex transport and the capacity to present viral peptides to CD8 T lymphocytes was restored. The revertant virus regained wild-type properties in vivo and fulfilled the second requirement by proving the causal linkage between targeted deletion and biological phenotype. As with HCMV infection in humans, the primary infection of mice even with wild-type MCMV is usually asymptomatic. Newborn mice and mice that are a few days old are much more sensitive than adult mice to tissue culture–grown virus, due to the immaturity of the NK cell response 44 . In neonates, the infection with 10 2 PFU causes a high percentage of mortality and runting in survivors. The attenuating effect of the m152 gene deletion resulted in a higher number of survivors and an earlier cessation of runting. The third requirement was also fulfilled: loss of the phenotypic difference between deletion mutant and revertant virus in the absence of the host immune function affected by the viral gene product. gp40 blocks the export of nascent MHC class I molecules already loaded with viral peptides. The predictable consequence is the inhibition of CD8 T cell priming and CD8 T cell effector function. Loss of the m152 gene should lead to an increased sensitivity of the virus to lymphocytes. Indeed, the virus mutant grew to smaller titers in the various tissues tested. This attenuation did reflect a more stringent control of the deletion mutant by T cell functions, as elimination of T cells resulted in comparable tissue titers of mutant and revertant virus. Furthermore, the attenuating effect of the m152 deletion mutant was absent in C57BL/6 mice that failed to form the functional MHC class I molecules due to the lack of β2-microglobulin expression and also in mice that have a defect in the maturation of MHC class I–restricted CD8 + T cells due to the deletion of the CD8 gene. Altogether, this study proves for the first time that in their natural host, herpesviruses benefit from functions that inhibit antigen presentation in the MHC class I pathway in vivo. It remains open whether the observed function is the only function of the m152 gene product in vivo. MHC class I molecules activate CD8 + T cells and, at the same time, inhibit NK cells 45 46 . Accordingly, a prediction of the transport block of MHC class I molecules due to m152 gene expression is the susceptibility of MCMV-infected cells for NK cell–mediated destruction in vivo. A deletion of the m152 gene and the restoration of MHC class I molecule transport should result in an enhanced resistance of infected cells to NK cell control in vivo. Our data do not support this assumption. Preliminary studies suggest that the lack of the m152 gene certainly does not make the virus more resistant to control by NK cells (Krmpotic, A., B. Polic, and S. Jonjic, unpublished data). Both HCMV and MCMV genes code for glycoproteins that show homology to MHC class I molecules, UL18 in HCMV 47 and m144 in MCMV 27 48 . It has been hypothesized that these viral MHC class I homologues are capable of engaging NK cell inhibitory receptors to protect cells from lysis due to the downregulation of MHC class I expression. Attenuation of MCMV harboring a deletion in the m144 gene has been explained by enhanced control by NK cells in vivo 48 . However, a more recent study on UL18 functions failed to confirm the inhibitory function of viral MHC class I homologues on NK cells 49 . Therefore, the potential interaction of m152 with m144 needs to be addressed. Another explanation is that the remaining functions of the genes m04 and m06 fully complement the expected NK cell effect of m152 . The genes m04 and m06 have an effect on MHC class I molecules. Both genes are expressed later than m152 during the MCMV replication cycle, and both genes encode glycoproteins that bind tightly to MHC class I molecules. gp34, encoded by the m04 gene, forms a complex with MHC class I molecules that can be detected on the surfaces of infected cells, but the functional consequence is not clear 17 . The m06 gene product gp48 binds to MHC class I molecules in the ER and reroutes them to lysosomes for rapid proteolytic degradation 18 . This leads to the downregulation of MHC class I surface expression in the late phase of the replication cycle 18 23 . Here, we show that the m04 and m06 genes cannot fully compensate all aspects of the loss of the m152 function. Thus, the interaction between the m152 gene product and other viral gene functions is not yet clear and remains to be tested. We are in the process of constructing double and triple deletion mutants to determine the individual contribution of each of the MHC class I–reactive genes and MHC class I homologues in immune evasion. To this end, we have recently pioneered the cloning of infectious herpesvirus genomes and have developed targeted and random mutagenesis techniques 50 51 . Our results show for the first time that genes that inhibit antigen presentation in the MHC class I pathway provide a significant growth advantage for CMV during primary infection. What is the potential benefit for the virus? The conditions of primary infection define the load of latent viral genomes and the risk of recurrence of the CMV infection 39 . Accordingly, we predict that the m152 gene allows a higher number of MCMV genomes to establish a latent infection, thereby enhancing the chance for reactivation and transmission to the next host and thus escaping extinction. | Study | biomedical | en | 0.999995 |
10544201 | SAM-19 cells were derived from the autonomously growing mouse CTL/rat thymoma hybrid PC60 25 that cannot be activated by IL-2 on its own to express perforin 26 . We noticed a weak IL-2 response of the perforin gene in a subline designated SA. Further analysis indicated that the SA line, but not the other lines, contained cells constitutively expressing the mouse IL-2Rβ chain. Constitutive expression of mouse IL-2Rγ was equivalent in all lines. After cloning these cells at the single-cell level, one clone, SAM-19, was obtained that expressed IL-2Rβ mRNA at levels equivalent to those in the CTL effector–like CTLL-2 line. Neither resting nor IL-2–stimulated cells expressed detectable levels of mouse IL-2Rα protein or mRNA. As described for T cells exclusively expressing IL-2Rβ and IL-2Rγ 27 , SAM-19 responded to recombinant mouse IL-2 at concentrations >100 U/ml with the induction of the mouse perforin and granzyme B genes. Half-maximal levels of perforin mRNA were detected after ∼14 h of induction. Maximum levels were induced after ∼48 h. Cell counts of SAM-19 cultures maintained in the presence of 500 U/ml IL-2 versus its absence did not significantly differ from each other, suggesting that IL-2R signaling has little or no effect on the growth and survival of SAM-19. The cells were maintained in IMDM supplemented with 10% FBS, 5 × 10 −5 M β-ME, and 50 μg/ml gentamycin. Total RNA was extracted and analyzed as described 28 . Purification and RNase treatment of nuclei as well as the elongation reactions and their purifications were performed as described 29 using ∼2.5 × 10 7 nuclei and 240 μCi of α-[ 32 P]UTP (800 Ci/mmol) per reaction (15 min at 30°C). Hybridizations were carried out with 1.6 × 10 7 /ml TCA-precipitable counts for 36 h at 65°C in 10 mM Tris, pH 7.5, 10 mM EDTA, pH7.5, 0.3 M NaCl, 1% SDS, 250 μg/ml tRNA, 1× Denhardt's solution, and 0.5% nonfat dry milk. Filter washes, including an RNase A digestion, have been described 29 . Target probes were obtained by PCR of genomic DNA and cloned as follows. The β 2 -microglobulin probe and the 5S ribosomal RNA probe were cloned into pCRII (Invitrogen Corp.). The perforin 5′ probe containing the short first exon and the beginning of the first intron and the perforin 3′ probe containing part of the third exon were cloned into M13 mp18, and single-stranded phage DNA containing perforin antisense DNA was produced. 10 μg denatured DNA was slot blotted for each target. All constructs were confirmed by sequencing. The two genomic clones depicted were obtained by screening a commercially available human placenta pWE15 cosmid library (Stratagene Inc.) on duplicate filters with a human perforin exon III probe and a human perforin promoter probe. They were further analyzed by restriction mapping and Southern hybridization to these two probes. All analyzed fragments matched the results of genomic Southern blots. Also, the sequence of a promoter fragment of both clones was identical to the one we reported previously 30 . The initial reporter gene construct was assembled by cloning a KpnI–SfiI fragment (−15,600 to −277) into the promoterless pGL3 basic reporter vector (Promega Corp.), in which the remaining human perforin promoter, exon I, intron I, and the untranslated sequences of exon II had been fused to the firefly luciferase gene. Constructs without the first intron used an EcoRV site at +59. The progressive deletion constructs were obtained by exonuclease treatment of a KpnI (−15,600)- and HindIII (−13,300)-digested plasmid, followed by Klenow and T4 DNA polymerase treatment, gel purification, and religation. All constructs were transformed by electroporation into DH10B cells (Life Technologies). Other deletions, including internal deletions, were created by restriction enzyme digestion followed by T4 DNA polymerase treatment and religation of the vector. The analysis of the upstream enhancer also involved the cloning of PCR fragments, all of which were verified by sequencing both strands of the enhancer. Mutations of the signal transducer and activator of transcription (Stat) 1 elements were introduced using the QuickChange site-directed mutagenesis kit from Stratagene Inc. After verification of the sequences on both strands of the enhancers, they were recloned into a fresh vector to eliminate second-site mutations outside of the sequenced DNA. The dominant negative Stat5a expression vector was generated similarly. Vector backbone–free DNA for stable transfections employed NotI sites flanking the inserts of the pWE15 cosmid clones or the NotI and SalI sites of the pGL3 reporter vectors. For stable transfections, an ∼5:1 molar excess of the perforin transgene over a selection cartridge containing a TK promoter driving the neomycin gene in a total of 40 μg DNA was transfected into 400 μl of 2.5 × 10 7 /ml SAM-19 in DMEM supplemented with 4.5 g/liter glucose and 25 mM Hepes by electroporation of a 4-mm gap cuvette with an ECM600 system (BTX) set to 230 V, 3,000 μF, and 24 Ω. The washed cells were cultured for 15–17 h in 2 ml of complete growth medium, washed again, and selected as 2 × 10 4 /ml live cells (1.0 ml per well in a 48-well plate) in the presence of 1.3 mg/ml active G418 (Life Technologies). 2–3 wk later, G418-resistant clones were expanded and screened by PCR for the presence of human perforin promoter sequences. PCR-positive clones were further analyzed by genomic Southern blot of restriction-digested DNA side-by-side with digested human genomic DNA to determine the integrity of the transgenic constructs and to estimate the copy numbers of the transgene. Transgenic mice, generated as we described previously 24 , were analyzed accordingly. Electroporation conditions for transient transfections were identical but used less DNA and included an internal control plasmid, pRL-CMV (Promega Corp.) in which a CMV promoter drives the renilla luciferase gene. After the electroporated cells were washed, they were split into two 500-μl aliquots (three aliquots for activations using pharmacological agents), rested for 1–3 h, and then supplemented with 500 μl of medium with or without recombinant mouse IL-2 (and/or pharmacological agents), providing a final concentration of 600 U/ml. After overnight incubation for 15–17 h, the cells were washed with PBS and lysed and extracted in 50–80 μl passive lysis buffer (Promega Corp.) by two rounds of freeze thawing. Reporter gene activities were determined in three 10-μl aliquots of each extract using the dual luciferase assay system from Promega Corp. and a ML2250 96-well plate luminometer (Dynex Technologies). Signals were integrated for 10 s for both luciferase activities. The ratios of firefly luciferase activity to renilla luciferase activity varied by <5% in the triplicate measurements. Their average was used to represent the analysis of each independently transfected sample. An important limitation in the study of antigen- and/or growth factor–dependent CTL clones is that they downregulate their overall RNA and protein synthesis and begin to apoptose when deprived of stimuli. In fact, the time periods required to “downregulate” the perforin gene to study its “activation” considerably overlap with this generalized cell shutdown. Therefore, we established a novel CTL tissue culture model, designated SAM-19, to facilitate the study of perforin gene induction by IL-2R signaling. SAM-19 is an autonomously growing, growth factor–independent clone derived from a mouse CTL–rat thymoma hybrid (see Materials and Methods). These cells respond to high doses of mouse IL-2 with the induction of mouse perforin and granzyme B mRNAs with kinetics similar to those observed in primary cells 3 . The induction of the perforin gene by IL-2 did not depend on newly synthesized proteins , and the mRNA induction was not regulated at the posttranscriptional level (data not shown), properties analogous to those of primary CTLs 3 . In addition, perforin mRNA induction in SAM-19 was not blocked by rapamycin , as reported for primary cells 31 . Taken together, SAM-19 appears to comprise a reasonable in vitro model with which to study perforin gene regulation by IL-2R signals. Before undertaking more detailed studies, we considered the possibility that IL-2–unresponsive constitutive expression by the mouse perforin promoter in transgenic mice 24 could have been due to a regulation at the level of transcription elongation. As assayed by nuclear run-on analysis, however, transcription of the 5′ and 3′ ends of the perforin gene were induced by IL-2 to similar levels . These data indicate that IL-2R signals regulate the perforin gene at the level of transcription initiation via regulatory domains other than its promoter. An expression screening strategy was used to identify putative regulatory domains other than the promoter. This approach was facilitated by the compact dimension of the human perforin gene , the ability to distinguish the human and the mouse perforin mRNAs from each other, and the transfectability of SAM-19. In initial experiments, two suitable human gene locus DNAs from a cosmid library were stably transfected into SAM-19. The expression of both the −16,500 and −20,700 DNAs was regulated by IL-2 in several independent clones . This suggested that these DNAs contained IL-2–responsive cis-acting sequences in their overlapping regions. Further efforts focused on their 5′ flanks, because we failed to detect transcriptionally relevant DNA in transient transfections of the intragenic DNA and the 3′ flank of the −20,700 gene locus DNA. Indeed, a restriction fragment comprising most of the cloned 5′ flank fragment also drove a transgenic luciferase reporter gene in an IL-2–responsive manner , in contrast to a promoter construct (−277). Interestingly, an intermediate construct (−5,300) responded at intermediate levels, which was shown in further investigations to be due to the presence of two regulatory domains rather than one. Unlike the inducibility of the transgenes, their levels of expression in relation to the transgenic copy numbers varied considerably in all clones, suggesting that these DNAs do not contain additional constitutive enhancers, silencers, or a locus control region. To extend the results from the stable transfections of SAM-19 to normal T cells, a −15,600 to +59 perforin/luciferase construct was analyzed in transgenic mice. Two founders did not express luciferase activity at all, neither in resting nor activated peripheral lymphocytes. The other two founders expressed the transgene, with its expression regulated by IL-2R as well as TCR signals, including the cross-linking of CD3 or PMA plus ionomycin ( Table ). The rates of induction for luciferase protein observed in the transgenic mice were higher than those in the SAM-19 system. Based on our experience with a transgenic CD4 reporter gene 24 , this may reflect a global increase in the translation efficiency upon activation of freshly obtained primary T cells. More importantly and unlike the previously investigated promoter transgenes, the −15,600 to +59 perforin/luciferase-transgenic T cells clearly responded to activation signals. To address exactly where the IL-2–responsive cis-acting DNA was located, progressive deletions of the construct analyzed in transgenic mice were assayed in transient transfections. A consistent pattern for their IL-2 responses was noted . The most 5′ deletion (from −15,600 to −13,300) resulted in an impaired IL-2 response. This response was abolished once sequences downstream of −1,450 were deleted. This finding suggested the presence of two cis-acting DNAs, consistent with the intermediate levels of response seen previously in the stable transfections of the reporter construct of intermediate length . This interpretation was also consistent with the reporter analysis of internal deletions . The deletion of the sequences residing between the two putative domains as well as the spacing between the two domains did not impair the levels of regulation observed by the entire 5′ flank. Finally, the far-upstream regulatory domain on its own also conferred an IL-2 response to the unresponsive perforin promoter . Both regulatory domains required the context of a promoter (data not shown), indicating that neither comprised a second promoter. The responses by each individual domain appeared to be additive, suggesting that their functions were independent of each other within the experimental context. The far-upstream regulatory DNA identified in the transient transfections comprised the very end of the −15,600 construct, whereas both of the original gene locus DNAs extended farther upstream and appeared to respond somewhat more to IL-2 . Therefore, the remainder of the cloned DNA was analyzed to delineate the 5′ and 3′ borders of the far-upstream regulatory DNA . This led to an NsiI–BalI fragment that retained the maximal response of the far-upstream regulatory DNA . This fragment also functioned in the context of the heterologous SV40 promoter . IL-2 increased the transcriptional activity of the constructs over the promoter levels irrespective of the orientation of the regulatory DNA. These attributes were consistent with an IL-2–inducible enhancer, whose required core was contained within ∼150 bp . The analogous experiments for the upstream regulatory domain recapitulated these findings and localized this enhancer within ∼130 bp . Consistent with the rapamycin insensitivity of perforin mRNA induction by IL-2 , the activation of the enhancers was not blocked by rapamycin at concentrations that abolished granzyme B induction . Because perforin mRNA can also be induced in primary CTLs by TCR signals 2 , SAM-19 was activated with pharmacological agents known to mimic TCR signals, namely phorbol ester and ionomycin. These agents induce perforin mRNA in SAM-19 in the absence of detectable IL-2 (data not shown). Regarding the involvement of the identified enhancers in this process, the far-upstream enhancer, but not the upstream enhancer, was significantly activated in SAM-19 . This activation was sensitive to cyclosporin A (CsA), 1 indicating that calcineurin participated in the activation of the enhancer. These findings suggest that the far-upstream enhancer may also respond to TCR signals. The T and NK cell–restricted expression of perforin raised the additional question of whether activation of the enhancers by pharmacological agents might be lineage specific. There was no substantial activation of either enhancer in the J588L B cell or the L929 fibroblast model . These data suggest that the enhancers can be activated primarily in T cells. Lastly, participation of the Jak/Stat signaling pathway from the IL-2R was investigated, because both enhancers contained Stat-like elements. Coexpression of a dominant negative signal transducer and activator of transcription (Stat)5 molecule 32 blocked the activation of both enhancers by IL-2R signals in a dose-dependent manner, in contrast to the cotransfections with the parental wild-type Stat5 expression vector . These data imply that the activation of Stat5 plays an important direct or indirect role in the activation of the perforin enhancers by IL-2R signals. Visual and computer-aided inspection 33 of the sequenced enhancer cores revealed several similarities to known binding sites or their cores . Potential binding sites present in both enhancers at similar positions included two Ets cores, an activator protein (AP)-1 site and, most strikingly, two identically spaced Stat-like elements. One closely resembled the consensus dyad symmetry . The other element was less well conserved . A third less well conserved Stat-like element occurred only in the far-upstream enhancer and overlapped with the highly conserved element (STAT). To determine whether these elements were indeed of functional relevance, mutant enhancers were analyzed. Minimal mutations were designed to selectively interfere with the potential Stat elements . Regarding both enhancers, a mutation of their 3′ STAT-n sites reduced their transcriptional activation by IL-2 nearly to the levels of unactivated cells . A mutation of their STAT sites or both sites together (double mutant) completely abolished their enhancer function. In contrast, mutation of the 5′ STAT-n site present only in the far-upstream enhancer did not impair enhancer activation by IL-2 . These results demonstrate a cooperative requirement for both the STAT and the 3′ STAT-n site in each enhancer. Combined with the observed inhibition of enhancer activation by a dominant negative Stat5 molecule, these results suggest that Stat molecules may directly target these elements. To address whether Stat molecules could transactivate the enhancers via the putative elements, a constitutively active Stat5 molecule was coexpressed with the perforin enhancer reporter constructs. We focused on Stat5 rather than Stat3 because we could not detect the activation of Stat3 in SAM-19 upon IL-2R signaling under conditions where activated Stat5 molecules were readily detectable (data not shown). To that end, a chimeric Stat5-VP16-Jak2 molecule that is autoactivated and leads to Stat5-specific DNA binding and strong transactivation was used 34 . Cotransfection with the respective expression vector, but not the empty expression vector, strongly hyperactivated the wild-type enhancer, but not the SV40 promoter, in the absence of IL-2 . Regarding the upstream enhancer, the transactivation was absolutely dependent on the intact STAT site and to a lesser extent on the 3′ STAT-n site . Additional IL-2R signaling provided somewhat higher levels, suggesting that additional transcription factors were induced. The analogous transactivation studies of the far-upstream enhancer were complicated by its additional 5′ STAT-n site. As described above, this site was physiologically irrelevant because its mutation did not at all impair the IL-2 response. It may have served, however, as an alternative binding site for Stat5-VP16-Jak2 when the partially overlapping STAT site had been mutated, because all three sites had to be mutated to completely abolish the transactivation (triple mutant). Regardless, the data presented for both enhancers strongly suggest that their activation by IL-2R signals is dependent on the binding of activated Stat molecules to a tandem element, which may be comprised of a higher affinity site (STAT) and a lower affinity site (3′ STAT-n). Scrutiny of the regulation of the perforin gene is biologically important and offers a valuable model to shed molecular light on the activation/differentiation of CTLs. This line of investigation comprises a largely unexplored area in comparison to analogous investigations of cytokine genes, which have become widely used as a paradigm toward an understanding of Th activation/differentiation. In our study toward deciphering the control of perforin expression by IL-2, >45 kb of the human perforin gene locus was surveyed in a transgenic tissue culture system that mimics important aspects of perforin gene induction in primary CTLs . This analysis led to two IL-2–responsive enhancers in the perforin gene locus , which also promoted inducibility in T cells derived from transgenic mice, at least in the context of their intervening genomic sequences ( Table ). The activation of both enhancers by IL-2 required not only the activation of the Stat pathway but also the participation of tandem Stat-like elements that could be transactivated by Stat5 molecules in our CTL differentiation model . These findings suggest that the activation of Stats by IL-2R signals, in particular the activation of Stat5 molecules, plays an important role for the generation of CTLs. The regulation of the perforin gene by IL-2R signals via Stat proteins is consistent with several observations besides the well documented activation of Stat5 and Stat3 by IL-2R signals 35 . Stat proteins are latent transcription factors 36 and, accordingly, the onset of perforin mRNA induction by IL-2 does not require newly synthesized proteins in our model cell line nor in primary cells 3 . Similarly, the activation of Stat5 37 , the induction of perforin mRNA , and the activation of its enhancers , as well as perforin mRNA induction by IL-2 in primary cells 31 , are all resistant to rapamycin. Conversely, TGF-β, which has been suggested to block the activation of the Jak/Stat pathway in T cells, including the activation of Stat5 38 39 , also prevents the induction of perforin by IL-2 40 . Importantly, the analysis of Stat5 knockout animals, as well as the phenotypes of animals lacking the Stat docking sites of IL-2Rβ, have recently indicated that this IL-2R signaling pathway may target the perforin gene in vivo and may be essential for the generation of cytotoxicity. Stat5 exists in humans and mice as two closely related genes with overlapping expression patterns and, therefore, has an often redundant role in vivo for both nonlymphoid and lymphoid tissues 41 42 . Nevertheless, immunologically relevant phenotypes, albeit perhaps rather discrete, have been reported for certain single-deficient animals. Stat5a-deficient animals fail to upregulate IL-2Rα in response to IL-2R signals 43 . Stat5b-deficient splenocytes, on the other hand, poorly generate CTLs in response to IL-2 or IL-15. This defect, which is accompanied by profoundly reduced but still detectable levels of perforin mRNA, led to the proposal that perforin is a Stat5-regulated gene 44 . This notion is supported by our results. Similarly, splenocytes of mice expressing an IL-2Rβ lacking the Stat docking sites fail to generate CTLs in response to IL-2 45 . The redundancy of Stat5a and Stat5b may relate to the incomplete block of perforin gene induction by IL-2 or IL-15 in Stat5b-deficient splenocytes inasmuch as Stat5a-deficient cells also expressed slightly reduced levels of perforin 44 . The inability of double-deficient T cells to enter the cell cycle 42 does not allow us to readily address this issue experimentally due to the relatively long stimulations required for significant inductions. It is also possible that Stat5 molecules are facilitators rather than the essential players for perforin gene activation or that they are redundantly used with Stat3 molecules. Whereas the latter pathway was not functional in our SAM-19 model (see Results), another report published while this manuscript was in preparation indicates that the upstream enhancer of the perforin gene could also be activated by Stat3 molecules 46 . The authors identified a constitutively enhancing fragment similar to the upstream enhancer of our analysis by transient transfections of ∼1,400 bp of the 5′ flank into YT cells, a constitutively perforin-expressing NK cell–like lymphoma. The function of this DNA in YT cells was dependent on what is referred to in Fig. 6 as the STAT element, whereas the 3′ STAT-n site was not investigated. Regardless, the STAT element was shown to bind constitutively activated Stat3 molecules present in the YT lymphoma and Stat5 molecules when extracts of primary NK cells exposed to IL-2 were applied, suggesting that Stat5 and Stat3 molecules may regulate the upstream enhancer of the perforin gene. It is conceivable that genes are regulated redundantly or activation stage specifically by Stat5a, Stat5b, and Stat3 molecules in T cells, because an impaired upregulation of IL-2Rα in response to IL-2R signals has been described not only for Stat5-deficient animals 42 43 but also for Stat3-deficient T cells 47 . Notably, Stat5 proteins may also play an essential role in the development and/or maintenance of lineages that constitutively express perforin, i.e., NK cells 2 and γ/δ T cells 48 49 50 . NK cells are absent in animals deficient in both Stat5 proteins 42 , as well as in animals deficient in the ability to activate Stats in response to IL-2R or IL-15R signals 45 . The latter animals also lack γ/δ intraepithelial lymphocytes, a lineage that so far has not been investigated in the Stat5 double-deficient animals. The biological relevance of Stat5 molecules and the identified enhancers for the constitutive expression of perforin by these lineages remains to be analyzed. A constitutively active Stat signaling pathway in NK cells in vivo, and thereby also an activation of the perforin gene by the identified enhancers, could be envisioned based on the expression of IL-15 in virtually all tissues and the absolute requirement of IL-15R signals for NK development and/or maintenance 51 52 . The perforin enhancers and the enhancer of the human and mouse IL-2Rα genes 53 54 55 56 57 are presently the only suspected Stat5 and Stat3 targets in vivo among genes specifically expressed by lymphocytes. Interestingly, they are also similarly organized. Just like the perforin enhancers, the IL-2Rα enhancer requires a tandem Stat element that serves as a composite binding site for a tetrameric Stat5 complex binding individual sites of weaker affinity 55 57 . We are presently addressing whether the perforin sites, each of which can bind Stat5 in vitro (Schindler, U., and M.G. Lichtenheld, unpublished observation), also serve as targets for a tetrameric Stat5 complex, because the spacing of the Stat-like elements in the perforin enhancers exceeds the spacing of those in the IL-2Rα enhancers (17 vs. 11 bp). The loss of enhancer function by an individual mutation of either site is consistent with a tetrameric assembly, but these experiments do not address the mechanism for the observed cooperation. The other element with a major function in the mouse and human IL-2Rα enhancers entails an Elf-1–binding Ets core located 14 bp 3′ of their tandem Stat elements 54 56 . This organization is exactly like that of the far-upstream enhancer of the perforin gene . An analogous element in the upstream enhancer is located 10 bp 3′ of the tandem Stat-like elements. The STAT sites of both perforin enhancers as well as the 3′ STAT-n site of the upstream enhancer incorporate an Ets binding motif sequence core similar to that of the human IL-2Rα enhancer. This element has been suggested to repress the enhancer in unactivated cells via an unidentified Ets family protein 54 . Unlike the IL-2Rα enhancer and the upstream enhancer of the perforin gene, the far-upstream enhancer may respond not only to IL-2R but also to TCR signals that have been implied as a second pathway for perforin gene induction 1 2 3 , because this enhancer also responded to PMA and ionomycin in a CsA-sensitive manner in SAM-19 . This observation could suggest a possible role for a nuclear factor of activated T cells (NFAT)-like element in the far-upstream enhancer whose sequence resembles the “IL140” element of the IL-3 enhancer, which is known to strongly activate transcription independently of AP-1 proteins 58 . The physiological activation of the perforin enhancers is likely to involve transcription factors in addition to Stat5. The combined control of the perforin enhancers remains to be experimentally established and compared with that of the IL-2Rα enhancer. Based on the inspection of the perforin sequences , other participating transcription factors could be IL-2 inducible, namely AP-1, cAMP-responsive element binding proteins, and nuclear factor κb 59 60 61 , or differentially expressed during lymphoid development, namely Ikaros proteins 62 and proteins recognizing the E-box 63 . In summary, this study investigates the differentiation processes of CTLs by working backwards from the perforin gene. It indicates that the induction of the perforin gene by IL-2R signals involves at least two enhancers whose activation is dependent on Stat elements that can be targeted by Stat5 molecules. These results are consistent with and complement the ongoing findings of the reverse genetics analysis of the IL-2R Jak/Stat signaling pathway. | Study | biomedical | en | 0.999998 |
10544202 | DO11.10 mice, which are transgenic for the TCR recognizing OVA peptide 323–339 (pOVA 323–339 ) 17 , were provided to us on a BALB/c background by Ken Murphy (Washington University, St. Louis, MO) and were bred in our facilities. IL-4–deficient BALB/c mice (The Jackson Laboratory) were bred in our facilities. Cells were transferred into 6–12-wk-old BALB/c mice (The Jackson Laboratory) or IFN-γR −/− mice (provided by J. Aguet, Molecular Biology Institute, Zurich, Switzerland; backcrossed six generations onto BALB/c). To generate Th1 or Th2 cells from DO11.10 mice, CD4 T cells were isolated by negative selection as previously described 18 using mAbs to CD8 (clone 53-6.72, clone 2.43; reference 19 ), class II MHC I-A d , and anti-Ig–coated magnetic beads (Collaborative Research, Inc.). Naive CD4 T cells were further isolated from this population by positive selection with a biotinylated anti–L-selectin antibody (Mel-14; PharMingen) and streptavidin microbeads using MACS™ (Miltenyi Biotec). Syngeneic T cell–depleted splenocytes were used as APCs and prepared by negative selection using antibodies to CD4 (GK1.5; reference 20 ), anti-CD8, anti-Thy1 21 , and treatment with rabbit complement. APCs were mitomycin-C treated. To generate Th1 cells, cultures contained pOVA 323–339 at 5 μg/ml, IL-12 at 5 ng/ml (Genetics Institute), IL-2 at 10 U/ml (Collaborative Research, Inc.), and anti–IL-4 (11B11; reference 22 ) at inhibitory concentration. To generate Th2 cells, cultures contained pOVA 323–339 at 5 μg/ml, IL-4 at 200 U/ml (Collaborative Research, Inc.), IL-2 at 10 U/ml, and anti–IFN-γ (XMG1.2; reference 23 ) at inhibitory concentration. All cultures were set up in flasks containing equal numbers of CD4 T cells and APCs at a final concentration of 5 × 10 5 cells/ml and were maintained for 4 d. Cultured Th1- or Th2-like cells were harvested after 4 d and washed with PBS, and cells were injected intravenously into syngeneic recipients. 1 d after transfer of cells, mice were challenged with inhaled 1% OVA in PBS as previously described 15 for 20 min daily for a total of seven days over a period of nine days (four consecutive days exposed, two days rested, and three consecutive days exposed). Control mice received inhaled OVA only. Mice were analyzed 24 h after the final exposure to antigen. At the time of transfer, an aliquot of Th1- or Th2-like cells was retained for restimulation. 5 × 10 5 CD4 T cells/ml and 5 × 10 5 freshly isolated BALB/c APCs per milliliter were cultured with pOVA (5 μg/ml). Supernatants were collected after 48 h. Bronchoalveolar lavage (BAL) cells obtained from individual mice were restimulated in vitro at 2 × 10 6 cells/ml in the presence of pOVA (5 μg/ml). IFN-γ, IL-4, and IL-5 levels from cell supernatants were determined by ELISA (Endogen). Assays were standardized with recombinant IFN-γ, IL-5, IL-10 (Endogen), and IL-4 (Collaborative Research, Inc.). The lower limit of sensitivity for each of the ELISAs was 0.6 ng/ml (IFN-γ), 5 pg/ml (IL-4), 0.010 ng/ml (IL-5), and 200 pg/ml (IL-10). For BAL fluid cytokines, mice were exposed to aerosolized OVA on days 1 and 2, BAL was performed on day 3, and fluid was analyzed by ELISA for cytokines IL-4, IL-13 (R & D Systems, Inc.), IL-5 (Endogen), and IFN-γ (Biosource International, Inc.). Cell populations were stained with anti-CD4 (Quantum Red-L3T4; Sigma Chemical Co.), the biotinylated anticlonotypic antibody KJ1-26 24 , and FITC–avidin D (Vector Labs.). KJ1-26 is specific for the transgenic TCR in the DO11.10 mice. Th1 and Th2 cell populations transferred were >97% CD4 and KJ1.26 positive. After a period of inhalational exposure, BAL cells were analyzed by FACS™ using these antibodies. BAL was performed by cannulation of the trachea and lavage with 1 ml of PBS. Total cell counts were performed, cytospin preparations of BAL cells were stained with Dif-Quik (Baxter Healthcare Corp.), and differentials were performed based on morphology and staining characteristics. Lungs were prepared for histology by perfusing the animal via the right ventricle with 20 ml of PBS. Lungs were then inflated with 1.0 ml of fixative instilled through a tracheostomy tube. Samples for paraffin sectioning were formalin fixed and sectioned in the coronal plane at 5 μm, ensuring that central airways were visible. Sections were stained with hematoxylin and eosin and periodic acid-Schiff (PAS). Histological mucus index (HMI) was performed as previously described on PAS-stained sections and is equivalent to the linear percent of epithelium positive for mucus 15 25 . This index was calculated for each mouse lung, and then the mean HMI was calculated for each experimental group. We previously showed that TCR-transgenic Th1 or Th2 cells generated in vitro and transferred into recipient mice are recruited to the lung after inhaled antigen challenge and retain their polarized cytokine profile in vivo, and that both Th1 and Th2 cells induce airway inflammation 15 . Th2 cells stimulate inflammation with lymphocytes and eosinophils and induce airway epithelial mucus production ( Table ). Th1 cell activation leads to lymphocytic inflammation, but Th1 cells fail to stimulate eosinophilia or mucus production. It was unclear if the differences in Th1 and Th2 cell effects resulted from Th1 cells lacking the cytokines necessary to induce eosinophilia and mucus or if Th1 cells actively inhibited these processes. To investigate the mechanism by which Th1 cells failed to stimulate eosinophilia and mucus production and if Th1 cells could inhibit Th2-induced inflammation, we performed mixing experiments by transferring both Th1 and Th2 cells together into recipient mice. Th1 and Th2 cells were each generated in vitro from CD4 T cells isolated from TCR-transgenic, DO11.10 mice that were cultured with APCs, pOVA 323–339 , and polarizing cytokines as previously described 15 . At the time of transfer, Th1 cells secrete high levels of IFN-γ and no IL-4 and IL-5, whereas Th2 cells produce very low levels of IFN-γ but high levels of IL-4 and IL-5 . Mice received transfer of Th1, Th2, or a mixture of Th1 and Th2 cells (Th1 + Th2) and were exposed to inhaled antigen. Airway eosinophilia, which is consistently present after transfer of Th2 cells and exposure to inhaled OVA, is markedly reduced when Th1 + Th2 cells are transferred . The inhibitory effects of Th1 cells on airway eosinophilia are dependent on the number of Th1 cells transferred, as when fewer Th1 cells are transferred into the mice, more eosinophils are present in the BAL. When equal numbers of Th1 and Th2 cells were transferred together and mice were exposed to inhaled antigen, eosinophils were consistently inhibited sevenfold or greater in experiments that employed transfer of a range of different cell numbers (10 6 –5 × 10 6 cells). Mice exposed to inhaled OVA after no cells were transferred or after Th1 cells were transferred did not exhibit BAL eosinophilia. Th2 cells induce a marked increase in mucus staining in the bronchial epithelium, but when Th1 + Th2 cells are transferred, there is a reduction in airway epithelial mucus staining . This inhibition of mucus staining in the airway epithelium is modest when airway inflammation is severe (after transfer of 2.5 × 10 6 cells). When fewer Th2 cells are transferred into recipient mice, resulting in less inflammation, mucus staining is markedly inhibited by equal numbers of Th1 cells . When two times the number of Th1 cells are transferred with Th2 cells (2:1), there is complete inhibition of mucus staining. Thus, Th1 cells inhibit Th2-induced mucus in a dose-dependent fashion. Mice that were exposed to inhaled OVA and received transfer of no cells or Th1 cells showed mucus staining in <5% of bronchial epithelial cells. Therefore, when Th1 and Th2 cells are transferred together into recipient mice, Th1 effects dominate, resulting in the inhibition of Th2-induced airway eosinophilia and mucus production. It was possible that Th1 cell inhibition of Th2-induced inflammation resulted from decreased Th2 cell activity, either by blocking recruitment of cells to the lung or by direct suppression of Th2 cell activation. To determine if Th2 cells were active in the lungs of mice after transfer of Th1 + Th2 cells, we examined BAL fluid recovered after transfer of cells and exposure to inhaled OVA. In mice that received Th1 + Th2 cells and inhaled OVA, IL-4, IL-5, and IL-13 were present in the BAL fluid at levels comparable to those in BAL fluid recovered from mice that were exposed to OVA after transfer of Th2 cells alone . IFN-γ levels were similar in mice that received Th1 + Th2 cells or Th1 cells alone and inhaled OVA. BAL cells recovered from mice after exposure to inhaled OVA and transfer of Th1 + Th2 cells produced IL-4, IL-5, IL-13, and IFN-γ (data not shown), again indicating that both the transferred, OVA-responsive DO11 Th1 and Th2 cells are present in the respiratory tract. These data suggest that Th1 inhibition of Th2-induced inflammation does not result from inhibition of Th2 cell activation in the respiratory tract but at steps downstream of cytokine secretion. In mice that received Th1 + Th2 cells and inhaled OVA, airway inflammation, as measured by the total number of BAL cells recovered from mice, was not increased when compared with mice that received Th2 cells and inhaled OVA ( Table ). This was independent of the number of transferred cells, as shown. These data indicate that cotransfer of Th2 and Th1 cells, doubling the number of transferred cells, does not lead to increased airway inflammation yet results in suppression of airway eosinophilia and mucus induction. To determine the mechanism by which Th1 cells inhibit Th2-induced inflammatory responses, we investigated the role of the Th1 cytokine IFN-γ, as IFN-γ had been shown previously to have inhibitory effects on Th2 cell functions in vivo, including inhibition of CD4 T cell migration, eosinophilia, and IgE production 26 27 28 29 . We transferred DO11 Th1, Th2, or Th1 + Th2 cells into IFN-γR +/+ or IFN-γR −/− mice. Although the transferred DO11 Th1 cells were able to secrete IFN-γ at the time of transfer, IFN-γR −/− recipient mice were unable to respond to IFN-γ. After exposure to inhaled OVA, IFN-γR −/− mice that received Th1 + Th2 cells showed marked airway eosinophilia, with levels of eosinophils equivalent to those in IFN-γR −/− mice that received transfer of Th2 cells and inhaled OVA . Thus, Th1 cells no longer inhibited Th2-induced eosinophilia in the absence of IFN-γ signaling. IFN-γR +/+ mice that received transfer of the same population of Th1 + Th2 cells had 18-fold fewer eosinophils in the BAL. After transfer of Th2 cells and exposure to inhaled antigen, IFN-γR −/− recipients had four times as many eosinophils in the BAL than IFN-γR +/+ recipients, indicating that even during heavily skewed Th2 responses, the inhibitory effects of IFN-γ on airway eosinophilia are present. IFN-γR −/− mice that received Th1 cells and inhaled OVA or mice that received inhaled OVA alone did not exhibit significant eosinophilic airway inflammation. Therefore, the induction of airway eosinophilia requires activated Th2 cells and can be inhibited by IFN-γ secreted by Th1 cells. When Th1 + Th2 cells are transferred into IFN-γR −/− mice and mice are exposed to inhaled OVA, mucus staining is present at levels similar to mucus staining in IFN-γR −/− mice that received Th2 cells alone . Th1 cell inhibition of Th2-induced mucus is no longer present in IFN-γR −/− mice, suggesting that IFN-γ inhibits mucus production by airway epithelial cells. Yet when Th1 cells were transferred into IFN-γR −/− mice and mice were exposed to inhaled OVA, mucus was also induced , indicating that Th1 cells are capable of inducing mucus when IFN-γ signaling is absent. IFN-γ not only inhibits Th2-induced mucus production, but the absence of mucus in IFN-γR +/+ animals that received Th1 cells appears to be a result of active inhibition by IFN-γ. These data suggest that Th2 cells are not essential for mucus induction. It was still possible that the induction of mucus production by DO11 Th1 cells in IFN-γR −/− mice was a result of Th2 cytokine activation, as blockade of IFN-γ during a Th1 cell response can induce the cell population to produce IL-4 30 . To test if the cytokine pattern of the DO11.10 Th1 population had shifted in vivo, we harvested BAL cells from mice that received Th1 cells and inhaled OVA and restimulated the cells in vitro with pOVA 323–339 . BAL cells recovered from IFN-γR −/− recipient mice secreted high levels of IFN-γ and minimal IL-4, IL-5, and IL-13, similar to the levels of cytokines secreted by BAL cells recovered from IFN-γR +/+ recipient mice ( Table ). Thus, Th1 cells, after recruitment and activation in IFN-γR −/− mice, remain polarized to Th1 yet are capable of stimulating mucus production. In summary, airway epithelial mucus production and airway eosinophilia are stimulated by different mechanisms; recruitment of eosinophils to the airway requires Th2 cells, whereas mucus can be induced by both Th1 and Th2 cells, as long as IFN-γR signaling is absent. Despite different mechanisms of induction, both airway eosinophilia and mucus production can be inhibited by activation of Th1 cells producing IFN-γ. Th1 and Th2 cytokines are both present after transfer of Th1 + Th2 cells and exposure to inhaled OVA , suggesting that the downregulatory effects of Th1 on Th2 cells are not through direct inhibition of Th2 cell activation. The mechanism of action of Th1 cells is precisely defined in transfer experiments using IFN-γR −/− recipient mice . In IFN-γR −/− mice, the transferred DO11.10 Th2 cells express the IFN-γR and are capable of responding to IFN-γ secreted by Th1 cells. If Th1 cells were directly blocking Th2 cell function, then inhibition of eosinophilia and mucus production would persist in IFN-γR −/− mice. Yet the inhibitory effects of Th1 cells on Th2 cell–induced eosinophilia and mucus production are completely abolished in IFN-γR −/− mice. These studies suggest that IFN-γ produced by Th1 cells inhibits Th2-induced eosinophilia and mucus production despite ongoing Th2 cytokine secretion, through effects requiring IFN-γR in recipient mice. As different inflammatory pathways stimulate airway eosinophilia and mucus production, it is likely that IFN-γ inhibits these processes through distinct mechanisms that are induced in target tissue. Recent studies have established that CD4 Th2 cells and their cytokines initiate an inflammatory response in the respiratory tract with many features of asthma. In contrast, Th1 cells lead to inflammation but exhibit none of the asthmatic pathology. As both Th1 and Th2 cells have been identified in the lungs of asthmatic patients, we investigated whether Th1 cells could regulate allergic airway pathology. In this report, we show that coactivation of Th1 and Th2 cells in the lung leads to a dominance of Th1 effects, inhibiting both airway eosinophilia and mucus production. Th1 cells, through the production of IFN-γ, inhibit these Th2-induced effects, not by regulating Th2 cell activity, as previously suggested, but by blocking downstream pathways induced by Th2 cytokines. Furthermore, the marked inhibitory effects of Th1 cells occur without an increase in airway inflammation. Thus, it appears that Th1 cells block critical pathologic changes that contribute significantly to morbidity and mortality in asthma. Our data show that IFN-γ inhibits airway eosinophilia even during polarized Th2-type responses, indicating that Th1 cells in the airways of asthmatics may be active in controlling disease. In addition, these studies are the first to show that the development of inflammatory pathology in asthma can be differentially controlled. Whereas airway eosinophilia depends on the presence of activated Th2 cells in the lung, mucus can be induced by different types of inflammatory infiltrates as long as IFN-γR signaling is blocked. Th1 cells fail to stimulate mucus because IFN-γ inhibits its production. Th1 cells do not induce eosinophilia, most likely due to a lack of IL-5 31 . Th1 responses have been proposed to protect against asthma. This theory is based on Th2-dominant lymphocyte populations in the airways of asthmatics and evidence that Th1 responses protect in asthmatics and in animal models of asthma 2 7 11 12 13 . Th1 cells may inhibit Th2 cell function at different stages in the effector response. Th1 cells, through the production of IFN-γ, have been shown to inhibit Th2 cell cytokine production and Th2 cell proliferation in vitro 32 33 . In mice, the Th1 cytokine IFN-γ has inhibitory effects on Th2-induced airway eosinophilia and AHR. When administered before inhaled antigen challenge, IFN-γ reduced the number of CD4 T cells in the respiratory tract 26 28 29 or reduced Th2 cytokine secretion 26 34 . These effects may result from inhibition of Th2 cell recruitment by IFN-γ. Once Th2 cells are present in the respiratory tract, IFN-γ suppresses the resolution of Th2-induced inflammation, as shown in IFN-γR −/− mice that had prolonged eosinophilia and Th2 cytokine production 35 . In this report, we show another role of IFN-γ in the regulation of Th2 responses. Th1 cell production of IFN-γ blocks Th2-induced inflammatory pathways downstream of cytokine production. This may occur by direct effects on eosinophils and epithelial cells or through an intermediate cell derived from the recipient mice. This mechanism of inhibition is of potential importance in asthmatic airways, where Th2 cells are chronically present, as we show that Th1 cells can inhibit Th2 cell effects while Th2 cells are actively secreting cytokines. The inhibitory pathways induced by Th1 cells require more than a few days for induction. In our studies, the inhibitory effects of Th1 cells were seen in mice exposed to antigen over 9 d. These effects were not observed when we killed mice after just 2 d of antigen challenge: BAL eosinophilia was similar after transfer of Th2 cells or Th1 + Th2 cells and inhaled antigen (data not shown). It is possible that a short period of antigen exposure explains, in part, why other investigators did not observe a similar reduction in eosinophilia after recruitment and activation of Th1 and Th2 cells in the respiratory tract 36 37 . This delay in inhibition of eosinophilia and mucus production by Th1 cells may point to a biological pathway that requires either time or stimulation with higher levels of IFN-γ to induce inhibition. In defining the functional effects of Th1 cells on Th2-mediated airway inflammation, our studies provide insights into potential mechanisms governing symptom control in conventional allergy immunotherapy. These mechanisms need to be analyzed, as we have shown, by assessing the lymphocyte populations present at sites of inflammation where cytokines exert their effects. Studies of grass pollen immunotherapy for atopic skin disease and allergic rhinitis showed a reduction in late phase responses and eosinophil accumulation in the skin and nasal mucosa. These effects were associated with increased IFN-γ–expressing cells, yet cells positive for IL-4 and IL-5 were unchanged 9 10 . In addition, persistent Th2 cytokine production with increased IFN-γ–producing cells may explain why IgE levels and skin prick testing were not reduced, but IgG1/IgG4 levels increased in successful immunotherapy of pollen-allergic individuals 38 . Increasing the population of activated Th1 cells at sites of allergic inflammation should be a focus of new techniques of immunotherapy, as IFN-γ can exert its effects despite ongoing Th2 cell activation. These effects may be sustained, as there is evidence that potent, long-term stimulation with Th1 cytokines can shift a Th2-predominant population toward Th1 39 40 41 . Increasing Th1 cell activation during immunotherapy in asthma bears the potential risk of increasing inflammation. Successful immunotherapy for atopic skin disease and rhinitis in allergic patients has not borne out these concerns 10 41 . In our studies and others, airway inflammation was not increased when both Th1 and Th2 cells were recruited to the lung in wild-type recipient mice 42 . This may relate to normal regulation of T cell proliferation that is typically present in intact, immunocompetent mice 43 . Hansen et al. 42 showed that Th1 and Th2 cells transferred into SCID mice, which lack these regulatory elements, leads to unrestrained proliferation and overwhelming airway and parenchymal inflammation. Furthermore, reducing airway eosinophilia leads to a reduction in tissue damage and cellular infiltration 44 45 46 . Blocking recruitment of eosinophils to the airway results in a reduction in proinflammatory factors that potentiate inflammation. Thus, as these studies show, the net effect of recruiting Th1 cells to the lung is that total inflammation is relatively unchanged. Airway inflammation has long been associated with excess mucus production. In chronic bronchitis, cystic fibrosis, and asthma, mucus hypersecretion is associated with different characteristic immune responses in the lung. Unlike asthmatics, patients with cystic fibrosis or chronic bronchitis typically do not show activated Th2 cells in their airways. In a model of asthma, we recently showed that Th2 cells stimulate airway mucus production 15 . Mucus induction requires IL-4Rα but is independent of IL-4, IL-5, eosinophils, and mast cells 47 . Other recent studies showed that IL-13 and IL-4 are important mediators in mucus production 48 49 50 51 52 . Here we also show that Th1 cells stimulate mucus production in the absence of IFN-γR signaling. The neutrophil-predominant inflammatory response in these mice and the relationship of neutrophilia with mucus hypersecretion in cystic fibrosis and chronic bronchitis suggests that neutrophils may be involved in mucus hyperproduction. Neutrophil elastase is a potent mucus secretagogue 53 , yet the ability of this enzyme to stimulate increased mucin production has not been determined. We have shown that mucus induction is not due to a shift of the transferred Th1 cell population to a Th2 phenotype in IFN-γR −/− recipient mice. There still remains the possibility that very low levels of IL-13 secreted by Th1 cells stimulate mucus production in IFN-γR −/− mice. In summary, we have shown that mucus can be induced by an inflammatory response that is not dominated by production of IL-13 and IL-4. Although different inflammatory responses stimulate mucus production, Th1 cells, through production of IFN-γ, inhibit mucus induced by both Th1 and Th2 cells. Inhibitory pathways for mucus have not been previously demonstrated. IFN-γ has been shown to have inhibitory effects on some epithelial functions. In gastric epithelial cells, mucus secretion was inhibited by IFN-γ 53 . IFN-γ also inhibited growth of a human bronchial epithelial cell line and reduced barrier function and chloride secretion in intestinal epithelial cells 54 55 . Beyond these limited studies, the inhibitory effects of IFN-γ on airway epithelium are not known. Interestingly, mucus production is not a feature of Th1-mediated pulmonary diseases in humans. Mycobacterium tuberculosis infection and sarcoidosis are diseases in which IFN-γ–producing CD4 T cells have been identified in the lung biopsies and in BAL 56 57 58 . It is possible that the lack of mucus production in these conditions results from IFN-γ suppression. IFN-γ has many proinflammatory effects in the lung, most notably on macrophages, activating production of reactive nitrogen and oxygen species. The inhibitory effects of IFN-γ could therefore be through the production of inhibitory mediators by inflammatory cells or by direct effects on goblet cells. These studies establish the first known natural inhibitor of mucus production, one that can be active in different inflammatory settings. In summary, using a transfer system that we developed to study the role of CD4 Th1 and Th2 cells in airway disease, we have defined two different pathways by which Th1 cells can regulate airway inflammation. Th1 cells, through an effect mediated by IFN-γR, block the recruitment of eosinophils to the airway and inhibit airway epithelial mucus production. In the absence of IFN-γR, Th1 cells induce mucus production but do not stimulate airway eosinophilia. Thus, Th1 cells have differential effects on stimulating these inflammatory responses. The inability of Th1 cells to stimulate eosinophilia likely results from a lack of IL-5. The mechanism by which mucus production is stimulated by Th1 cells in the absence of IFN-γR signaling is not yet known. As we learn how these inflammatory responses are regulated, we will identify new targets for directed immunotherapy for asthma and mucus hypersecretion. | Study | biomedical | en | 0.999997 |
10544203 | C57BL/10 mice at 5–6 wk of age were obtained from the animal department of the Netherlands Cancer Institute. Mice were handled at all times in accordance with institutional guidelines. Peptides were produced using standard g-fluorenylmethoxycarbonyl (FMOC) chemistry. Soluble fluorochrome (PE or allophycocyanin [APC])-labeled MHC tetramers were produced as described previously 17 19 and stored frozen in Tris-buffered saline/16% glycerol/0.5% BSA. Influenza A viruses A/NT/60/68 and A/HKx31 were provided by Dr. R. Gonsalves, National Institute for Medical Research, London, UK. Influenza virus B/Lee/40 was obtained from the American Type Culture Collection. Mice were killed at indicated time points after infection, and organs were removed for further analysis. Inflamed lung tissue and spleens were minced in single chamber mesh filters. The single cell suspensions obtained were treated with NH 4 Cl solution to get rid of contaminating erythrocytes, before staining for flow cytometry purposes. The influenza A virus (A/NT/60/68) NP–derived H-2D b -restricted CTL epitope, ASNENMDAM, was introduced into EL4 tumor cells by retroviral insertion as a COOH-terminal fusion with the enhanced green fluorescent protein (eGFP) gene product (Wolkers, M.C., manuscript in preparation). In all instances, mononuclear cells were stained with directly labeled mAbs or MHC tetramers. Analysis was performed on a FACSCalibur™ (Becton Dickinson) using CELLQuest™ software (Becton Dickinson). Before staining, propidium iodide (PI) was added to gate for PI-negative (living) lymphocytes. Cytolytic activity of sorted CD8 + T cells derived from inflamed pulmonary tissue was determined in a standard 5-h 51 Cr-release assay. EL4 target cells were preincubated with peptides for 1 h at 37°C. Percent specific lysis was calculated from the equation: [(experimental 51 Cr release – spontaneous 51 Cr release)/maximal 51 Cr release – spontaneous 51 Cr release)] × 100%. Spleen-derived mononuclear cells were stained with anti-CD8 mAb in the presence of PE-labeled MHC tetramers containing increasing concentrations of MHC monomers. Cells were subsequently analyzed by flow cytometry. Intracellular cytokine staining was performed as described 20 . In brief, spleen cells were incubated with peptide (0.5 μM) for 5–6.5 h at 37°C in the presence of recombinant human (rh)IL-2 (50 U/ml) and Brefeldin A (0.1 μl/ml). After incubation, cells were surface stained with anti-CD8a–APC mAb (PharMingen), incubated in Cytofix/Cytoperm solution (PharMingen) for 20 min on ice, washed, and stained for intracellular cytokine with anti–IFN-γ–FITC (PharMingen) or FITC-labeled isotype control antibody (PharMingen). Analyses were performed on a FACSCalibur™ (Becton Dickinson) using CELLQuest™ software. Isotype control antibodies resulted only in background staining (data not shown). Mice were injected subcutaneously with 100 μg peptide in IFA, 4–6 wk after a primary influenza A virus infection. On days 0, 1, and 2, 100 μg anti-CD40 mAb (FGK.45) was injected intravenously 21 . On day 10 after peptide immunization, spleen cells were used for flow cytometry analysis. To assess the effect of random antigen variation on the dynamics of T cell responses in vivo, we infected mice with pairs of influenza A viruses. These viruses expressed either the same NP 366–374 epitope or epitope variants. The specificity of the resulting T cell repertoire was assessed by two-parameter MHC tetramer staining, and association of MHC tetramers to NK receptors was ruled out through analysis of CD8b-expressing cells only. When mice are infected once or twice with either influenza virus strain A/NT/60/68 or A/PR/8/34, which differ in the sequence of the immunodominant NP CTL epitope at positions 7 and 8 (ASNENM DA M vs. ASNENM ET M), the vast majority of the resulting NP-specific T cells selectively recognize the epitope of the strain encountered and not that of the opposite strain . This dominant role of the peptide side chains at positions 7 and 8 in ligand recognition by the majority of T cells is in accord with the prominent contribution of p8 and especially p7 to the TCR-exposed surface of this peptide–MHC complex 22 . This virus strain specificity of the NP-reactive CTLs contrasts sharply with the apparent lack of strain specificity during secondary responses against a variant strain. When mice that had recovered from a previous infection with influenza virus strain A/NT/60/68 are challenged with viral strain A/PR/8/34, most if not all NP PR -reactive T cells are fully cross-reactive between the two NP variants . This phenomenon is reciprocal: in mice that have previously experienced infection with strain A/HKx31 (a reassortant strain with the NP gene from A/PR/8/34), the NP-reactive T cell population that emerges upon infection with A/NT/60/68 is fully cross-reactive between the two viral strains . Although somewhat variable between individual mice, the extent of binding of the two MHC tetramers appears independent of the order in which the epitopes were encountered over a large series of experiments. To compare the affinity for primary and secondary antigen in a more direct manner, competition studies were performed. These experiments demonstrate that the binding of fluorochrome-labeled MHC tetramers can be inhibited by similar concentrations of ASNENMDAM- and ASNENMETM-containing monomers, indicating equal affinity of the cross-reactive TCRs for either antigen . In addition, these data rule out the possibility of dual TCR expression 23 24 by the cross-reactive T cells, since both MHC monomers compete for binding of ASNENMDAM-containing MHC tetramers. The above results indicate that the subsequent encounter of variants of a T cell epitope results in the expansion of a cross-reactive T cell population, as established by biochemical assays. In fact, this expansion inhibits the expansion of the largely strain-specific population observed during a regular primary response. However, T cell recognition of ligands with similar affinities can have drastically different functional outcomes, due to differences in off rates of TCR–ligand interactions 25 26 27 28 29 . Therefore, we examined the functional behavior of this biochemically cross-reactive T cell population towards both the primary and the mutant epitope. The cross-reactive T cells that are observed during a recall influenza A virus infection lyse target cells pulsed with both the primary and the recall antigen directly ex vivo to the same extent over a wide range of E/T ratios, and over a range of peptide concentrations . As an independent test of functional cross-recognition, we measured the ability of these cells to initiate IFN-γ synthesis upon stimulation with the original or variant epitope. Since simultaneous staining of cells with MHC tetramers and intracellular IFN-γ staining is technically difficult, cells were stimulated in the presence of the original and variant peptide separately or simultaneously . The proportion of IFN-γ–positive CD8 + T cells is similar in all three cases, indicating that the T cell populations that recognize the two epitopes are largely overlapping and therefore cross-reactive. We conclude that the T cell population that emerges upon encounter of an antigenic variant is biochemically cross-reactive, and this cross-reactivity is fully reflected in their functional behavior. To better understand the ontogeny of the cross-reactive T cell population, we examined the kinetics and composition of this T cell pool. The cross-reactive cytotoxic T cell population appears 2–3 d earlier at the site of infection (i.e., pulmonary tissue; data not shown) than the antigen-specific T cells during infection of naive mice 15 16 17 , suggesting that these cells originate from a preexisting memory T cell population. Several studies have shown a narrowing of the antigen-specific polyclonal TCR repertoire during recall infection, due to the preferential outgrowth of a subpopulation of memory cells 30 31 32 33 . In naive mice that are infected with influenza virus A/PR/8/34, the repertoire of NP-specific T cells involves a variety of BV elements . The slight preferential usage of the BV8.3 element reported previously for C57BL animals infected with influenza virus A/PR8/34 was not observed in these experiments 34 . In contrast, in A/NT/60/68-primed mice that are infected with influenza virus A/PR/8/34, the repertoire of A/PR/8/34-specific T cells is highly restricted . This narrow T cell repertoire is likely to reflect the affinity maturation observed previously 33 compounded by the low number of cross-reactive T cells within the original memory population. In certain animals, the oligoclonal nature of the cross-reactive T cell population appears to be directly visible from double-tetramer analyses of the influenza-reactive CD8 + T cell population. In these mice, the expanded cross-reactive T cell population appears as two to three separate populations , which may reflect slightly distinct affinities for the primary and secondary antigen. Several reports have indicated that the requirements for activating memory T cells differ from those for activating naive T cells, making them more susceptible to low-affinity TCR triggering or cytokine-mediated stimulation 35 36 37 38 39 . To address the possible contribution of aspecific or more broadly reactive T cells in the formation of the cross-reactive T cell population, influenza A/NT/60/68-primed mice were infected with the antigenically unrelated influenza B virus (B/Lee/40). Both at day 4 (data not shown) and day 8 after infection , no expansion of the influenza A/NT/60/68-specific memory T cell population is observed. This is in agreement with results by others showing that bystander activation during an acute viral infection is minimal 20 40 41 . Furthermore, the influenza A virus–specific T cells in mice that underwent sequential infections with A/NT/60/68 and A/PR/8/34 do not bind peptide–MHC tetramers that contain an adenovirus E1A-derived CTL epitope . Thus, the cross-reactive T cell population that expands upon infection of primed mice does require a significant structural homology between two antigens that are encountered sequentially, and is specific for these two peptide–MHC complexes. Finally, selective outgrowth of cross-reactive T cells does not take place to an appreciable extent when the two related antigens are introduced simultaneously . This suggests that the selective advantage of cross-reactive cells relies on quantitative or qualitative traits of the T memory population, such as homing properties or the requirements for costimulatory signals. To provide a first estimate of the extent of structural homology between two antigens that is required for the selective expansion of cross-reactive T cells, we searched the National Center for Biotechnology Information (NCBI) GenBank database for other natural variants of the H-2D b –restricted influenza NP epitope. Thus far, 10 variants of this epitope have been identified, 7 of which contain mutations that affect TCR-exposed side chains (positions 6, 7, and 8) (reference 22, and Table ). Because influenza A virus is not a common mouse pathogen, this set of mutants should be random with respect to T cell recognition. Synthetic peptides corresponding to these mutants were generated, and mice that had previously been exposed to influenza A/NT/60/68 or A/HKx31 were challenged by subcutaneous immunization. To circumvent the need of helper T cells for the induction of an effective CTL response, mice were treated with anti-CD40 mAb (FGK.45) after vaccination 21 42 43 44 . These experiments establish that in most if not all cases where influenza A virus–primed mice are confronted with antigens that contain a single mutation within the T cell epitope, an influenza-specific T cell population emerges that is fully cross-reactive between the primary and secondary antigen ( Table ). This phenomenon is not only observed for conservative substitutions, but also for more drastic amino acid changes (e.g., Ala8→Asn). For variants that contain multiple alterations in TCR-exposed residues, cross-reactivity is observed for some sequences (e.g., ASNENM DA M→ASNENM ET M), but not for others (e.g., ASNEN MD AM→ASNEN VE AM). Both the type of mutation and the contribution of the mutated residues to the TCR-exposed surface of the peptide are likely to be determining factors in this regard. For one of the mutant epitopes (ASNEN V ETM), the functional behavior of the cross-reactive T cells was also tested. In line with the biochemical data, intracellular IFN-γ staining of spleen cells that were stimulated with the primary antigen (ASNEN M ETM) or the peptide variant used for vaccination revealed an identical percentage of responding cells (data not shown). Cross-protection or the lack thereof by an epitope-specific T cell population cannot readily be tested through the use of mutant or recombinant viral strains. Even in settings where B cell immunity is absent, such as in μ-MT mice, it is difficult to exclude a possible contribution of T cell responses against subdominant epitopes that are conserved between the primary and the recall strain. To circumvent these difficulties, we developed a model system in which only the T cell epitope under study is shared between the primary and secondary antigen encounter. The NP 366–374 CTL epitope of influenza virus strain A/NT/60/68 was introduced into the EL4 tumor cell line as a COOH-terminal fusion with the eGFP gene product. Although the parental cell line grows progressively when injected subcutaneously in C57BL/10 mice, the NP epitope–expressing tumor is rejected over a 2–3-wk time period. Furthermore, tumor rejection is paralleled by the appearance of an NP 366–374 –specific T cell population and is markedly enhanced by simultaneous infection with influenza virus A/NT/60/68 (Wolkers, M.C., manuscript in preparation). Thus, the introduced NP epitope appears to function as a bona fide (neo)tumor antigen in this setting, as recognition of this antigen is correlated with tumor rejection. To examine the in vivo effects of cross-reactive T cell populations, mice that had previously been exposed to influenza virus A/NT/60/68 (carrying the homologous NP epitope), A/HKx31 (reassortant of A/PR/8/34; carrying a variant NP epitope), or an unrelated influenza B virus were challenged with EL4-NP 366–374 tumor cells. After tumor cell injection, blood samples were taken from individual mice and the frequency of virus strain-specific and cross-reactive CD8 + T cells was measured. In mice that had been infected with the unrelated influenza B virus , tumor growth is comparable to that in uninfected mice (data not shown). As expected, mice that were previously exposed to influenza virus A/NT/60/68, which shares the CTL epitope with the EL4-NP 366–374 tumor, showed a strong reduction in tumor growth . This protection is accompanied by a massive and rapid increase in the number of A/NT/60/68-monospecific T cells, which were apparently reactivated from the memory T cell pool by the NP epitope–expressing tumor . Importantly, intermediate tumor growth was seen in mice that had previously been infected with variant virus A/HKx31 . In addition, the reduced tumor outgrowth in these mice is accompanied by the expansion of a large population of CD8 + T cells, which cross-react between the NP 366–374 –expressing EL4 tumor and the viral strain used for priming . These results illustrate that the presence of a structurally related T cell antigen promotes the expansion of infrequent cross-reactive T cells even in the absence of any further noticeable homology between primary and secondary challenge, and this expansion is accompanied by a significant reduction in tumor outgrowth. The ability of cytotoxic T cell populations to cross-react between different viral strains was first appreciated by Townsend and Skehel in 1984 18 . They showed that repeated in vitro restimulation protocols of splenic lymphocytes derived from influenza A virus–primed mice could lead to the selection of NP-specific T cell lines that cross-reacted between different influenza A viral strains. At that time, it was unknown whether these T cell lines cross-reacted at the level of the (variable) immunodominant NP 366–374 CTL epitope, or whether shared subdominant epitopes were recognized. However, with the benefit of hindsight, these experiments can be said to have revealed for the first time the capacity of cytotoxic T cells to productively recognize viral variants. In spite of the early recognition of T cell cross-reactivity in in vitro assays, the biological in vivo significance of this cross-reactivity for the immune system to cope with viral variants has since remained unclear. Our findings now document that prior antigen exposure dramatically affects the repertoire of T cells used in a subsequent response to antigenic variants in vivo. The propensity for cross-reactivity between two T cell antigens appears roughly proportional to the sequence similarity between the epitopes tested, and seems to be a common event for antigens that are closely related. These cells that expand in vivo are phenotypically indistinguishable from conventional effector T cell populations (CD44 high , CD62L low ; data not shown) and are functional ex vivo and in vivo. This process appears to be due to the selective expansion of cross-reactive T cells present in the memory T cell pool, and is not dependent on shared B or T helper epitopes between primary and recall antigen. In recent years, several groups have studied the impact of epitope variants on antigen-specific T cell responses (for reviews, see references 6 , 45 , and 46 ). These variant epitopes were generally isolated as immune escape variants, or were identified in in vitro assays by their aberrant recognition by T cell clones. These selected antigen variants function as either partial agonists or antagonists of antigen-specific T cell responses not only in vitro but also in vivo. We sought to examine whether this type of T cell antagonism or partial agonism is a common phenomenon when a polyclonal T cell repertoire is confronted with antigenic variation. To this purpose, we studied the development of antigen-specific T cell repertoires after encounter of random natural variants of influenza A viruses. We conclude that during such encounters, the T cell repertoire generally reacts with the outgrowth of a T cell population for which the variant epitope is a full agonist. The TCRs encoded by these T cells bind with equal affinity to MHC molecules complexed with wild-type and variant epitopes. Furthermore, the functional capacity of this T cell population towards target cells expressing the original or the variant antigen is indistinguishable both in vitro and in vivo. How do these data fit in with previous observations that mutations in T cell epitopes can lead to CTL escape by the virus? During chronic HIV and HBV infections 10 11 , and also in a murine lymphocytic choriomeningitis virus (LCMV) model 12 , the mechanism that we have identified apparently does not operate efficiently. In theory, this could be due to the type of amino acid changes in the T cell epitopes involved. However, examination of the type of mutations in the T cell epitopes in those cases and the mutations studied here does not point towards obvious differences (not shown). Alternatively, the impaired ability to react to emerging antigenic variants in those settings may be due to alterations at the T cell level. Specifically, repetitive antigen-specific T cell stimulation results in a narrowing of the reactive T cell repertoire 33 . Indeed, the antigen-specific T cell expansions observed during chronic HIV infection are oligoclonal 47 . It may be hypothesized that for such restricted antigen-specific T cell populations, a single antigenic variant could antagonize a substantial part of the antigen-specific T cell response, and it will be a challenge to test this notion in a direct manner. Why do cross-reactive T cells dominate the response against antigenic variants over the largely strain-specific response observed normally during a primary T cell response? The cross-reactive T cells that we have identified appear to originate from the preexisting memory T cell pool, and naive and memory T cells differ both in quantitative and qualitative terms. Specifically, even though cross-reactive cells are infrequent in the original memory pool, these cells may still outnumber the strain-specific T cells that—due to their specificity—failed to get activated in the primary response. In addition, the activation requirements of memory T cells are less stringent than those of naive T cells 38 48 49 . This is reflected in a lessened requirement both for costimulatory signals and for prolonged antigenic stimulation. Finally, the ability of memory T cells to enter peripheral tissues may promote the early encounter of viral antigens 50 . Indirect evidence for functional T cell cross-reactivity during subsequent viral infections has been obtained previously. Experiments by Welsh and colleagues 51 have suggested that a measure of heterologous immunity may occur even for apparently unrelated viruses. Although in that case the antigenic determinants involved were not identified and cross-reactive cells were of low avidity, these results do suggest that the selective expansion of cross-reactive memory T cells could be a more general phenomenon. We conclude that a polyclonal T cell repertoire responds to the encounter of random variants by the expansion of memory T cells that are best fit for the recognition of the variant antigen. These findings suggest that random mutations in T cell epitopes are unlikely to result in CTL escape, and that a polyclonal T cell memory population can provide protection not only against the index sequence, but also against a swarm of related sequences. | Other | biomedical | en | 0.999997 |
10544204 | Antibodies used for these studies included the following: FITC-conjugated Ly9.1, anti–Thy 1.2, anti-CD8, anti-Fas, anti-IgM, anti-CD4, anti-CD5, and anti-IgD; PE-conjugated anti-CD4, anti-CD3∈, anti-CD44, anti-CD43, anti-CD23, and anti–IL-2 and anti-B220; biotin-conjugated anti–TCR-α/β, anti-IgD, anti-CD19, and anti-CD25; allophycocyanin-conjugated anti–TCR-α/β, anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-CD40, and anti-IgM antibodies and Cy5-conjugated streptavidin, all from PharMingen; and anti–extracellular signal regulatory kinase (ERK)1 and anti-ERK2 antibodies from Santa Cruz, anti–TCR-ζ and anti-ZAP70 antibodies supplied by Drs. Larry Samelson (National Institutes of Health, Bethesda, MD) and André Veillette (McGill Cancer Centre, Montreal, Quebec, Canada), respectively; and antiphospho-SAPK/JNK and anti-SAPK/JNK antibodies (reactive against all SAPK/JNK isoforms) from New England Biolabs. Texas red–conjugated streptavidin was purchased from GIBCO BRL. Monoclonal hamster anti–mouse CD3∈ was produced by the 145-2C11 hybridoma (provided by Dr. R. Miller, Ontario Cancer Institute) and purified from the supernatant by protein G chromatography; polyclonal rabbit anti-WASp antibody was derived by immunization with a polylysine-conjugated peptide corresponding to a putative nuclear localization signal within the WAS cDNA; anti–murine β-actin mAb, PMA, ionomycin, and murine recombinant IL-2 were purchased from Sigma Chemical Co.; rabbit anti–hamster and anti–mouse IgG antibodies and FITC-conjugated streptavidin were purchased from Jackson ImmunoResearch Laboratories. Murine recombinant IL-4 was obtained from PharMingen, [ 3 H]thymidine from Dupont/NEN, 7-amino-actinomycin D (7-AAD) from Calbiochem, Indo-1 from Molecular Probes, myelin basic protein (MBP) from Upstate Biotechnology, Inc., and an IL-2 ELISA kit from Genzyme. Brefeldin A was a gift from Dr. R. Miller. A WAS gene targeting vector was derived by subcloning a 3.3-kb segment of the WAS gene encompassing ∼2 kb of the 5′ flanking sequence upstream of the initiation codon through exon 4 of the gene into the Ssc8387I site of the pPNT expression cassette 46 and a 3.4-kb fragment encompassing the XbaI site in exon 11 through to a BamHI site within intron 11 into the pPNT XbaI-BamHI sites. Embryonic stem (ES) cells from the male-derived R1 ES cell line (129/Sv) were electroporated with this targeting vector and selected with neomycin and gancyclovir 47 . Surviving clones were screened for homologous recombination at the WAS locus by PCR using the following primer pair: 5′-GTGAAGGATAACCCTCAGAAGTCC-3′ (forward primer, S1, derived from sequences within exon 2 of the WAS gene) and 5′-CGGAGCAGAATCTAGATGGCAGAGT-3′ (reverse primer, S2, representing sequences in the 3′ region downstream from exon 12 of the WAS gene) . Targeted clones were verified by Southern blotting with a probe derived from a 450-bp segment external to the 5′ region of homology . Two independently derived WAS − / − clones were then either aggregated with CD1 eight cell stage embryos or microinjected into 3.5-d recombinase activating gene 2–deficient ( RAG-2 − / − ) blastocysts 48 , and the aggregates or blastocysts were then implanted into pseudopregnant foster mothers. WAS genotypes of the chimeric progeny were confirmed using a PCR assay including the forward primer (G1; 5′-ACTGAAGGCTCTTTACTATTGCT-3′), derived from a sequence within the neomycin resistance gene, and a reverse primer (G2; 5′-ACTGAAGCCTCTTTACTATTGCT-3′), corresponding to a sequence within exon 11 of the WAS gene. Chimeric male mice carrying the mutation in the germline were bred to the C57BL/6 background by backcrossing over six generations. Cells were resuspended in immunofluorescence staining buffer (PBS containing 1% BSA and 0.05% NaN 3 ) and incubated with the appropriate fluorochrome-conjugated antibodies for 30 min at 4°C. For three- and four-color staining, Texas red– or Cy5-conjugated streptavidin was used after staining with biotin-conjugated antibodies. Stained cells were analyzed using a FACScan™ with CELLQuest™ software (Becton Dickinson). For detection of expression of the early activation marker CD69, thymocytes (2 × 10 6 cells/ml) were cultured with medium alone, with plate-bound anti-CD3 antibody (10 μg/ml), or with plate-bound anti-CD3 plus anti-CD28 antibodies (10 and 0.2 μg/ml, respectively) for 24 h. Cells were stained with FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and biotinylated anti-CD69 antibodies followed by Cy5-conjugated streptavidin. Expression of CD69 was measured on gated CD4 + CD8 + thymocytes. For analysis of cell proliferation, single cell suspensions were prepared from thymi, lymph nodes, and spleen of age-matched WAS − / − and WAS +/+ (C57BL/6) mice bred at the Samuel Lunenfeld Research Institute, and the cells were then subjected to erythrocyte lysis in ammonium chloride buffer. Thymocytes and lymph node cells were cultured in 96-well flat-bottomed microtiter plates (2 × 10 6 cells/ml) for 48 h in culture medium alone or in the presence of either plate-bound (0–25 μg/ml) or soluble (2 μg/ml) anti-CD3∈ antibody with or without anti-CD28 antibody (0.2 μg/ml) or with soluble anti-CD3 antibody plus PMA (1 μg/ml and 5 ng/ml, respectively), Con A (1 μg/ml) or PMA plus ionomycin (5 μg/ml and 250 ng/ml, respectively), or soluble anti-CD3 antibody plus IL-2 (1 μg/ml and 50 U/ml, respectively). Splenocytes (2 × 10 6 cells/ml) were cultured for 48 h with medium alone, LPS (5 μg/ml), IL-4 (2 ng/ml), or IL-4 plus anti-IgM antibody F(ab′) 2 fragment (1.25 and 5 μg/ml), or anti-CD40 antibody (2 μg/ml). Cultured cells were pulsed with [ 3 H]thymidine (1 μCi/well) for 16 h before terminating the incubation. Incorporated radioactivity was measured using an automated β scintillation counter. Alternatively, for biochemical assays, 4 × 10 7 thymocytes or lymph node T cells were resuspended in 100 μl 2% FCS–containing RPMI and incubated for 30 min at 4°C in the presence or absence of biotin-conjugated anti-CD3 antibody (25 μg/ml). After removal of unbound antibody, the cells were resuspended at a concentration of 2 × 10 8 cells/ml, warmed to 37°C for 1 min, and incubated for varying periods with 10 μg/ml streptavidin. To evaluate IL-2 secretion, supernatants were collected from unstimulated and TCR-stimulated cells and the amount of IL-2 was quantified by ELISA (Genzyme). Numbers of IL-2–producing cells were assayed by incubating stimulated and unstimulated lymph node T cells (2 × 10 6 ) in 24-well plates precoated with anti-CD3 antibody (5 μg/ml) with or without addition of soluble anti-CD28 antibody (4 μg/ml) and in the presence or absence of Brefeldin A (5 μM) at 37°C for 8 h. Cells were harvested, washed with immunofluorescence buffer, and labeled with an FITC-conjugated anti-CD4 antibody. Cells were then washed again and incubated for 15 min in 150 μl of 4% paraformaldehyde at room temperature. Cells were then washed, permeabilized, and labeled by 2-h incubation with a PE-conjugated anti–IL-2 antibody in 100 μl of 0.1% saponin–containing immunofluorescence buffer. Cells were washed twice with permeabilization buffer, resuspended in immunofluorescence buffer, and analyzed by flow cytometry. Splenocytes and thymocytes (2 × 10 6 cells) from WAS +/+ and WAS − / − mice were lysed in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 1 μg/ml each of leupeptin, pepstatin, and aprotinin as protease inhibitors. After 15 min incubation in cold lysis buffer, unlysed cells were removed by centrifugation and the lysates were then boiled in the presence of 6× reducing sample buffer, electrophoresed through 10% SDS-polyacrylamide, and transferred to nitrocellulose (Schleicher & Schuell). The filters were incubated at 4°C for at least 1 h in TBST solution (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.05% Tween 20) plus 3% gelatin. Filters were then incubated for 2 h at room temperature with anti-WASp antibody (1 μg/ml) in TBST, followed by the addition of goat anti–mouse antiserum labeled with peroxidase (Amersham Pharmacia Biotech) and horseradish peroxidase conjugate (Bio-Rad Laboratories). The blot was stripped and reprobed with an anti–β-actin antibody in order to assess loading. For immunoprecipitation, lysates prepared from unstimulated or TCR-stimulated thymocytes or lymph node T cells (4 × 10 7 ) were precleared by incubating an equal amount (100–250 μg) lysate protein with protein A–Sepharose (Amersham Pharmacia Biotech) for 1 h at 4°C and for an additional 1 h with 40 μl rabbit preimmune sera. Lysates were then incubated for 3 h at 4°C with specific antibody or rabbit preimmune serum and 25 μl packed protein A–Sepharose beads, and the immune complexes were then collected by centrifugation, washed four times in lysis buffer, and resuspended in SDS sample buffer for immunoblotting analyses. Alternatively, ERK1/ERK2 immune complexes immunoprecipitated using a mixture of anti-ERK1 and anti-ERK2 antibodies were washed in MAPK buffer (5 mM Hepes, pH 7.4, 10 mM MgCl 2 , 100 mM NaVO 4 ) and resuspended in 50 μl reaction buffer (30 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 , 2 mM MnCl 2 containing 5 μg MBP [Upstate Biotechnology], 1 mM cold ATP, and 10 μCi [γ- 32 P]ATP [Dupont/NEN]). After 15 min incubation at 30°C, reactions were terminated by addition of 12 μl 6× SDS-PAGE loading buffer, and the samples were boiled, electrophoresed through 12% polyacrylamide gels, and transferred to nitrocellulose. The phosphorylated MBP bands were visualized by autoradiography, and ERK1 and ERK2 expression levels were determined by anti-ERK2 immunoblotting analysis. Thymocytes and lymph node T cells (5 × 10 6 cells/ml) were labeled with Indo-1 (5 μM) and incubated at 37°C in the dark for 30 min. Cells were washed, resuspended in RPMI containing 2% FBS and 10 mM Hepes, pH 7.4, and incubated with biotinylated anti-TCR antibody (5 μg/ml) for 15 min at 4°C. After washing, the cells were resuspended in RPMI buffer at a concentration of 10 7 cells/ml and stimulated with 5 μg/ml of streptavidin. Calcium levels were detected by flow cytometric analysis of Indo-1 violet-blue fluorescence ratio 49 . Freshly isolated thymocytes were plated at a density of 2 × 10 6 cells/ml in 96-well tissue culture plates precoated with either anti-CD3 antibody (20 μg/well), anti-CD3 plus anti-CD28 (each 20 μg/well), anti-Fas antibody (0.1, 1, or 5 μg/ml), or PMA plus ionomycin (10 and 500 ng/ml, respectively). After 24 h, the cells were harvested, washed with PBS, and stained with FITC-conjugated anti-CD4, PE-conjugated anti-CD8, and 7-AAD (10 μg/ml) for 30 min on ice 50 . After three washes, the cells were analyzed using the Becton Dickinson FACScan™ and CELLQuest™ software. 10 6 cells were incubated with 1 μg/ml of biotinylated anti-TCR (thymus) or 1 μg/ml of biotinylated anti–mouse IgD (spleen), for 30 min on ice. After washing, cells were labeled for 30 min on ice with 1 μg/ml streptavidin-FITC. Cells were washed and resuspended in 100 μl of RPMI and incubated at 37°C for 5 min. The cells were pelleted in a microcentrifuge for 15 s and then fixed for 15 min in 2% paraformaldehyde. After washing twice in 0.5% BSA–containing PBS, the cells were cytospun onto slides and mounted with SlowFade Light (Molecular Probes). The cells were visualized with a confocal fluorescence microscope (MRC-600; Bio-Rad Laboratories). The average percentage of capped cells was obtained from 10 microscope fields per sample (100–200 cells/field). Internalization of the TCR was assayed by incubating 10 6 lymph node T cells with anti-CD3 antibody (1 μg/ml) for 30 min on ice. After washing, cells were incubated with biotinylated goat anti–hamster antibody (2 μg/ml) for 30 min at 4°C and then warmed at 37°C. Aliquots were removed at varying time points thereafter, transferred to ice, and the reaction was stopped by addition of 0.1% NaN 3 . Cells were then stained with FITC-conjugated streptavidin, fixed for 15 min in 4% paraformaldehyde, and washed before analysis by flow cytometry. Thymocytes were incubated with anti-CD3∈ (1 μg/ml) for 30 min on ice. After washing, the primary antibodies were cross-linked using goat anti–hamster Ig (1 μg/ml) for 5 min. The control cells were incubated only with the cross-linking antibody. Activation was terminated by addition of 4% paraformaldehyde. After fixation, cells were incubated with FITC-conjugated phalloidin (Sigma Chemical Co.) for 30 min, washed three times in PBS, and analyzed using a FACSCalibur™. Long bones (humerus, femur, tibia) were removed from selected mice, and the ends were then clipped and flushed using a 27-gauge needle and ice-cold HBSS. Clumps of marrow were then broken up by repeated pipetting, and the cells were spun at 500 g at 4°C for 5 min, resuspended in HBSS in a 15-ml polypropylene tube, and underlaid with a discontinuous Percoll gradient (52, 65, and 75% Percoll solution). Cells were then centrifuged at 1,500 g for 30 min at 4°C, and the neutrophil-enriched fraction (at the interface of the 65 and 75% gradients) was harvested, diluted with an equal volume of HBSS, and spun down in a microfuge for 10 s. These cells were resuspended in 1 ml of RPMI and counted using a Coulter counter. To evaluate phagocytosis, cells were mixed with opsonized zymosan (10 particles/cell) and lucifer yellow at 0.5 mg/ml, pelleted in a microfuge tube, and then incubated for 5 min at 37°C. Cells were then washed three times in ice-cold PBS, and the number of cells with lucifer yellow–containing phagosomes was counted using a fluorescence microscope. To derive ES cells carrying a disruption of the WAS gene, a targeting construct was developed in which a 3.5-kb segment encompassing intron 4 to exon 11 of the WAS gene was substituted with the pPNT expression vector. This vector was then introduced into R1 ES cells, and two clones carrying a WAS mutant allele were then aggregated with CD1 eight cell embryos. Once germline transmission of the targeted allele was confirmed by PCR and Southern analysis , the WAS mutation was bred into the C57BL/6 background. The two WAS − / − ES cell clones were also injected into RAG − / − blastocysts so as to derive WAS − / − RAG − / − chimeric mice which were used in addition to the WAS − / − mice for some studies of lymphocyte function. As illustrated by immunoblotting analysis, male mice carrying the WAS mutant allele showed no WASp expression in either thymic or splenic cells despite the presence of surface (s)Ig + B cells and mature CD4 + CD8 − and CD4 − CD8 + T cells . Although lymphoid cells were present in the WAS − / − mice, an analysis of cell numbers in these animals revealed lymphocyte cellularity to be reduced in all lymphoid organs examined. A reduction in lymphoid cell number was most evident in the thymus, where the number of thymocytes was about one quarter of that observed in wild-type mice ( Table ). Reduced thymic cellularity appeared to reflect a decrease in the total and relative numbers of CD4 + CD8 + double-positive thymocytes, an abnormality which, in turn, was associated with relative increases in the numbers of double-negative CD4 − CD8 − and single-positive CD4 + CD8 − and CD4 − CD8 + cells . Although this defect was not detected in a previous analysis of WASp-deficient mice 45 , the current findings are consistent with several lines of evidence revealing lymphocyte maturation to be altered in WAS patients 17 18 22 . To further examine this maturation defect, the CD4 − CD8 − double-negative precursors, identified by virtue of negative staining for CD4, CD8, B220, TCR-α/β, and TCR-γ/δ, were evaluated for expression of the maturation markers CD25 and CD44. These latter markers have been previously shown to identify four double-negative thymocyte subsets, CD44 + CD25 − , CD44 + CD25 + , CD44 − CD25 + , and CD44 − CD25 − , which demarcate progressive stages of thymocyte maturation 51 . As shown in Fig. 1 D (lower panel), among these four double-negative subsets, the CD44 − CD25 + population was relatively increased and the CD44 − CD25 − population relatively decreased in the WAS − / − compared with wild-type mice. Thus the reduction in total thymocyte numbers observed in the absence of WASp appears to reflect impaired progression of the CD44 − CD25 + to the CD44 − CD25 − stage and by extension, reduced transit from the double-negative to the double-positive stage. Interestingly, this same perturbance in thymocyte maturation has also been detected in animals deficient for the CD45 52 , Lck 53 , and Vav 54 55 signaling effectors, an observation which strongly suggests that this defect reflects diminished signaling, most likely via the pre-TCR 56 . WAS − / − thymocytes were also examined for expression of TCR-α/β, CD3, and the maturation marker CD5, all of which were found to be expressed normally on the mutant thymocytes (data not shown). Similarly, analysis of splenic and bone marrow B cell populations revealed expression of CD43, CD19, CD23, CD86, CD25, B220, sIgM, and sIgD to be comparable in WAS − / − and wild-type mice (data not shown). However, for thymocytes, total numbers of peripheral CD4 + CD8 − and CD4 − CD8 + T cells and peripheral B220 + sIgM + B cells were reduced by ∼50% in the WAS − / − chimeric compared with wild-type animals ( Table ). Together, these data indicate lymphopoiesis to be impaired, albeit not arrested, in the context of WASp deficiency. WASp effects on lymphoid ontogeny appear to be predominantly realized in the thymic compartment at the double-negative stage of thymocyte differentiation, and it may be for this reason, as well as sensitivity of this defect to strain background 54 , that impaired thymic ontogeny was not detected in an independently derived WAS − / − mouse 45 . Importantly, however, in the latter mice, as well as in some WAS patients 6 , significant reductions of peripheral T lymphocyte number have been observed. Thus, the available evidence strongly suggests a role for WASp in driving the maturation and/or expansion of T and possibly mature B cell populations. Whether this latter role is subserved by WASp modulation of the proliferation/expansion and/or survival of lymphoid precursors remains to be determined. Alternatively, the reduction in lymphocyte number observed in the WAS − / − mice might reflect perturbation in the transit of immature precursors from the bone marrow to other lymphoid organs. In addition to lymphocytes, platelets were also found to be reduced in the periphery of WAS − / − mice. As shown in Table and consistent with the detection of thrombocytopenia in virtually all WAS patients, platelet number was reduced by ∼40% in the WAS − / − relative to wild-type mice. At present, the basis for this finding remains unclear, with preliminary data revealing that platelet survival is normal in these animals (data not shown). The physiologic significance of this defect is also unclear, as the WAS − / − mice, in contrast to classical WAS patients, show no overt signs of a hemorrhagic diathesis. In view of previous data suggesting that antigen receptor–evoked lymphocyte activation is impaired in WAS patients 9 10 12 , thymocytes and peripheral B and T lymphocytes from the WAS − / − chimeras were next studied with respect to their responses to antigen receptor ligation. The results of these analyses revealed that anti-CD3 antibody–evoked proliferation was markedly reduced in WAS − / − relative to WAS +/+ thymocytes at all anti-CD3 antibody concentrations tested (data not shown). Similarly, Con A stimulation elicited significantly less proliferation in WAS − / − thymocytes than wild-type cells. By contrast, the mutant cells proliferated normally in response to PMA and ionomycin, a maneuver which triggers proliferation in the absence of antigen receptor engagement. In addition, anti-CD3 as well as anti-CD3 plus anti-CD28 antibody–induced upregulation of the CD69 early activation marker 57 58 were markedly diminished in the mutant cells . Similar to WAS − / − thymocytes, WASp-deficient peripheral T cells were also observed to have reduced proliferative responses to anti-CD3 antibody, but a normal response to PMA and ionomycin . However, WAS − / − peripheral T cells also responded, albeit less than wild-type cells, to anti-CD3 antibody stimulation combined with PMA treatment, whereas addition of CD28 could partially restore proliferation of these cells . Anti-CD3–induced proliferative responses of the WASp-deficient cells were also restored to almost normal levels by the addition of IL-2 . To investigate the basis for this latter finding, anti-CD3–induced production of IL-2 was also evaluated in the WAS − / − T cells. As shown in Fig. 2 E, wild-type T cells secreted only a small amount of IL-2 after anti-CD3 antibody treatment, but dramatically increased their IL-2 production in conjunction with anti-CD28 antibody–mediated costimulation. However, in the WAS − / − cells, IL-2 production was not only negligible after anti-CD3 antibody stimulation, but also increased by much less (50%) in these cells than in wild-type cells after CD28 costimulation . This defect in the induction of IL-2 does not reflect impairment of IL-2 secretion, as an evaluation of IL-2 production based on intracellular staining of T cells stimulated with anti-CD3/anti-CD28 antibodies in the presence of the secretion blocker Brefeldin A 59 60 revealed the numbers of IL-2–staining cells to be ∼50% less in the WAS − / − relative to wild-type cell cultures . By contrast, in the absence of Brefeldin A, IL-2 accumulation was not detected in the mutant or wild-type cells, indicating the IL-2 secretion pathway to be intact in the context of WASp deficiency. Thus, these observations indicate that WASp effects on TCR signaling are modulated by costimulatory signals and also suggest that the T cell proliferative defect conferred by WASp deficiency is due, at least in part, to a defect in IL-2 production. Taken together, these data confirm the participation of WASp in the transduction of activation signals via the TCR and suggest that the effects of WASp on TCR-mediated activation are most profound in immature T cells and may be tempered by concomitant activation signals delivered via CD28, IL-2, and possibly other stimulatory receptors. The data also identify potential functional differences between the T cells of the WASp-deficient mice studied here and those of WAS patients in whom anti-CD3–induced proliferative responses appear to be only partially restored by exogenous IL-2 10 . While further studies are required to determine whether this discrepancy reflects the use of transformed WAS patient cell lines, the current data provide definitive evidence of a role for WASp in promoting the coupling of TCR stimulation to T cell activation and to the IL-2 production integral to normal T cell mitogenesis. In contrast to thymocytes and peripheral T cells, WASp-deficient splenic B cells showed only a marginal reduction in response to antigen receptor ligation, a finding that was not altered by varying the concentrations of anti-Ig antibody . As observed in WAS − / − T cells, this mild reduction in responsiveness of the WASp-deficient B cells to mitogenic stimulus appears to be antigen receptor specific, as these cells proliferated normally in response to LPS, IL-4 , and the combination of IL-4 with anti-CD40 antibody (data not shown). Thus, WASp deficiency appears to be associated with impairment in T cell and to a much lesser extent, B cell antigen receptor (BCR) signaling. While a defect in BCR signaling was not reported in a previous study of WASp-deficient mice 45 , the current findings are consistent with data indicating that antigen receptor–evoked B cell proliferative responses are impaired in patients with classical WAS 9 . However, it is currently unclear whether the divergent effects of WASp deficiency on TCR versus BCR signaling reflect differences in the biochemical requirements required for signal delivery by the respective receptors or the capacity of another effector, such as N-WASp, to adequately subserve WASp functions in B, but not T cells. In view of these data identifying a role for WASp in coupling antigen receptor engagement to lymphocyte proliferation, the possibility that this protein also modulates antigen receptor capacity to activate cell death cascades was also investigated. Specifically, WAS − / − and wild-type double-positive thymocytes were cultured for 24 h in the presence of anti-CD3 antibody alone or combined with anti-CD28 antibody, and the viability and rate of cellular apoptosis were then evaluated in CD4 + CD8 + cells by immunofluorescence analysis of 7-AAD staining. As shown in Fig. 2 F, results of this analysis revealed that viability of the anti-CD3–stimulated WAS − / − CD4 + CD8 + cells was ∼35% greater than that of wild-type cells. This difference was even more marked in the context of anti-CD3/anti-CD28 stimulation. By contrast, cell viability after anti-Fas antibody or PMA/ionomycin treatment was similar in the mutant and wild-type cells. Therefore, these data indicate that WASp specifically modulates the apoptotic as well as mitogenic signaling cascades evoked by TCR engagement. At present, the physiologic significance of these findings remains unclear, although the involvement of WASp in TCR-mediated apoptosis implies a potential role for this protein in facilitating negative selection events in the thymus, a possibility which might be etiologically relevant to the autoimmune phenomena frequently expressed by WAS patients 6 . To elucidate the biochemical basis for the T cell functional defects conferred by WASp deficiency, WAS − / − T cells were evaluated with respect to the signaling events elicited by TCR engagement. As indicated by the antiphosphotyrosine immunoblots shown in Fig. 3a and Fig. b , the levels of TCR-ζ, ZAP70, and total cellular protein tyrosine phosphorylation detected in resting and TCR-stimulated WAS − / − thymocytes and peripheral T cells were essentially identical to those observed in similarly treated wild-type cells. Along similar lines, TCR-evoked increases in MAPK activation and in phosphorylation, and presumably activation of SAPK/JNK were comparable . By contrast, the increase in intracellular calcium levels induced by TCR ligation was less sustained in the WAS − / − compared with wild-type cells, with intracellular calcium concentrations 20 and 25% reduced in the WAS − / − relative to wild-type cells at the 600- and 700-s time points, respectively . At present, it is unclear whether this quantitative difference in intracellular calcium mobilization observed in wild-type compared with WAS − / − cells translates to a sufficiently significant perturbance of other downstream signaling events, such as NF-AT translocation to the nucleus, so as to account for the very significant impairment in TCR-induced IL-2 expression by the mutant cells. As WASp has also been implicated by many lines of evidence in the regulation of cytoskeletal architecture, the relevance of WASp to lymphocyte cytoskeletal rearrangements induced by antigen receptor engagement was also investigated. To this end, WAS − / − lymphocytes were studied with respect to the induction of antigen receptor capping, a phenomenon that requires de novo actin polymerization and microfilament rearrangement. As indicated in Fig. 4 A and illustrated by the representative micrograph , a defect in ligand-induced capping was detected in WAS − / − thymocytes, the number of these cells showing T cell antigen receptor clustering to be ∼40% less than that of wild-type thymocytes. As is consistent with a defect, albeit mild in BCR-induced proliferation, anti-Ig–mediated capping of the BCR was also impaired in the context of WASp deficiency. In addition, immunofluorescence analysis of anti-CD3–treated phalloidin-labeled WAS − / − thymocytes, an assay that selectively detects expression of polymerized F-actin, revealed that antigen receptor–mediated actin polymerization was markedly reduced in WAS − / − thymocytes and lymph node T cells (data not shown). Together, these data demonstrate a requirement for WASp in the regulation of cytoskeletal rearrangement in response to antigen receptor engagement. In addition to patch and cap formation, internalization of antigen receptors after their engagement appears to require cytoskeletal rearrangement, as endocytosis of these receptors is abrogated by cytoskeleton-disrupting agents such as dihydrocytochalasin B 61 . In view of this observation, together with data from our lab (Siminovitch, K., unpublished observations) and another 62 revealing the capacity of WASp and the Las17/Beel yeast WASp homologue, respectively, to interact with proteins implicated in endocytosis, the possibility that WASp plays a role in ligand-triggered TCR endocytosis was investigated by assaying the levels of biotinylated TCR present on WAS − / − T cells at varying times after cell stimulation. As illustrated in Fig. 4 C, the results of this analysis revealed the rate of TCR endocytosis to be markedly reduced in the context of WASp deficiency, with 50% of the receptors internalized in wild-type cells, but only 10% internalized in WAS − / − cells at 1 h after stimulation. Therefore, these results suggest the involvement of WASp in the process of receptor endocytosis. At present, the level at which the endocytic pathway is disrupted by WASp deficiency remains unclear. However, this defect may also impact on the properties of nonlymphoid cells, as opsonized zymosan particle internalization was also found to be significantly reduced in WAS − / − compared with wild-type neutrophils . Together, these data raise the possibility that the altered endocytosis of the TCR and/or other receptors and consequent changes in levels of surface receptor expression impact on a broad range of immune cellular functions, including not only cell activation, but also maintenance of cell polarity, antigen presentation, and/or the uptake of extracellular nutrients and microorganisms. In summary, the current data reveal that transduction of TCR-evoked proliferation, apoptosis, and actin polymerization–inducing signals are impaired in WAS − / − T cells. WASp deficiency also engenders a defect in thymocyte maturation, by impairment of the progression of CD4 − CD8 − progenitors from the CD44 − CD25 + to a CD44 − CD25 − stage. This developmental defect may reflect diminished signaling capacity of the pre-TCR in the absence of WASp, as has been previously proposed in relation to the similar, albeit more severe, maturational defect detected in Lck-deficient thymocytes 53 . Similarly, the reduction in peripheral T cell numbers conferred by WASp deficiency may reflect not only impaired thymopoiesis, but also diminished proliferative/expansion potential consequent to defective transduction of TCR mitogenic signals. As is consistent with the immunologic abnormalities described in WAS patients, the early stages of B cell ontogeny appear intact and BCR-induced proliferation is only modestly diminished in the context of WASp deficiency. However, the reduction in splenic B cell numbers and in capping capacity of stimulated antigen receptors on WAS − / − B cells implies that the modest diminution in antigen receptor–evoked B cell proliferation is physiologically relevant and may engender impaired expansion of B cells within the peripheral compartment. Despite the plethora of functional defects detected in WAS − / − T cells and thymocytes, many of the biochemical events involved in signal relay through the TCR (such as tyrosine phosphorylation and MAPK and SAPK/JNK activation) remain intact in these cells. In this context, the WAS − / − cells are strikingly similar to the lymphoid cells of mice genetically rendered deficient for Vav 63 64 65 , a guanine nucleotide exchange factor for the Rho family of GTPases 66 67 . Although the effect of Vav deficiency on B cell ontogeny and function is more prominent than that of WASp deficiency, Vav − / − mice manifest defects in T cell maturation, TCR-evoked proliferation, cytokine production, and actin polymerization that closely resemble those observed in the WAS − / − mice. In addition, as with WAS − / − T cells, Vav − / − T cells show little or no abnormalities with respect to the early activation events induced by TCR ligation. At present, the biochemical basis whereby both the structural (receptor clustering and actin polymerization) and physiologic (proliferation and cytokine production) sequelae of TCR ligation are disrupted by WASp or Vav deficiency remains unclear. However, a relationship between these facets of T cell activation has become increasingly well recognized and is strongly supported by recent data indicating a pivotal role for cytoskeletal rearrangement in reorienting the T cell–APC interface so as to create a framework for the incorporation of signaling molecules into supramolecular activation clusters (SMACs) that amplify signal delivery 68 69 . Taken together with the T cell defects observed in WAS − / − mice, these observations suggest that while TCR activation signals can be initiated in the absence of cytoskeletal-driven events such as receptor clustering, signal delivery in this context is insufficient to achieve optimal activation and cytokine production. Thus, by virtue of its capacity to interact with both cdc42 and the Arp2/3 complex, WASp may act to promote the spatial rearrangements required for SMAC formation and thereby facilitate the juxtaposition of activated antigen receptors and signaling molecules that is required for efficient transduction of activation signals. Accordingly, the defective actin polymerization and TCR clustering engendered by WASp deficiency would predictably translate to signaling of insufficient amplitude and/or duration for optimal activation and cytokine production. This hypothesis is supported by preliminary data revealing that T cell microtubule organizing center (MTOC) polarization toward the T cell–APC contact site is reduced in stimulated WAS − / − T cells (Burkhardt, J., and K. Siminovitch, data not shown) and, if correct, might explain the disparate effects of WASp deficiency on B cell versus T cell activation, the latter of which appears more highly dependent on proper actin filament assembly 70 71 . In view of data indicating CD28–B7 interaction to be both necessary and sufficient for inducing movement of the cortical actin cytoskeleton toward the TCR–ligand interface 68 , the capacity of CD28 costimulation to partially normalize TCR-evoked proliferative responses of WAS − / − T cells suggests that WASp effects on cytoskeletal rearrangement may be particularly important for the propagation of TCR-initiated signals evoked in the absence of costimulatory signal. The capacity of WASp to interact with Nck may also be of some relevance to WASp effects on TCR signaling, as the Nck adaptor protein has been shown to promote TCR-mediated NF-AT activation by virtue of an interaction with the Pak1 serine/threonine kinase 40 . As Pak1, like WASp, appears to act downstream of cdc42 activation in a JNK-independent fashion, these observations suggest that WASp effects on TCR-evoked IL-2 production may reflect not only WASp modulation of upstream cytoskeletal-mediated TCR rearrangements, but also WASp interactions with both activated cdc42 and Nck. While these hypotheses require further investigation, the current data indicate that mice with WASp-deficient lymphoid cells provide a valuable system for dissecting out the complex structural and biochemical properties of WASp and delineating the molecular events that couple antigen receptor stimulation to cytoskeletal rearrangement and lymphocyte activation. | Other | biomedical | en | 0.999996 |
10544205 | 5C.C7 T cell lines derived from 5C.C7 TCR transgenic mice (specific for moth cytochrome c [MCC]/I-E k ; reference 31 ) were maintained in RPMI supplemented with l -glutamine, nonessential amino acids, sodium pyruvate, penicillin/streptomycin, and 10% fetal bovine serum. T cell lines were stimulated every 2 wk with irradiated splenocytes and MCC peptide (88–103). 2 d after the addition of peptide and splenocytes, the medium was supplemented with 5 U/ml of IL-2. Jurkat T cells, MCC9 T cell hybridoma (specific for MCC/I-E k 32 ), and CH27 were grown in the RPMI medium using 10% bovine calf serum. Primary B cell cultures were maintained in RPMI supplemented with 3% fetal bovine serum. Wild-type and mutant Y . pseudotuberculosis 33 were initially grown overnight to plateau phase in 2× YT at 26°C. Cells were diluted 1:50 into 2× YT supplemented with 20 mM sodium oxalate and 20 mM magnesium chloride and grown for 2 h at 26°C, then shifted to 37°C for 2 h. Bacterial numbers were determined by OD 600 = 1 × 10 9 bacteria/ml. MCC9 T cells, extensively washed with antibiotic-free medium, were exposed to the specified Y . pseudotuberculosis strain at a multiplicity of infection (MOI) of 10 for 1 h at 37°C. Gentamicin (final concentration 100 μg/ml) was added to kill the bacteria followed by T cell activation assays. T cells in 96-well plates were activated by either MCC peptide (88–103) complexed with glycosyl phosphatidylinositol–linked I-E k 34 , 1 × 10 5 CH27 cells and MCC peptide, or 1 μM ionomycin and 25 ng/ml PMA in 200 μl of supplemented RPMI medium. After overnight incubation at 37°C, 100 μl of medium was assayed for IL-2 production by standard sandwich ELISA (JES6-1A12, JES6-5H4; PharMingen) and streptavidin-europium detection (Wallac). For calcium flux, experiments were carried out by exposing 5C.C7 T cells to Y . pseudotuberculosis at an MOI of 50 for 1 h. Cells were loaded with the calcium-sensitive dye, Fura-2 (Molecular Probes), washed, and stimulated by an MCC peptide–loaded, confluent layer of I-E k –expressing Chinese hamster ovary cells, and monitored as described previously 35 . Jurkat T cells were exposed to specified Y . pseudotuberculosis for 1 h at an MOI of 50 at 37°C, followed by the addition of anti-CD3 (OKT3 ascites, 1:100). After 5 min, cells were spun down at 4°C and lysed with cytoplasmic extract buffer (1% NP-40, 150 mM NaCl, 10 mM Tris [pH 8.0], 2 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM β-glycerol phosphate, 5 mM dinitrophenyl phosphate, 100 μg/ml leupeptin, 100 μg/ml pepstatin, and 1 μM PMSF). Nuclei were removed by centrifugation at 4°C. The cytoplasmic extract (1 × 10 6 cell equivalents) was diluted into 2× reducing Laemmli buffer and run on 10% SDS-PAGE. Phosphotyrosine proteins were detected by Western blotting, using the unconjugated antiphosphotyrosine antibody, 4G10 (Upstate Biotechnology), followed by an anti–mouse Ig–horseradish peroxidase conjugate for detection. Blots were developed with enhanced chemiluminescence (Amersham Pharmacia Biotech). Each blot was subsequently stripped and reprobed for β-actin. CD3ζ immunoprecipitations were conducted as described 36 . B cells were isolated from MD4 anti–hen egg lysozyme (HEL) Ig transgenic mouse spleens by negative depletions with anti-Thy1, anti-CD4, anti-CD8, and anti–Mac-1 (Caltag) as described 37 . Cells were >98% B220 + as assessed by FACS ® . Anti-HEL B cells were exposed to wild-type or mutant Y . pseudotuberculosis at an MOI of 50 for 1 h at 37°C, followed by the addition of HEL protein (500 ng/ml). After 3, 10, and 30 min, the B cells were lysed with cytoplasmic extract buffer, nuclei were removed by centrifugation, and 1 × 10 6 cell equivalents were diluted into 2× reducing Laemmli buffer and run on 10% SDS-PAGE. Phosphotyrosine proteins were detected by Western blot as described above. For B7.2 upregulation, freshly isolated anti-HEL Ig B cells were first incubated at 37°C with the specified Y . pseudotuberculosis at an MOI of 20. After 1 h, gentamicin was added to a final concentration of 100 μg/ml. Splenic B cells from anti-HEL Ig transgenic or C57BL/6 mice were stimulated with HEL (500 ng/ml) or anti-IgM (3 μg/ml; Jackson ImmunoResearch Laboratories), respectively, for 12 h at 37°C. B cells were then stained for FACS ® analysis using anti-B220–FITC (RA3-6B2; PharMingen) and anti-B7.2–PE (GL1; PharMingen). Propidium iodide was included to assess cell viability. The development of a specific immune response is essential to resolve Yersiniosis. Since T cells are integral components of the specific immune response, we investigated whether Yersinia infection could significantly alter T cell activity by monitoring their cytokine production in response to antigenic activation. In the initial experiment, we preexposed either the APC, CH27, an I-E k –expressing B cell lymphoma, or MCC9, an MCC/I-E k specific T cell hybridoma, to either wild-type Yersinia or a Yersinia lcr mutant. The lcr mutant possesses the virulence plasmid, pYV, but is unable to produce or secrete Yops due to a mutation in a Yop regulatory locus 38 39 . After a 1-h incubation with Yersinia at an MOI of 10, the cells were treated with gentamicin (to kill the bacteria) and then mixed with MCC peptide and the appropriate untreated cells to achieve antigen activation. As shown in Fig. 1 , in both cases, IL-2 production was significantly decreased after exposure to wild-type Yersinia but not to the lcr Yersinia mutant. This indicates that one or more secreted Yop(s) was responsible for the observed cytokine inhibition. Interestingly, the Yersinia inhibitory effect on cytokine production is much more pronounced when T cells were preexposed to Yersinia , suggesting that Yersinia 's effect is greater on T cells than on CH27 cells. However, to definitively test if the bacteria were directly affecting T cells, we needed to exclude the possibility that, when the two cell types are mixed together, the bacteria interact with the other cell type before gentamicin killing takes effect. Therefore, plate-bound I-E k /MCC peptide (88–103) protein complex, instead of CH27 cells, was used in subsequent T cell activation assays. MCC9 T cells exposed to wild-type, but not a virulence plasmid–deficient (pYV − ) or an lcr mutant (data not shown) Yersinia , showed a dramatic reduction in IL-2 production compared with unexposed controls . These data suggest that one or more components of the Y . pseudotuberculosis virulence plasmid can directly inhibit T cell activation. To determine which Yop(s) are necessary for the suppression of IL-2 production, several Yersinia Yop mutants were screened in the T cell activation assay . YopO/YopJ- and YopE-deficient bacteria, but not YopH-deficient bacteria, were able to inhibit IL-2 production as efficiently as wild-type bacteria. In addition, the YopH mutant was unable to inhibit IL-2 production when MCC9 was stimulated by APCs . These results suggest that YopH is necessary for the inhibition of cytokine production in T cells. Similarly, exposure to wild-type, but not pYV-cured or YopH-deficient mutant, Yersinia inhibited the MCC/I-E k –specific production of IL-3, IL-4, and IFN-γ by T cell lines derived from the 5C.C7 TCR transgenic mice (data not shown). Cell viability, as assayed by propidium iodide staining and trypan blue exclusion, did not significantly change as a result of exposure to Yersinia in any of the above experiments (data not shown). These data suggest that YopH may target a common component in cytokine activation pathways. YopH has tyrosine phosphatase activity, and T cell activation through the antigen receptor is known to require the rapid phosphorylation of the tyrosine kinases, CD3ζ chain, and linker/adaptor molecules 40 . The phosphorylation and clustering of these molecules are critical to T cell activation, and these events precede the activation of protein kinase C as well as the rapid changes in intracellular calcium concentration accompanying T cell activation. Protein kinase C activation and the intracellular calcium flux are, in turn, required for induction of cytokine production. As shown in Fig. 2 , the YopH inhibitory effect can be reversed by the addition of PMA/ionomycin, indicating that the YopH-mediated cytokine suppression was upstream of protein kinase C activation and calcium flux. Consistent with this supposition, the ability of Yersinia -exposed 5C.C7 T cells to flux calcium in response to antigen was impaired in a YopH-dependent manner (data not shown). These results suggest that the effect of YopH is largely limited to early tyrosine phosphorylation events in T cell signaling. The biochemistry of antigen receptor signaling has been well characterized in the human T cell Jurkat line (for a review, see reference 40). Hence, we evaluated whether the induction of the tyrosine phosphorylation cascade in response to TCR cross-linking by the mAb OKT3 is altered when Jurkat cells are exposed to Yersinia . As shown in Fig. 3 , many tyrosine-phosphorylated proteins induced after TCR cross-linking, including CD3ζ, were severely reduced or absent in T cells exposed to wild-type or YopE-deficient, but not pYV − or YopH-deficient, mutant Yersinia compared with the sham-exposed T cells. These results suggest that YopH targets some of the earliest initiators of the T cell signaling complex. The antigen receptor on B lymphocytes (BCR) consists of the membrane-bound Ig and associated Igα and Igβ molecules. Tyrosine phosphorylation of Igα and Igβ is required for the initiation of the tyrosine phosphorylation signaling cascade after antigen binding 41 . Because of the signaling similarities between T cells and B cells, we tested whether the initial signaling events after BCR stimulation are affected by exposure to Y . pseudotuberculosis . As in T cells, we found that exposure to wild-type Yersinia interfered with the induction of the early tyrosine phosphorylation signaling cascade in response to antigen receptor engagement in a YopH-dependent manner . Anti-HEL Ig transgenic B cells were exposed to wild-type, pYV − , or YopH-deficient Yersinia and activated with HEL. B cells exposed to wild-type but not pYV − or YopH-deficient Yersinia showed a drastic reduction in the surface expression of B7.2 and CD69 (data not shown). Similarly, the induction of B7.2 and CD69 on splenic B cells isolated from C57BL/6 mice in response to anti-IgM cross-linking was also impaired when the B cells were exposed to wild-type but not pYV − or YopH-deficient Yersinia (data not shown). This effect was not due to Yersinia -induced cell death since the number of live cells, as assessed by propidium iodide, was not significantly different among samples (data not shown). In this study, we have examined the interaction between Yersinia and the adaptive immune system by monitoring the response of T and B lymphocytes to antigen after a brief exposure to Yersinia . Our results provide evidence that the Y . pseudotuberculosis tyrosine phosphatase, YopH, inhibits the signaling cascades associated with T and B cell antigen receptor activation. This is one of the first examples of a bacterial pathogen directly manipulating lymphocyte signaling and activation. Continuation of this inhibitory effect may explain why Yersinia infection, in some cases, is characterized by a chronic infection of lymphatic organs. The consequences of the YopH dephosphorylation activity may affect a wide range of T and B cell–mediated immune responses, including cytokine production. In fact, Yersinia infection dramatically suppresses the induction of IFN-γ and TNF-α production in some mouse strains 42 43 . When these cytokines are replaced exogenously, the mice are better able to survive infection. Since infection with an lcr − Yersinia triggers IFN-γ and TNF-α production in infected mice, one or more Yops are likely responsible for the suppression of cytokines. Recent experiments suggest that YopJ/P suppresses TNF-α production in macrophages 20 21 22 , whereas we found that IFN-γ production was reduced in T cells infected with Yersinia . Thus, the inhibition in cytokine production observed after infection with wild-type Yersinia may be due to the combined action of YopH and YopJ/P on different cell types. The pathological significance of our finding is not limited to a bacteria-induced suppression of cytokine production. The development of a T cell–mediated autoimmune disease, reactive arthritis, has long been associated with Y . pseudotuberculosis and Y . enterocolitica infection in genetically susceptible individuals. Inappropriate T cell signaling has been postulated as a factor in the development of autoimmune diseases. Thus, T cells exposed even briefly to wild-type Y . pseudotuberculosis may signal aberrantly, if at all, for a significant period of time. If a stimulus is delivered to the TCR during a partially responsive period, the T cell may respond inappropriately, leading to the development of abnormal immune responses. In addition, complete T cell activation requires a signal delivered through TCR–peptide/MHC complex and the engagement of costimulatory molecules such as CD28/B7.2. A failure to induce normal levels of B7.2 on APCs has been associated with the induction of T cell nonresponsiveness 44 . Although many other factors are likely to be involved in the development of reactive arthritis, we believe that the ability of Yersinia to disrupt TCR and BCR signaling cascades may contribute to this condition in some individuals. It has been suggested that the catalytic domain of YopH at the COOH-terminal end of the molecule selectively targets tyrosine-phosphorylated sites that contain the D/EpYxxP motif 45 . In addition, a domain in the NH 2 -terminal region of YopH may be required for the efficient recognition of substrates. This substrate-binding domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 domain 13 . Although the target(s) of YopH in lymphocytes is not known, we observed that most of the tyrosine kinases and adapter/linker proteins that are normally phosphorylated after T and B cell antigen receptor stimulation were either not phosphorylated or rapidly dephosphorylated in the presence of YopH. The three known YopH targets identified in epithelial and Hela cells, paxillin, p125 FAK , and p130 cas 12 13 14 , and its closely related homologue, p105 casL , are also found in lymphocytes 46 . Further, p105 casL , associated with the cell membrane and clustered integrins, is known to be phosphorylated after TCR ligation 47 . Thus, as p105 casL may be involved in both TCR and integrin signaling, it is tempting to speculate that, in addition to the CD3ζ-ZAP70 in T cells or the Igα/Igβ pathway in B cells, a second pathway involving paxillin-p125 FAK -p105 casL contributes to lymphocyte activation. An abrogation of p105 casL phosphorylation may have a significant inhibitory effect on T cell function. Alternatively, YopH may also target one member or members of the known T cell signaling cascade, including protein tyrosine kinases such as ZAP70 and Syk and tyrosine-phosphorylated adapter/linker proteins such as linker for activation of T cells (LAT), B cell linker protein (BLNK), and SH2 domain–containing leukocyte protein of 76 kD (SLP-76), which all contain sequences similar to the optimum YopH target consensus sequence. It is possible that a rapid dephosphorylation of these proteins may destabilize the TCR complex, which may lead to its disassembly and exposure of most or all components of the TCR complex, including CD3ζ, to phosphatases. We are currently characterizing the cellular distribution and protein targets of YopH in lymphocytes to distinguish among these possibilities. The Yops play a crucial role in Yersinia virulence. Most past experiments have focused on the effects of these Yops in epithelial cells and macrophages. Yet, our work clearly demonstrates the rapid and essential role of YopH in inhibiting T cell and B cell signaling. Importantly, the redistribution and reorientation of the cytoskeleton are necessary early events for lymphocyte activation 48 . Yet, both YopE and YopO, which have been shown to disrupt actin filaments leading to alterations in the cytoskeleton of both phagocytes and epithelial cells 16 49 , have no demonstrable effect in our assays. Furthermore, although YopJ/YopP induces apoptosis in macrophages 17 19 , neither primary B cells, T cell lines, or T cell hybridomas appear susceptible to this virulence factor. YopH activity has been shown to mediate several effects in epithelial cells as well as macrophages, including: inhibition of Fc receptor–mediated oxidative burst in macrophages 50 , inhibition of neutrophil functions 51 52 , FA disassembly in epithelium cells, and inhibition of phagocytosis in conjunction with YopE. These effects are all very different from the one we report here. Thus, it appears that different Yops target different host cell types involved in defense against Yersinia infection. By the same token, the enteropathogenic Yersinia harbor several different adhesion molecules that mediate their attachment to host cells. There is experimental evidence to suggest that some of the adhesins are better adapted to deliver specific Yops to certain host cells types. Invasin, which targets β1 integrins 53 54 , is essential for Peyer's patch translocation across the epithelial barrier, but not as efficient for delivering Yops to macrophages. Perhaps the plasmid-mediated adhesin, YadA, suffices in delivering Yops into macrophages. Taken together, it appears that Yersinia have evolved to produce effectors that are specifically designed for the different cell types that the bacteria encounter in the course of an infection. The study of pathogen–host interactions provides a fascinating insight into the strategies used by microbes to manipulate normal host functions to their own benefit. The Yops are part of a complex secretion–translocation apparatus that responds to host cell cues and results in the delivery of an array of proteins into the host cell cytoplasm. Homologues of the secretory arm of this complex, the type III secretion system, have been found in a growing number of pathogenic gram-negative microorganisms. It is likely that other pathogenic microorganisms secrete molecules analogous to Yops to alter host cell function and cause immune suppression or an alteration in the immune response to the benefit of the microbe. It is instructive to see, for example, that Salmonella species have two fully functional type III secretory systems, one involved in entry and the other involved in intracellular replication, that are essential for virulence. Moreover, one of these Salmonella type III secretory pathways delivers an effector protein, SptP/SptA, into the host cell cytoplasm. SptP/SptA has a COOH-terminal phosphatase domain similar to that found in YopH and an NH 2 -terminal domain that is homologous to the YopE cytotoxin 55 56 . Curiously, infection with Salmonella can also trigger reactive arthritis in humans. Similarly, Shigella and Chlamydia and other pathogens have chromosomal islands containing type III secretory and effector homologues, and these bacteria are likewise associated with the development of autoimmune disease subsequent to infection. Is this the result of an underlying common strategy employed by many bacteria to alter the host immune response? Regardless, studying the molecular mechanisms of bacterial infection and host immune responses will reveal additional subtleties of bacterial pathogenicity and provide new information about the development of immune response. | Study | biomedical | en | 0.999995 |
10544206 | Reagents were purchased from the following sources: RPMI 1640, PBS, and FCS from GIBCO BRL; Immu-Mount from Shandon; OCT compound from Miles Labs; N -cetyl- N , N , N -trimethyl-ammonium bromide (TAB), Genziana violet, BSA, chloral hydrate, dianisidine dihydrochloride, erythrosine, FITC-conjugated bovine albumin (FITC-albumin), and heparin from Sigma Chemical Co.; human TNF-α from Genzyme Corp.; human IL-1β from Dompè; human monocyte chemotactic protein 3 (MCP-3) from Peprotech; Diff-Quik from Baxter Dade AG. CD1 male mice (25–30 g; Charles River) were housed with free access to food and water, under a 12-h light–dark cycle with constant temperature (21–23°C) and humidity (60 ± 5%). Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with international laws and policies . All the antibodies, unless otherwise specified, were rat anti–mouse mAbs and were used as sterile purified immunoglobulins. BV11 and BV12 mAbs were generated in our laboratory following a previously described procedure and characterized as described 20 22 . BV12 recognizes a different JAM epitope than BV11 and is devoid of biological activity 20 . Fab fragments of BV11 were prepared by standard procedures 23 . Other antibodies used in this study were as follows: (a) HB-151, anti–HLA-DR5, used as isotype-matched control of BV11 (American Type Culture Collection); (b) MEC 7.46 to PECAM 22 ; (c) RB6-8C5 to granulocyte-differentiation antigen (CD15) was obtained from Dr. R.L. Coffman (DNAX, Palo Alto, CA). Endotoxin content of all the mAbs was tested by the Limulus assay with chromogenic detection (BioWhittaker). Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated rabbit anti–rat IgG from Sigma Chemical Co. was used as secondary antibody in immunofluorescence microscopy. A polyethylene cannula was permanently implanted in the right lateral ventricle (0.5 mm lateral and 1.0 mm posterior to bregma) 4 d before the onset of the experiment, as described elsewhere 24 . At time 0, the animals were treated intravenously with mAbs at a standard dose of 100 μg/mouse in a final volume of 100 μl or with 100 μl of sterile, pyrogen-free saline. The dose of 100 μg/mouse for the mAbs was selected as the optimal dose to inhibit monocyte chemotaxis in vivo 20 . After 10 min, mice were subjected to ICV treatment with a mixture of TNF-α (3 U/g) and IL-1β (1.25 U/g) dissolved in saline in a final volume of 2.5 μl (sham-injected animals received the same volume of saline). In a different set of experiments, animals were ICV-treated with 1 μg/mouse of MCP-3. After 6 h of treatment (except where otherwise specified), mice were anesthetized intraperitoneally with chloral hydrate (350 mg/kg), and 5–10 μl of CSF was drawn from the cisterna magna using a glass capillary with a tip of ∼300 μm 24 . Careful surgery was conducted in order to avoid blood contamination of CSF. Mice were then decapitated, and brains were immediately cooled by liquid nitrogen and stored at −80°C until measurement of myeloperoxidase (MPO) activity or included in OCT compound, frozen in isopentane cooled by liquid nitrogen, and stored to −80°C, until preparation of brain tissue sections for immunofluorescence microscopy. CSF aliquots (2 μl) were immediately diluted in cool RPMI (containing 10% FCS) and kept in ice until cytospin centrifugation. Differential leukocyte counts of CSF samples were determined on Diff-Quik–fixed and erythrosine-stained cytospun slides by phase–contrast microscopy. Total blood leukocyte counts were determined by phase–contrast microscopy in heparin-anticoagulated blood diluted with Turk solution (1:20 ). Infiltrated leukocytes and vascular antigens were localized in brain tissue sections by immunofluorescence microscopy 20 24 . Serial cryostat sections (20 μm) were cut horizontally from the brain, placed on glass coverslips, and dried overnight at room temperature 24 . Tissue sections were fixed in cool methanol (5 min at −20°C), rinsed two times with PBS, incubated in PBS containing 2% of BSA (30 min at room temperature), and subsequently incubated for 1 h at room temperature with TRITC-conjugated anti–rat IgG and rinsed (three times, 5 min each) in PBS. Dried coverslips were then mounted in Immu-Mount, observed in a Zeiss Axiophot microscope, and images were recorded on Kodak TMax P3200 films. MPO activity was measured as described previously 25 26 . In brief, the frozen tissues were weighed and homogenized in 20 volumes of 5 mM PBS, pH 6.0, at 4°C and then centrifuged at 30,000 g (30 min at 4°C). Supernatants were discarded, and pellets were extracted in 10 volumes of 0.5% TAB in 50 mM PBS, pH 6.0, at 25°C. Samples were frozen on dry ice (three freeze–thaw cycles) and then sonicated for 10 s at 25°C. Samples were subsequently incubated for 20 min at 4°C, centrifuged at 12,500 g for 15 min at 4°C, and MPO assay was performed as described 26 27 . The absorbance change (ΔA) at 460 nm was measured over 2 min in the thermoregulated flow cell (25°C) of a Uvikon 860 spectrophotometer (Kontron Instruments). The data were derived using a kinetic computer program 26 . Sham- or cytokine-treated mice (with or without mAb pretreatment) were injected intravenously with FITC-albumin (1.5 mg/mouse) 1 h before time of killing. CSF was collected as usual, and 4 μl was immediately diluted in 100 μl of ice-cold phosphate buffer and kept at 4°C. Samples were then placed in a 96-well microtiter plate, and fluorescence content was measured in a fluorimeter at 492-nm absorbance and 520-nm emission wavelengths, respectively. Vascular JAM staining was observed in brain tissue sections of sham- or cytokine-treated animals that had received mAb BV11 in vivo. BV11 staining was evident in different sized blood vessels that penetrate the brain parenchyma and in those of choroid plexus . No specific staining with the secondary antibody was observed when the animals were treated with saline; when the mice were treated with BV12 or when brain slices were treated with BV11 in vitro, we obtained a similar immunofluorescence pattern as that reported in Fig. 1 (not shown). No major difference in JAM staining was observed in cytokine- or sham-treated animals. These data confirm previous work 20 showing that, despite the complex organization of the TJs in the brain microvasculature, JAM is located more superficially and can be available to mAb staining. Mice ICV-treated with TNF-α (3 U/g body wt) and IL-1β (1.25 U/g body wt) showed a time-dependent CSF recruitment of both neutrophils and monocytes . Maximal neutrophil recruitment at 6 h was almost 90% of the total leukocytes. Monocyte infiltration did not exceed 14% of the total recruited leukocytes. A comparable time course of leukocyte recruitment, measured as MPO activity, was observed in brain extracts of cytokine-treated mice . After 6 h of treatment, cytokines induced a 3.7-fold increase of MPO activity compared with sham-treated animals. As reported in Fig. 2 B, panel a, the CSF of sham-treated animals was virtually devoid of circulating cells. In contrast, CSF of cytokine-treated mice contained a large number of recruited leukocytes . This response was markedly reduced by intravenous administration of BV11 mAb . mAb BV11 was able to reduce neutrophil accumulation in the CSF by ∼76%. This activity was retained by its Fab fragment, whereas an isotype-matched control, mAb HB-151, was ineffective . mAb BV12, which is able to bind JAM to a different epitope than BV11, did not show a significant inhibitory activity . Monocyte recruitment was also reduced by ∼50% by BV11 or its Fab fragments, whereas HB-151 was ineffective . At a longer time, 9 h after treatment, inhibition was still apparent, even if slightly lower, reaching 52% for neutrophils (not shown). The inhibitory effect of BV11 was observed also when mice were injected ICV with MCP-3 (1 μg/mouse 28 ). Monocytes and neutrophils were efficiently recruited by MCP-3 ICV, and their number was significantly reduced in mice pretreated with BV11, but not with HB-151 and BV12 . In all of the experiments reported above, the number of circulating leukocytes was never substantially altered by infusion of any type of mAb in cytokine- or sham-treated mice. Blood samples were taken by puncture of the left heart ventricle. In a typical experiment, in animals treated ICV with IL-1β and TNF-α for 6 h, circulating leukocytes were as follows: 9.1 ± 2.2 × 10 6 /ml in saline; 8.3 ± 1.3 × 10 6 in HB-151; 10.1 ± 2 × 10 6 in BV11; and 10.5 ± 1.8 × 10 6 in BV12-treated animals. In comparison, in animals treated ICV with saline for 6 h, circulating leukocytes were as follows: 8.6 ± 1.2 × 10 6 /ml in saline; 8.8 ± 1.1 × 10 6 in HB-151; 9.9 ± 1.9 × 10 6 in BV11; and 9.5 ± 1.2 × 10 6 in BV12-treated animals. In addition, we were unable to detect increases in neutrophil trapping in lungs, spleen, liver, and kidneys by immunofluorescence analysis of CD15-stained tissue sections in animals treated with any of the mAbs used in this study. As further control, we measured MPO activity (see below) in lungs of mice treated for 6 h with saline, BV11, BV12, HB-151 (100 μg/mouse), or BV11 Fab fragments (200 μg/mouse). The values, expressed as ΔA/min/g, were not significantly different: 34.6 ± 0.3 in saline; 27.6 ± 0.2 in HB-151; 27.1 ± 0.7 in BV12; 29.6 ± 0.6 in BV11; and 26.8 ± 0.7 in BV11 Fab–treated animals. Neutrophil infiltration of the brain was also evaluated by histological means. Brain tissue sections of sham-treated mice showed essentially no staining of neutrophils with the anti-CD15 mAb RB6-8C5 . In cytokine-treated mice, infiltrated neutrophils could be recognized in the surrounding areas of cerebral blood vessels and in submeningeal spaces . Pretreatment of the mice with mAb BV11 but not with HB-151 (not shown) visibly reduced neutrophil extravasation. To quantify leukocyte infiltration, we measured MPO activity in brain extracts. The basal MPO activity was increased by about three times in animals treated with TNF/IL-1 and was reduced in mice pretreated with mAb BV11 but not with HB-151 . As another marker of acute inflammatory reaction in the brain, we tested the effect of mAb BV11 on the extravasation of FITC-albumin. Cytokine treatment induced a 3.4-fold increase of FITC-albumin extravasation in CSF . This enhancement of permeability was significantly reduced in mice pretreated with mAb BV11 but not with the control mAb, HB-151. As shown previously by others 29 , the direct injection of TNF-α and IL-1β ICV induces acute inflammatory reactions in the brain parenchyma largely reminiscent of bacterial meningitis. Reduction of inflammation in meningitis can decrease mortality 3 7 30 31 . Inhibition of leukocyte adhesion to endothelial cells by β2 integrin–blocking mAbs in bacterial meningitis reduces CSF protein accumulation 7 8 and improves the survival rate. Furthermore, in mice deficient in endothelial P- and E-selectins, leukocyte and protein influx in the CSF upon inflammatory cytokine ICV injection was virtually abolished 21 . In this paper, we extend these observations by showing that inhibition of a novel leukocyte ligand, JAM, which regulates leukocyte transmigration 20 , is effective in reducing inflammatory reactions in experimental meningitis. Intravenous pretreatment of mice with the anti-JAM mAb BV11 strongly reduced leukocyte extravasation and albumin permeability in the CSF and brain parenchyma promoted by ICV administration of cytokines. The effect of BV11 was specific and not influenced by leukocyte Fc receptor, since BV11 Fab fragments retained the activity and an isotype-matched control mAb was ineffective. In addition, BV12, an mAb directed to JAM but recognizing a different epitope 20 , was devoid of activity. Although in previous work 20 we observed that JAM was important in controlling monocyte extravasation, in the present model we confirm and extend this observation to neutrophils, which constitute the majority of the cellular exudate. The effect of JAM appears to be specific for leukocyte transmigration, since BV11 does not inhibit monocyte or neutrophil adhesion to endothelial cells 20 . In addition, BV11 also reduced the effect of MCP-3 on CSF accumulation of both cells types. MCP-3 does not induce endothelial inflammatory reactions like IL-1β/TNF-α, and therefore these data are in favor of a specific effect of the mAb on leukocyte recruitment. Despite reduction in albumin influx in CSF, it is unlikely that BV11 acts on endothelial permeability directly since it did not modify basal or IL-1/TNF–increased endothelial permeability in vitro in the absence of leukocytes ( 20 ; and data not shown). A more likely hypothesis is that for its localization at intercellular junctions, JAM binds leukocytes and directs their migration through the junctions. Since leukocyte transmigration through cytokine-activated endothelial cells may lead to an increase in permeability 32 33 , JAM inhibition would cause an indirect protection of blood–brain barrier breakdown. A similar mechanism has been indicated for PECAM 19 , and it is possible that these two proteins constitute a new class of agents which collaborate in promoting cell movement through endothelial cell junctions. JAM is located at TJs at the most apical domain of the intercellular cleft 20 , whereas PECAM is found at a more basal site of the junctions 34 . JAM may be necessary for the first interaction of circulating cells, which would then move along the cleft. An important issue is JAM counterreceptor in monocytes and neutrophils. Monocytes and neutrophils obtained from the peritoneal fluid of thioglycollate-injected mice 20 or neutrophils from femoral bone marrow bind very poorly mAbs BV11 and BV12. Therefore, it is conceivable that these cells recognize JAM through a heterophilic receptor and that this interaction is inhibited by BV11. In conclusion, the data reported here support the concept that JAM could constitute a novel target for modulating leukocyte extravasation at sites of inflammation. Therefore, manipulation of the molecular organization of endothelial junctions may be an effective approach to control vascular permeability and leukocyte traffic. | Other | biomedical | en | 0.999994 |
10544207 | The wild-type TCR β chain (Vβ8.2-Jβ2.1) cDNA was used as a template for mutagenesis. Deletion of the 14 nucleotides forming the Cβ FG loop has been described 7 . Transgenic vectors have also been described previously 22 . Packaging cell lines GP+E-86 23 were transfected with retroviral vector LXSN expressing the Vβ8.2-Jβ2.1 + TCR-β or β-loop − chain or LXSP expressing the Vα4-Jα47 + or Vα14-Jα281 + TCR α chain cDNA. The TCR α chain (Vα14-Jα281) was cloned from NK1.1 α/β + T cell hybridoma total RNA provided by R. MacDonald (Ludwig Institute for Cancer Research, Lausanne, Switzerland). After appropriate selection of the packaging cells, the infectious supernatants were used to infect TCR − hybridomas 24 as previously described 25 . The TCR-β or β-loop − chain was first introduced into the hybridomas and, after neomycin selection (G418; 1 mg/ml), these cells were superinfected with TCR α chain by culturing them on packaging lines producing LXSP TCR-α Vα4-Jα47 or Vα14-Jα281. The hybridomas were then maintained in IMDM supplemented with 2% FCS, neomycin, and puromycin (10 μg/ml). TCR expression was tested by FACS™ as early as 4 d after selection. Stable transfectants were maintained in G418 and puromycin-containing medium. Hybridomas were lysed at 2 × 10 7 cells/ml in 1% Triton X-100 (Bio-Rad Labs.), 150 mM NaCl, 20 mM Tris/HCl, and 5 mM EDTA, pH 7.5, buffer containing complete protease inhibitors (Boehringer Mannheim) for 30 min at 4°C. Lysates cleared of cell debris were immunoprecipitated with purified mAb F23.1 (2 μg/ml) and protein G–Sepharose (Pharmacia). After washing with lysis buffer and PBS, the lyophilized pellets were resuspended in reducing SDS buffer, loaded on a 4–12% Bis-Tris precast gel (Novex), and transferred onto nitrocellulose membrane Hybond-C extra (Amersham). Blots were probed in PBS 6% blotting blocker nonfat milk (Bio-Rad Labs.) and 0.2% Tween with purified mAb H58 (anti-Cα), followed by goat anti–hamster horseradish peroxidase–labeled mAb (Southern Biotechnology Associates, Inc.) or biotinylated F23.1 (anti-Vβ8) mAb followed by streptavidin–horseradish peroxidase (Southern Biotechnology Associates, Inc.). The proteins were detected with a chemiluminescent detection system (Pierce Chemical Co.). BALB/c and C56BL/6 mice were purchased from IFFA-Credo. The TCR-β knockout mice have been described 26 and were bred in our specific pathogen–free animal facility with the TCR-β or TCR β-loop − transgenic mice. Cell suspensions from thymi were depleted of CD8 + T cells with anti-CD8 31M antibody 27 and complement treatment (Cedarlane Labs.), and liver cells were simply ficolled to eliminate red cells before immunofluorescence stainings, performed as previously described 28 . Flow cytometric analyses were performed on a FACSCalibur™ equipped with CELLQuest software (Becton Dickinson). The reagents used were mAbs 145-2C11 (anti-CD3∈), NKR-P1C (anti-NK1.1), H57-597 (anti-Cβ), RM4-5 (anti-CD4), IM7 (anti-CD44, Pgp-1), TM-β1 (anti–IL-2R β chain), MEL-14 (anti-CD62L) (all seven mAbs purchased from PharMingen), biotinylated F23.1 (anti-Vβ8.1,2,3), and second step reagent streptavidin–allophycocyanin (Molecular Probes, Inc.). Single NK1.1 + CD3 + cells were sorted into polycarbonated 96-well plates (one cell per well in 5 μl of PBS) and immediately frozen on dry ice and stored at −70°C. To prepare cDNA, the plate was heated up to 65°C for 1 min before adding into each well 10 μl of the reverse transcriptase (RT)-PCR mix (reverse transcriptase Superscript II; GIBCO BRL) for 1 h at 42°C under standard reaction conditions. After heat inactivation of the enzyme (2 min at 95°C), DNA amplification was carried out as described 29 . 75 μl of a PCR mix containing Taq polymerase and the primers necessary for DNA amplification of the Vα14 + TCR α chain (5′ Vα14 CTAAGCACAGCACGCTGCACA [reference 20]; 3′ Cα ATGGATCCTCAACTGGACCACAGCCTCA) and Vβ8.2 + TCR β chain (5′ Vβ8.2 CTTGAGCTCAAGATGGGCTCCAGGCTCTTC; 3′ Jβ2.1 CTGCTCAGCATAACTCCCCCG) were added to the wells for the first round of PCR (30 cycles). An aliquot from this PCR (1 μl) was used for a second round of PCR (35 cycles) to individually reamplify the Vα14 + TCR α chain or Vβ8.2 + TCR β chain using the same specific primers. To avoid any influence of the endogenous β locus on the expression of the mutated β chain, mice transgenic for the Vβ8.2 + TCR β chain lacking the Cβ FG loop (β-loop − ) were backcrossed to TCR-β −/− mice 26 . T cell development in these mice was compared with that in wild-type Vβ8.2 + TCR β chain transgenic mice, also with a β −/− background. As described in our previous study 7 , peripheral T cells from mice transgenic for the TCR β or β-loop − chain express equal levels of the TCR–CD3 complex, and whereas the anti-Vβ8 F23.1 mAb recognizes all T cells, the Cβ-specific H57 mAb does not stain cells expressing the TCR β-loop − chain . It is worth pointing out that in the absence of the Cβ FG loop, the anti-CD3∈ 2C11 mAb stains better, suggesting that the epitope recognized is more accessible, a result that might not be surprising, as one of the CD3∈ chains is physically adjacent to the β chain in the TCR–CD3 complex 5 . We consistently found that TCR β-loop − transgenic mice have significantly fewer NK1.1 α/β + T cells in the thymus, liver, and spleen (data not shown) in comparison to TCR-β transgenic mice or wild-type littermates . Thus, a mutation in the Cβ domain can notably alter development of NK1.1 α/β T cells. This result was puzzling, considering that conventional α/β T cells are normal in TCR β-loop − transgenic mice 7 . Furthermore, the mutated TCR β chain uses Vβ8.2, a variable region that is usually expressed by >40% of NK1.1 α/β T cells 30 . Hence, monoclonal expression of the wild-type Vβ8.2 + TCR β chain allows NK1.1 T cell development comparable to that of nontransgenic littermates. Characteristically, NK1.1 T cells express intermediate levels of TCR 8 . Interestingly, in TCR β-loop − transgenic mice, TCR expression on the few remaining NK1.1 T cells is even lower than in control animals; these cells express about four times less TCR than do those in wild-type β-transgenic mice . Otherwise, NK1.1 T cells in β-loop − transgenic mice express normal levels of CD4 and are CD44 + CD62 ligand (L) − and IL-2Rβ + , as expected for this T cell population 8 . CD1d, a β 2 microglobulin–associated molecule required for NK1.1 T cell development 9 10 , is also expressed at normal levels in TCR β-loop − transgenic mice (data not shown). To determine if development of NK1.1 α/β + T cells could be rescued by the expression of endogenous β chains, as has been described for other TCR-β transgenic mice 19 , we studied NK1.1 T cell frequency in TCR β-loop − transgenic mice on a β +/− background. We have already observed that in these mice, inhibition of β rearrangements via allelic exclusion is not total, and ∼10–20% of peripheral T cells can express endogenous β chains (data not shown). NK1.1 α/β + T cells expressing endogenous β and β-loop − chains could be distinguished by the Cβ-specific H57 mAb, which cannot stain T cells expressing the mutated β chain . As shown in Fig. 4 , expression of endogenous β chains can rescue NK1.1 T cell development to a certain extent. NK1.1 Cβ + cells appear in the livers of TCR β-loop − transgenic mice on a β +/− background. Yet these cells only account for about one-third of the whole NK1.1 T cell population. The NK1.1 Cβ − T cells are still predominant. There are two populations of NK1.1 Vβ8 + cells, which express either intermediate or low TCR levels in TCR β-loop − transgenic mice on a β 1/− background. Interestingly, expression of endogenous β chains accounts for most of the NK1.1 T cells expressing intermediate TCR levels. Thus, expression of endogenous β chains did rescue some NK1.1 T cell development and restore TCR expression to intermediate levels. This result strongly suggested that the Cβ FG loop is needed for efficient TCR assembly in NK1.1 T cells. As most NK1.1 α/β + T cells express an invariant Vα14-Jα281 TCR α chain 20 21 , and the mutant TCR β chain is expressed at normal levels by conventional α/β T cells but not by NK1.1 T cells , we assessed whether the mutant Vβ8.2 + TCR β chain could still pair with the Vα14 + TCR α chain. TCR − hybridomas were transfected with cDNAs coding for either the wild-type TCR β or β-loop − chain together with the Vα14 + TCR α chain or Vα4 + TCR α chain (the original partner of the nonmutated β chain) cDNAs. As shown in Fig. 5 A, the TCR β-loop − chain clearly pairs with and is expressed on the cell surface with the Vα4 + TCR α chain but is barely detectable on the cell surface with the Vα14 + TCR α chain. In contrast, the wild-type TCR β chain is expressed on the cell surface with both α chains . However, the Vα14 + TCR is expressed at lower levels than is the Vα4 + TCR. This observation may reflect the in vivo situation in which a TCR on NK1.1 T cells is expressed at lower levels than on conventional α/β T cells. To assess whether impaired cell surface expression of the TCR wild-type β and β-loop − chain together with the Vα14 + TCR α chain is due to a problem of pairing, TCRs from the transfectants were immunoprecipitated with anti-Vβ8 mAb. As can be seen in Fig. 5 B, the TCR α chain can be coimmunoprecipitated with the TCR β chain in all transfectants expressing the TCR on the cell surface. In contrast, the Vα14 + TCR α chain cannot be coimmunoprecipitated with the mutant β chain in detectable amounts. This result implies that the Vα14 + TCR α chain pairs very poorly with the Vβ8 + TCR β chain lacking the Cβ FG loop. It is worth pointing out that in the hybridomas producing the wild-type β and Vα14 + chains, many fewer assembled α/β dimers can be immunoprecipitated compared with control Vβ8.2/Vα4 dimers. This latter result suggests that mere physical constraints on the assembly of the β chain with the Vα14 + TCR α chain exist and is consistent with the low TCR expression on normal NK1.1 T cells. Next, we assessed whether the NK1.1 α/β + T cells that do develop in TCR β-loop − transgenic mice express the Vα14 + TCR α chain by performing RT-PCR on single NK1.1 CD3 + T cells sorted from TCR-β and β-loop − transgenic mice. As summarized in Table , the frequency of NK1.1 T cells expressing Vα14 is not significantly decreased in TCR β-loop − transgenic mice in comparison to wild-type TCR-β transgenic animals. One has to keep in mind, however, that there are few NK1.1 T cells in the mutant mice, and these express much lower levels of TCR . This, together with biochemical data, strongly suggests that in TCR β-loop − transgenic mice, both the impaired development of NK1.1 T cells and their weak TCR expression is due to the physical constraints on the assembly of the β chain lacking the Cβ FG loop with the Vα14 + TCR α chain. We have previously shown that in conventional T cells expressing Vβ8.2, deletion of the Cβ FG loop has no effect on Vα 7 and Jα repertoire usage (our unpublished data). This result suggested that no drastic conformational changes in the TCR β chain were created by the mutation. However, in this study we clearly show that expression of the Vα14 + α chain is sensitive to deletion of the Cβ FG loop. Therefore, deletion of the Cβ FG loop must create some subtle change in TCR β chain conformation. It seems that expression of the Vα14 + α chain does not allow much structural flexibility of the TCR, as it is particularly sensitive to TCR β chain conformation. Its expression might impose stringent constraints on α/β assembly. This could at least partially explain why the Vβ repertoire of NK1.1 T cells is relatively limited 30 . Pairing with the apparently conformation-sensitive Vα14-Jα281 TCR α chain could be the initial pressure on Vβ usage in NK1.1 T cells 19 . Recently, results obtained by using Vα14-transgenic mice suggested that selection was the main force in shaping the NK1.1 T cell repertoire 31 . Here we have shown that in addition to selection, differential Vα–Vβ pairing can also potentially influence the NK1.1 T cell diversity. In summary, our data show that subtle changes in the TCR β chain conformation (which do not seem to affect conventional Vβ8.2 + α/β TCRs) can substantially alter pairing with the Vα14 + α chain and impair NK1.1 T cell development. | Study | biomedical | en | 0.999998 |
10545491 | Kinesin-II is a heteromeric kinesin . It is isolated from green algae, nematodes, echinoderms, and vertebrates as a heterotrimeric complex containing two unique, though related, motor subunits that are members of the KIF3A/3B or KRP85/95 kinesin subfamily . The two motor subunits are typically associated with a nonmotor subunit known as kinesin-associated polypeptide . It appears that the heteromeric nature of kinesin-II allows the organism to treat it as a combinatorial protein. For example, at least three isoforms of the kinesin-II motor subunits (KIF3A, KIF3B, and KIF3C) are expressed in the mouse and rat. KIF3A is capable of forming heterodimers with either KIF3B or KIF3C, while KIF3B and KIF3C are unable to heterodimerize with each other . In addition, several isoforms of the associated nonmotor subunit, KAP, can be generated from alternative splicing of KAP mRNA . Kinesin-II, and thus far all NH 2 -terminal kinesins, display only plus end–directed microtubule-based in vitro motor activity, suggesting that in vivo kinesin-II–driven transport will be limited to plus end–directed movement. Both kinesin-II motor and nonmotor subunit isoforms display differential tissue expression and even differential subcellular localization in the mouse, suggesting that different combinations of these subunits have been adapted for specialized transport needs . Indeed, in frog melanophores, kinesin-II powers the dispersion of pigment granules , while in the unicellular Chlamydomonas and Tetrahymena , kinesin-II drives anterograde intraflagellar transport . In higher animals, kinesin-II has also been adapted for anterograde axonal transport. An axonal transport role for kinesin-II was hypothesized early in the studies of murine KIF3A/3B . Reported to be associated with membrane-bounded vesicles, the true nature of the axonal kinesin-II cargo has eluded researchers. Now, a likely kinesin-II cargo has been identified in the axons of Drosophila neurons . In these studies, two Drosophila kinesin-II motor subunit homologues, KLP64D (KIF3A/KRP85) and KLP68D (KIF3B/KRP95), were found to coexpress in cholinergic neurons. Mutations in KLP64D cause uncoordinated, slow movement and result in death at the larval or early adult stages of development. Interestingly, heterologous expression of the mouse homologue, KIF3A, results in rescue of the KLP64D mutations, indicating that these two proteins are functional homologues. Transport of choline acetyltransferase, ChAT, from the cell body to the synapse is also disrupted in these mutants, suggesting that ChAT is normally transported out to the synapse by kinesin-II. To test this model, these researchers exploited a mutant ( Klc - ) that is missing the light chains of conventional kinesin, a known anterograde axonal transport motor. The axons in segmental nerves of these mutant larvae contain periodic swellings caused by the accumulation of fast axonal transport cargoes . Some of the swellings contain both KLP64D and ChAT, while other swellings contain little of either protein, providing further evidence that kinesin-II may be transporting ChAT. These findings raise an interesting question. How does kinesin-II transport a normally soluble protein for long distances in the axon? Axonal forms of kinesin-II in the mouse appear to be associated with membranous vesicles . If the same is true for insect axonal kinesin-II, then ChAT may associate with these vesicles during axonal transport. Alternatively, kinesin-II in the insect may be transporting a single protein (ChAT) or a protein complex which contains ChAT. A precedence for kinesin-II–mediated transport of protein complexes comes from studies of intraflagellar transport. Initially identified in Chlamydomonas , intraflagellar transport (IFT) is the bidirectional movement of large proteinaceous rafts along the outer doublet microtubules of motile cilia and flagella and nonmotile sensory cilia . The anterograde movement of rafts out to the distal tip of these organelles is powered by kinesin-II, while the retrograde transport of the rafts back toward the cell body is mediated by cytoplasmic dynein 1b . The IFT rafts are composed of repetitive subunits made from ∼15 polypeptides that can be isolated as two large protein complexes known as IFT Complex A and IFT Complex B . Functional roles of IFT include both the assembly and function of cilia and flagella. Disruptions of kinesin-II function in Chlamydomonas , Tetrahymena , Caenorhabditis elegans , echinoderms, and the mouse have all resulted in severe inhibition of the assembly of cilia and flagella . There is also compelling evidence for the role of cytoplasmic dynein 1b as the retrograde IFT motor. Disruption of this dynein in Chlamydomonas results in the formation of very short (∼1 μm) flagellar stubs filled with kinesin-II and the IFT rafts . Furthermore, disruption of an 8,000-D Chlamydomonas dynein light chain results in a less severe flagellar assembly phenotype which is accompanied by a loss of retrograde IFT but not anterograde IFT . In the absence of retrograde IFT, the shorter-than-normal flagella are congested as they fill with both kinesin-II, and the IFT Complexes. This phenotype is similar to that observed in the che-3 mutation in C. elegans ; CHE-3 encodes the nematode cytoplasmic dynein 1b heavy chain. The hypothesis that CHE-3 dynein acts as the retrograde IFT motor in the nematode is strongly supported by Signor et al. 1999a . Signor et al. 1999a now show that kinesin-II can also be a cargo as well as a transporter. These researchers have examined intraflagellar transport in the sensory cilia found in the chemosensory neurons of C. elegans . They have used GFP fusions with the kinesin-II KAP subunit and two of the IFT raft subunits, OSM6 and OSM1, to visualize anterograde and retrograde transport of these proteins in dendrites and the nonmotile sensory cilia that extend out from the distal end of the dendrites. Using a fluorescence-based assay with wild-type organisms, KAP::GFP, OSM6::GFP, and OSM1::GFP were seen moving bidirectionally in both the dendrites and the sensory cilia of chemosensory neurons; the cilia of these cells are nonmotile axonemal structures that extend out from the dendrites and are exposed to the environment. Since the microtubules of the axoneme are unidirectional, with their plus ends emanating away from the dendrite toward the distal tip of the cilium, the anterograde transport of 0.7 μm/s is very likely to be driven by the plus end–directed kinesin-II. The retrograde transport of 1.1 μm/s, however, requires a minus end–directed motor, indicating that kinesin-II is acting as a cargo during retrograde IFT. To identify the retrograde IFT motor in the nematode, Signor et al. 1999a visualized IFT in a severe CHE-3 mutant that is effectively null for cytoplasmic dynein 1b. Severe mutations in the CHE-3 cytoplasmic dynein heavy chain completely abolish retrograde IFT along the cilium but do not interrupt anterograde IFT or the bidirectional dendritic transport of kinesin-II and IFT proteins. These results strongly suggest that CHE-3 cytoplasmic dynein serves as the retrograde IFT motor in the nematode and confirm earlier studies in Chlamydomonas that had implicated cytoplasmic dynein 1b as the retrograde IFT motor . It also stands to reason that kinesin-II actively transports the cytoplasmic dynein 1b out to the tip as it seems unlikely that simple diffusion could keep up with the rapid pace of retrograde transport (∼3.5 μm/s in Chlamydomonas and 1.1 μm/s in C . elegans ). In conclusion, the studies summarized above shed new light on the comings and goings of kinesin-II. Ray et al. 1999 provide evidence that kinesin-II is carrying the normally soluble ChAT through axons of cholinergic neurons while Signor et al. 1999a have shown that kinesin-II becomes the cargo during retrograde IFT. Through the combined studies in Chlamydomonas , C . elegans , and other model organisms, it is now clear that kinesin-II and cytoplasmic dynein 1b are integral and essential parts of an ancient and conserved intraflagellar transport system designed to assemble and maintain ciliary and flagellar organelles. Indeed, due to the intrinsic polarity of the axonemal microtubules and tight space restrictions, retrograde IFT is required to prevent a serious congestion of IFT rafts at the distal tip. Thus, kinesin-II and cytoplasmic dynein 1b act in concert to keep intraflagellar traffic flowing. Likewise, in the neuronal axons and dendrites, where kinesin-II and other kinesins have been adapted for anterograde transport, retrograde transport may be required to remove anterograde motors and other material from the distal ends of these cells and return them back to the cell body. | Study | biomedical | en | 0.999998 |
10545492 | Carbohydrates are major components of the outer surface of mammalian cells and these carbohydrates are very often characteristic of cell-types and developmental stages . One of such cell type–specific carbohydrates is sialyl Lewis X, NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAc→R. Sialyl Lewis X and Lewis X, Galβ1→4(Fucα1→3)GlcNAc, were originally discovered as differentiation antigens specific to granulocytes and monocytes . These oligosaccharides can be synthesized when α1,3-fucosyltransferase(s) is present . Erythroid cells, which lack the α1,3-fucosyltransferase, in contrast, express α1,2-fucosyltransferase. Resultant H-type oligosaccharides (O blood type), Fucα1→ 2Galβ1→4GlcNAcβ1→R, are converted to A and B blood group antigens by addition of α1,3-linked N -acetylgalactosamine or galactose through erythroid cell specific expression of two unique glycosyltransferases . These results show a typical example of cell type–specific oligosaccharides, of which synthesis is dependent on cell type–specific expression of a unique glycosyltransferase. Specific expression of unique oligosaccharides strongly suggests that these oligosaccharides may serve as cell surface markers. Indeed, rapid and explosive understanding of the roles of sialyl Lewis X and its variants as cell recognition molecules has been taking place recently. In this mini-review, we would like to focus on the roles of this group of carbohydrates and C-type lectins that recognize those carbohydrates . In mammals, lymphocytes circulate in the vascular and lymphatic compartments, allowing maximum exposure of lymphocytes to foreign pathogens. Lymphocytes leave the vascular compartment at lymph nodes, traverse the lymphatic organs, and then return to the vascular system. This directed flow of lymphocytes is dependent on carbohydrate ligands present on specialized endothelial cells, termed high endothelial venules (HEV) 1 . It was discovered that lymphocyte binding to HEV is dependent on sialic acid on HEV and can be inhibited by fucosylated sulfated oligosaccharides . When the homing receptor on lymphocytes, now called L-selectin, was molecularly cloned, its cDNA sequence predicted a carbohydrate-binding domain at its NH 2 -terminus. This carbohydrate-binding domain is similar to that of the hepatic lectin, which recognizes asialo plasma glycoproteins . Carbohydrate-binding activity of these lectins is dependent on Ca ++ , thus they are collectively called C-type lectin . Counterreceptors on HEV capture circulating lymphocytes via L-selectin–dependent adhesion, leading to transmigration . L-selectin was found to be required for this process . In parallel to this, two other adhesion molecules playing critical roles in the interaction between leukocyte-endothelial cells were identified. One of them, P-selectin, appears on the cell surface of platelets upon stimulation. Similarly, P-selectin, then E-selectin, another adhesive molecule, appears on the cell surface of endothelial cells after stimulation by inflammatory agents. E- and P-selectin also contain a carbohydrate-binding domain, which is highly homologous to that of L-selectin, having ∼60% identity at the amino acid levels among these molecules . Once these adhesion molecules were recognized as carbohydrate-binding proteins, carbohydrate-ligands for E- and P-selectin were immediately identified as sialyl Lewis X , previously shown to be present on neutrophils. During inflammation, leukocytes expressing sialyl Lewis X are recognized by P- or E-selectin and such initial adhesion results in the slowing down of leukocytes, a “rolling effect.” This rolling effect leads to vascular extravasation of leukocytes, similar to the process shown in Fig. 1 . The above results so far indicate that all selectins recognize sialyl Lewis X as ligands. How then could each interaction achieve a more specific interaction? First, glycoproteins presenting sialyl Lewis X oligosaccharides are specific for each cell-type, providing unique interactions to each cell-type. L-selectin carbohydrate ligands are presented by GlyCAM-1, CD34, MAdCAM-1, and other glycoproteins, all of which contain mucin-type O -linked oligosaccharides. P-selectin carbohydrate ligands are presented by PSGL-1, which also contains mucin-type O -linked oligosaccharides . In O -linked oligosaccharides of blood cells, the sialyl Lewis X capping structure is formed in core 2 branched structures, NeuNAcα2→3Galβ1→4(Fucα1→3) GlcNAcβ1→6(±NeuNAcα2→3Galβ1→3)GalNAcα1→ Ser/Thr . Core 2 branched oligosaccharides are synthesized by core 2 β1,6- N -acetylglucosaminyltransferase, C2GnT . Sialyl Lewis X is synthesized by sequential addition of α2,3-linked sialic acid and α1,3-linked fucose to its N -acetyllactosamine. The importance of α1,3-linked fucose in all selectin ligands was demonstrated by the generation of mutant mice defective with α1,3-fucosyltransferase VII . The most recent studies using the C2GnT knockout mice demonstrated that the majority of L- and E-selectin ligands on neutrophils were abolished after core 2 branched oligosaccharides became absent, more so than for P-selectin ligands . These results establish that the majority of selectin ligands on neutrophils are synthesized on core 2 branched oligosaccharides. Mucin-type O -glycans are presented as a cluster, thus presenting multiple sialyl Lewis X oligosaccharides to each selectin. Such a multiple presentation should increase binding to a selectin since selectin binding to a monovalent ligand is relatively low. This is more evident when one considers that selectins may be present as oligomers as shown for P-selectin , enabling their binding to multimeric ligands with much higher avidity. The specific interaction can also come from additional modifications such as sulfation. PSGL-1 contains three unique tyrosine residues close to the NH 2 -terminal mucin-like domain. Sulfation of one of these tyrosine residues in addition to sialyl Lewis X oligosaccharide(s) is essential for P-selectin binding to PSGL-1 . L-selectin, on the other hand, binds much better to sulfated forms of sialyl Lewis X oligosaccharides than non-sulfated forms . Most recently, one of the sulfotransferases, LSST, was molecularly cloned, demonstrating that LSST adds a sulfate to N -acetylglucosamine, resulting in the formation of sialyl 6-sulfo sialyl Lewis X, NeuNAcα2→3Galβ1→4(6-sulfo) (Fucα1→3)GlcNAcβ1→6[Galβ1→3]GalNAcα1→Ser/Thr . LSST preferentially acts on mucin-type core 2 branched O -glycans and the resultant ligands expressed on L-selectin counterreceptor CD34 enhance binding to L-selectin when assayed under shear force that mimics vascular flow, compared with non-sulfated sialyl Lewis X . Interestingly, 6-sulfo sialyl Lewis X formed mostly on N -glycans of CD34 by another sulfotransferase, GlcNAc-6-sulfotransferase , did not exhibit the same enhancing effect. These results reinforced the importance of sulfo sialyl Lewis X in core 2 branched mucin-type O -linked oligosaccharides as L-selectin ligand . In contrast, E-selectin binds to sialyl Lewis X oligosaccharides regardless of the presence or absence of additional sulfation. These results indicate that tyrosine sulfation and sulfation of sialyl Lewis X oligosaccharides provide specificity in P- and L-selectin recognition, respectively, achieving specific carbohydrate–protein interactions for each selectin. There are a few issues to be resolved in L-selectin ligand. First, lymphocyte homing in mice defective with C2GnT was only marginally impaired while L-selectin ligand functionality in neutrophils in the same mice was completely abolished . This finding indicates that either an additional C2GnT is present in HEV compensating for the loss of C2GnT or oligosaccharides other than core 2 branched O -glycans also serve as L-selectin ligands in HEV. Indeed, the cDNA encoding a novel C2GnT (called C2GnT-mucin type) has been cloned . This enzyme has also been found to be present in HEV (unpublished data). The second possibility was suggested by a report that some L-selectin ligands may be present in N -glycans or glycans resistant to digestion with O -sialoglycoproteinase, which cleaves mucin-type glycoproteins . Addressing these issues will likely reveal entirely new aspects of selectin ligands. The second point is that sulfation of sialyl Lewis X in HEV is more intricately regulated than we know now. Structural analysis of GlyCAM-1 oligosaccharides indicated the presence of 6′-sulfo sialyl Lewis X, NeuNAcα2→3(sulfo→6)Galβ1→4GlcNAc→R and possibly 6,6′- bi sulfo sialyl Lewis X, NeuNAcα2→3(sulfo→6) Galβ1→4(sulfo→6)GlcNAc→R, in addition to 6-sulfo sialyl Lewis X, in core 2 branched oligosaccharides . 6′-Sulfo sialyl Lewis X expressed on the cell surface supported the adhesion to L-selectin . A disulfated form of lactose inhibited L-selectin binding to GlyCAM-1 better than its monosulfated form . MECA-79 antibody that specifically detects HEV and inhibits the L-selectin-mediated binding in vivo and in vitro , recognizes a sulfated form of mucin-type O -glycans . Expression of LSST or GlcNAc-6-sulfotransferase in combination with other known sulfotransferases failed to form the MECA-79 antigen . These results suggest that 6,6′- bi sulfo sialyl Lewis X or other mono- and multiple sulfated sialyl Lewis X may play a role as an L-selectin ligand. It will be important to identify additional sulfotransferases that form such L-selectin ligands. The roles of C-type lectins is not limited to selectin-carbohydrate interactions. Indeed, various receptors were demonstrated as C-type lectins such as an IgE Fc receptor (Fc∈RII) and pulmonary surfactant . A C-type lectin domain of NK cell receptor(s) was shown to bind to fucoidan , which is also an inhibitor for L-selectin binding. It was also shown that melanoma cells densely expressing sialyl Lewis X oligosaccharides in short N -glycans after the transfection with an α1,3-fucosyltransferase can be targeted by NK cells, most likely through NK cell receptors of C-type lectin . On the other hand, B16 melanoma cells, which expressed moderate amounts of sialyl Lewis X in poly- N -acetyllactosamines long chain glycans after the same transfection, were highly metastatic probably through interaction with a C-type lectin on lung endothelial cells . This finding is consistent with the previous reports that carcinoma cells are enriched with sialyl Lewis X in poly- N -acetyllactosamines . These results provide a clear-cut example that a subtle difference in carbohydrate ligands results in entirely different biological consequences. The carbohydrate-recognition domain of a NK cell receptor binds to either MHC class I peptide or fucoidan . Similar and dual binding of C-type lectins was demonstrated in C-type lectin domains of proteoglycans such as brevican. In one of these cases, the C-type lectin domain binds to tenasin-R or HNK-1 glycan , another sulfated glycan uniquely present in neural and NK cells. The binding to one may preclude another from binding, providing another example of dual recognition by a C-type lectin. We expect that more examples will follow. In summary, this overview presents clear examples where carbohydrate-protein (C-type lectins) interaction plays a critical role in cell–cell interaction. The results demonstrate that the interaction of sialyl Lewis X oligosaccharides with a specific C-type lectin plays a critical role in cell–cell interaction. At the same time, modification, such as sulfation of sialyl Lewis X, its multiple presentation, scaffold of carbohydrates and the structure of the glycoprotein itself, all contribute to the specificity of the interaction, which ultimately regulates the biological function of sialyl Lewis X. Efforts to identify the precise structure of oligosaccharides recognized by a given C-type lectin involved in each case will provide critical understanding for the roles of oligosaccharides in cell–cell interactions. Such studies undoubtedly will enhance our understanding of the mechanisms dictating how cell–cell interactions can be finely tuned. | Study | biomedical | en | 0.999999 |
10545493 | mAb 6C4 was isolated in a screen of hybridoma cell lines which produce antibodies that recognize C . elegans embryos. Hybridoma cell lines were generated and ascites fluid was produced as described . Bristol strain N2 was cultured as described by Brenner 1974 . Embryos were prepared by hypochlorite treatment and attached to coverslips (type 1.5) pretreated with 3-aminopropyltriethoxysilane (Sigma Chemical Co.), and overlaid with an untreated coverslip. The coverslips were quick-frozen on dry ice and the overlaying coverslip rapidly removed. Fixation was in N,N -dimethylformamide or methanol at −20°C for 10 min. Slides were blocked for 30 min in PBS with 2 mM MgCl 2 , 0.1% Tween 20, and 3% BSA. Immunostaining was performed with mAb 6C4 ascites fluid at 1:500 dilution in blocking solution. Primary antibody was incubated for 1 h at room temperature. Coverslips were then washed three times in block for 5 min each before incubation with 10 μg/ml rhodamine-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Coverslips were then washed three times for 5 min each, with 0.05 mg/ml 4′,6-diamidino-3-phenylindole dihydrochloride (DAPI) included in the first wash. Coverslips were mounted on slides with 100% glycerol. Double labeling experiments were performed under similar conditions, except either an anti–β-tubulin antibody (Amersham Pharmacia Biotech), an anti–HCP-3 antibody , or MPM-2 antibody (DAKO) at 10 μg/ml was included with mAb 6C4. Detection was by rhodamine-conjugated goat anti–rabbit IgG, goat anti–mouse IgG, or fluorescein-conjugated goat anti–mouse IgM. Samples were examined by three-dimensional multiple wavelength fluorescence microscopy using Deltavision (Applied Precision). Images were collected at the indicated wavelengths for 30 0.2-μm optical sections per nucleus and were subsequently deconvolved mathematically . The limit of detection (resolution between two points) was 90 nm. Data were examined as either optical sections or as a projection of the entire stack. The mixed stage C . elegans cDNA library in λgt11 was a gift from Dr. Pete Okema (University of Illinois at Chicago, Chicago, IL). Immunoscreening of the cDNA library was carried out by the method of Young and Davis 1983 , using mAb 6C4 ascites fluid at 1:200 dilution. Detection of immunopositive plaques was by Vectastain ABC (Vector Labs Inc.). Oligos forward T7 (TAATACGACTCACTATAGGGggtggcgacgactcctgg), forward (ggtggcgacgactcctgg), reverse T7 (TAATACGACTCACTATAGGGttgacaccagaccaactgg), and reverse (ttgacaccagaccaactgg) were used to generate PCR products corresponding to the cDNA inserts obtained from the expression screening. This region included the COOH-terminal 324 amino acids of HCP-1 and 3′UTR. Each T7 oligo contains a T7 polymerase promoter (shown in capital letters) and sequence complementary to the flanking polylinker region of λgt11. Oligos HCP2-1 T7 (TAATACGACTCACTATAGGGgacctgaatgcgaagcttg), HCP2-1 (gacctgaatgcgaagcttg), HCP2-2 T7 (TAATACGACTCACTATAGGGgctggttgcagtttgagcgg), and HCP2-2 (gctggttgcagtttgagcgg) were used to PCR amplify a region of the HCP-2 mRNA corresponding to the COOH-terminal 384 amino acids of HCP-2, from total RNA, after first strand cDNA synthesis as described . 1 μg of PCR product was used to synthesize double-stranded RNA (dsRNA) using T7 polymerase as described . dsRNA was resuspended in water at a concentration of 5 mg/ml. dsRNA derived from the COOH-terminal region of HCP-1 or from the corresponding region of HCP-2 was injected into the syncytial gonad of wild-type hermaphrodites as described . We generated a mouse mAb, 6C4, that stains mitotic chromosomes in C . elegans . Holocentric chromosomes orient perpendicular to the spindle axis during metaphase and anaphase, with their respective kinetochores facing the spindle . Immunofluorescence with mAb 6C4 showed reactivity on the poleward face of metaphase chromosomes . This staining pattern suggests that mAb 6C4 recognizes a protein important for holocentric chromosome structure and function. To determine the mAb 6C4 staining pattern on individual chromosomes, we used deconvolution microscopy to generate high resolution images of the nuclei of two-cell embryos stained with mAb 6C4 and DAPI . The relative time of each nucleus in the cell cycle was inferred by comparing the staining patterns of the AB and P1 blastomeres; the AB blastomere division occurs before that of the P1 blastomere . The morphology of DAPI staining and presence of a nuclear membrane were used as additional landmarks of cell cycle progression. In these experiments, interphase nuclei had no detectable mAb 6C4 reactivity. In addition, nuclei that are in the earliest stages of chromosome condensation do not stain with mAb 6C4 (data not shown). Staining with mAb 6C4 was first observed in prophase nuclei that contain condensed chromosomes. At this stage, mAb 6C4 staining was observed as dots distributed throughout the nucleus; many of these dots colocalize with the chromosomes . These chromosome-associated dots are widely dispersed along the entire length of each chromosome. Later in prophase, mAb 6C4–stained structures located on opposite sides of each chromosome . In metaphase, when the chromosomes are aligned at the equatorial plane, chromosomes were oriented with each mAb 6C4–stained structure facing one centrosome . At anaphase, sister chromatids separated with the mAb 6C4–stained side on the poleward face of each sister chromatid . No mAb 6C4 staining was detectable by late anaphase. These results show that there is dynamic localization of the mAb 6C4 antigen to chromosomes throughout mitosis. During meiosis, C . elegans chromosomes lack a trilaminar kinetochore structure, and attachment to the meiotic spindle is mediated through the chromosome ends . We stained adult hermaphrodite gonads with mAb 6C4. No mAb 6C4 nuclear staining was detected in the mitotic region of the gonad of the hermaphrodite nor in oocytes arrested at diakinesis of prophase I (data not shown). After fertilization, embryos exit prophase I arrest and undergo two consecutive rounds of division to form two polar bodies. Staining with mAb 6C4 was detected on meiotic chromosomes after fertilization and before first polar body formation . The mAb 6C4 staining pattern was observed as a halo surrounding the meiotic chromosomes. To identify the antigen recognized by mAb 6C4, we screened a λgt11 C . elegans cDNA expression library with mAb 6C4. Three overlapping cDNAs were isolated that correspond to a predicted gene, ZK1055.1. We refer to this gene as hcp-1 . The hcp-1 gene can encode a 1,475–amino acid protein predicted to be composed primarily of coiled-coil domains . HCP-1 has a direct repeat of 132 amino acids that is 45% similar to a direct repeat of 179 amino acids present in CENP-F . A search of C . elegans sequence databases with the HCP-1 amino acid sequence revealed a second C . elegans coiled-coil protein, T06E4.2, that is 54% similar to HCP-1. We refer to T06E4.1 as HCP-2. The highest levels of similarity between HCP-1 and HCP-2 is seen at the two termini. HCP-2 lacks the tandem repeats observed in HCP-1 and CENP-F. A low level (<20%) of similarity is observed between HCP-1 and HCP-2 with several other coiled-coil proteins in the databases, probably due to conservation of the coiled-coil structure. To test whether hcp-1 encodes the antigen recognized by mAb 6C4, we characterized mAb 6C4 immunofluorescence in embryos in which HCP-1 expression has been reduced by RNA interference (RNAi) . RNAi has proved useful for studies of both maternally and zygotically expressed proteins. The hcp-1 (RNAi) embryos were fixed and stained with mAb 6C4. In these experiments, 77% of the hcp-1 (RNAi) embryos failed to stain with mAb 6C4. In contrast, only 0–2% of embryos from control injections with water or hcp-2 (RNAi) embryos failed to stain with mAb 6C4 . In embryos with no mAb 6C4 staining, all mitotic cells failed to stain, i.e., the staining was never observed to be mosaic. The failure of hcp-1 (RNAi) embryos to stain with mAb 6C4 was specific for this antibody, because the embryos stained positively with mAb K76, a P granule antibody . These results indicate that HCP-1 is the major antigen for mAb 6C4. The staining pattern observed by mAb 6C4 suggested that HCP-1 might be located at or near the kinetochore on C . elegans chromosomes. Currently, no proteins have been localized to the kinetochore in C . elegans . We tested several anticentromeric antibodies for cross-reactivity in C . elegans , and only MPM-2 was able to cross-react . MPM-2 recognizes a conserved phosphoepitope present during mitosis on many mitotic proteins. Using MPM-2, others found that the most intensely stained structure on metaphase chromosomes in a variety of organisms is the kinetochore . In C . elegans , MPM-2 identifies a structure adjacent to the condensed chromosomes at metaphase that is also recognized by mAb 6C4 . This structure is likely to be the kinetochore, since it is present only on the poleward face of metaphase chromosomes and MPM-2 has been observed to identify the kinetochore in a variety of organisms . To further determine whether HCP-1 is localized to the kinetochore, we used an antibody raised against a centromeric histone H3–variant, HCP-3, in a double labeling experiment with mAb 6C4. HCP-3 was identified as a histone H3–variant in C . elegans , that like the centromeric H3-like proteins, chromosome segregation (CSE) 4 in yeast, and CENP-A in mammals, is localized to the centromere and affects the fidelity of chromosome segregation . Anti–HCP-3 antibody staining is present as continuous lines of reactivity all along each chromosome . This is consistent with the centromere being present along the long axis of each holocentric chromosome . Staining of each chromosome with mAb 6C4 is also present along each chromosome and adjacent to the centromere, as identified by anti–HCP-3 antibody . Furthermore, this staining is restricted to opposite surfaces of the holocentric chromosome and is analogous to centromere/kinetochore staining of the primary constriction of mammalian chromosomes . The localization of HCP-1 to the region of the chromosome immediately adjacent to the centromere, and its colocalization with an antibody, MPM-2, which recognizes the kinetochore in many organisms, indicates that HCP-1 is likely to be a structural component of the C . elegans kinetochore. Embryos derived from hermaphrodites injected with hcp-1 dsRNA are viable and exhibit no visible phenotype other than loss of mAb 6C4 staining. We considered the possibility that HCP-1 might be functionally redundant with HCP-2. Wild-type staining with mAb 6C4 was observed with hcp-2 (RNAi) embryos which were also viable ( Table ). However, coinjection of both hcp-1 and hcp-2 dsRNAs resulted in 99% lethality of progeny embryos ( Table ). The synthetic lethality of hcp-1 (RNAi) and hcp-2 (RNAi), and the localization of HCP-1 to the kinetochore region of mitotic chromosomes, together suggest that both HCP-1 and HCP-2 function in mitotic chromosome segregation. To determine whether the synthetic lethality correlates with a defect in chromosome segregation, we stained hcp -1 (RNAi)/ hcp -2 (RNAi) embryos with an antitubulin antibody and DAPI. In these experiments, we observed that 80% ( n = 60) of all anaphases showed signs of defective chromosome segregation, including lagging chromosomes and anaphase bridges . This defect was observed as early as the first mitotic cleavage . In these anaphase nuclei, the spindle as observed by antitubulin staining appeared wild-type, in that a normal bipolar orientation is present . As observed by DAPI, chromosome condensation appeared to also not be grossly altered, as chromosomal structures are visible . In many instances, some chromosomes were not located between the centrosomes; for example, Fig. 6 C shows an early embryo in which the four AB xx blastomeres are in prophase. Several chromosomes are located outside of the spindles and along the previous spindle axis, indicating that they failed to segregate at the previous division . The hcp-1 (RNAi)/ hcp-2 (RNAi) embryos arrested with ∼50–100 cells. At this stage, the DNA content of individual nuclei was variable, suggesting that some nuclei have more and others have less than the diploid set of chromosomes . In contrast, DNA content in wild-type embryos was uniform among nuclei. These results further support the idea that HCP-1 and HCP-2 function together in chromosome segregation. We have identified mAb 6C4, which has a staining pattern on mitotic chromosomes in C . elegans that is analogous to that of anticentromeric antibodies in mammals. Anticentromeric antibodies stain two structures which flank mitotic chromosomes in mammalian cells . Similarly, mAb 6C4 recognizes two structures which flank mitotic chromosomes in C . elegans . These two structures are oriented towards the centrosomes at metaphase and anaphase in both mammals and C . elegans . This relationship of the C . elegans and mammalian staining patterns is consistent with the structural characterization of their chromosomes by EM. In mammals, the centromere, as defined by the presence of a kinetochore, is localized to the primary constriction . Conversely, in C . elegans the kinetochore is present on opposing sides and extends nearly the length of the chromosome . These similarities suggested that mAb 6C4 may be used to identify the C . elegans kinetochore. To test whether mAb 6C4 stains at or near the kinetochore, we performed a colocalization experiment using an antibody (MPM-2) previously shown to stain the kinetochore in many different organisms, and an antibody directed against a centromeric histone H3–variant, HCP-3, . MPM-2 recognizes a highly conserved phosphoepitope present on kinetochore-associated proteins during mitosis . MPM-2 antibody did cross-react in C . elegans , further indicating the conserved nature of its epitope. MPM-2 staining was observed to be strongest in the region adjacent to the mitotic chromosomes in a pattern overlapping with that observed for mAb 6C4. This overlap was specific for the region adjacent to the mitotic chromosomes, although both antibodies showed staining not associated with the chromosomes. Elimination of the mAb 6C4 antigen by RNAi did not affect the staining with MPM-2, indicating that the colocalization is not due to the presence of the MPM-2 epitope on HCP-1 (data not shown). Thus, the colocalization of MPM-2 and mAb 6C4 is likely the result of their epitopes being present at the same cytological structure, the kinetochore. Furthermore, mAb 6C4 staining is present adjacent, but not coincident with the staining of chromatin by anti–HCP-3 antibody. Both staining with anti–HCP-3 and mAb 6C4 is analogous to that observed on monocentric chromosomes by anticentromeric antibodies that recognize different regions of the trilaminar kinetochore . Taken together, these two colocalization experiments indicate that the staining pattern observed with mAb 6C4 along mitotic chromosomes in C . elegans represents the kinetochore of the holocentric chromosomes. We have used mAb 6C4 to identify two proteins, HCP-1 and HCP-2, which together are involved in kinetochore function. When we reduced the expression of both HCP-1 and HCP-2 by RNA-mediated inhibition, we observed that embryos arrested with ∼50–100 nuclei containing variable amounts of DNA. By observing embryos at earlier stages, we observed that chromosomes were often found to be unattached to the mitotic spindle. Not all chromosomes failed to attach to the spindle. This may reflect low levels of expression of HCP-1 or HCP-2. The inability of chromosomes to attach to the mitotic spindle does not appear to be the result of a defect in chromosome condensation. When viewed by DAPI staining, chromosomes are observed and appear normal when compared with wild-type. This is consistent with the localization of HCP-1 to the chromosomes occurring after chromosome condensation has begun, suggesting HCP-1 is not generally required for chromosome condensation. The hcp-1 (RNAi)/ hcp-2 (RNAi) defects are similar to those observed when human centromere components are inhibited, which include lagging chromosomes in anaphase and inefficient spindle attachment to chromosomes . These defects are also similar to defects observed when expression of the C. elegans homologue of the Zeste White 10 protein (CeZW10) is reduced by RNAi . Homologues of ZW10 have been localized to the kinetochore in both Drosophila and humans . Mutations in Drosophila zw10 result in aberrant chromosome segregation, which is observed during anaphase as lagging chromatids or chromosomes remaining in the vicinity of the metaphase plate during anaphase. Reduction of CeZW10 expression in C. elegans embryos results in a similar phenotype in which chromosomes are often observed to be improperly attached to both spindles and results in anaphase bridges during anaphase. Anaphase bridges and even lagging chromatids or chromosomes are observed with hcp-1 (RNAi)/ hcp-2 (RNAi) embryos, suggesting that like ZW10, HCP-1 and HCP-2 are required for proper kinetochore function during mitosis. The dynamic mAb 6C4 staining pattern on mitotic chromosomes suggests that there are intermediate stages of holocentric kinetochore assembly. As cells progress from early to late prophase, the mAb 6C4 staining pattern changes from discontinuous points dispersed along chromosomes to structures on opposite sides of each chromosome. The temporal relationship of these images indicates that the dispersed dots may be used to assemble the larger structures seen at late prophase. The visualization of a disperse mAb 6C4 staining pattern is consistent with a model in which there are discrete regions of C . elegans chromosomes that act as the primary points of spindle microtubule capture. This model is similar to the repeat subunit model proposed for the mammalian centromere, which proposes that the centromere is composed of short segments distributed along the DNA that bind spindle microtubules . These segments are brought into parallel register as a result of chromatin condensation to form the observed kinetochore structure. The C . elegans chromosomes may also contain such repeats discontinuously distributed along the entire chromosome. Our results support the model suggested by Albertson and Thomson 1982 that the holocentric chromosome may be thought of as an extended centromere. | Study | biomedical | en | 0.999996 |
10545494 | A full-length mouse pericentrin was constructed using a three piece cloning strategy. Pericentrin clone λpc1.2 was excised with restriction enzymes PvuI and EcoRV. The 5′ end of the final clone was amplified by PCR using VENT polymerase from clone PCR 1 using a 5′ primer (5′-CCGATATCAGATGGAAGACG-3′) with an EcoRV restriction enzyme site and a 3′ primer (5′-GTTTGGGAGGTAGAGGCT-3′) with a PvuI site. The amplified PCR product was digested with EcoRV and PvuI. Plasmid pcDNAI/Amp (Invitrogen Corp.) was used to construct a vector with 13 amino acids of hemagglutinin (HA) protein inserted at the HindIII site in the polylinker (a gift of Michael Green, UMass Medical School, Worcester, MA). The vector was linearized with EcoRV and ligated to form the full-length pericentrin, as described . The correct orientation of the fragments was confirmed by PCR using the T7 vector primer and the 5′-directed pericentrin primer. The sequence of the clone was confirmed using an automated sequencer (Bio-Rad Laboratories). The preparation of cDNAs encoding full-length rat p150 Glued , the human dynamitin , rat myc-tagged cytoplasmic dynein intermediate chain (DIC) 2C , and rat FLAG-tagged cytoplasmic heavy chain have been described previously. COS-7 cells were cultured as described (American Type Culture Collection) with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma Chemical Co.). Cells were grown on 12-mm round glass coverslips in 35-mm culture dishes (Falcon Plastics) and transfected with 2 μg of plasmid DNA (HA-tagged pericentrin [HA-Pc], β-galactosidase, pHAI, or no DNA) using lipofectamine (GIBCO BRL); transfection efficiency was ∼15%. Cells were fixed 35–42 h after transfection and processed for immunofluorescence staining, immunoprecipitation, metabolic labeling, or Western blotting. Cell viability was determined using mitotracker (Sigma Chemical Co.), which measures energy-dependent electron transport in mitochondria. Cell growth was determined by measuring the ratio of transfected cells to the total cell population; there was little change in this ratio over a 50 h time period. Affinity-purified rabbit IgG was prepared from sera raised against the COOH terminus of pericentrin and used at 1:1,000 for immunofluorescence microscopy and Western blotting. Anti-HA mAbs (12CA5) were obtained from Berkeley Antibody Co., Inc., and anti-HA polyclonal antibodies were a gift from Joanne Buxton . Antibodies to α and γ tubulin, mouse IgG, and rabbit IgG were obtained from Sigma Chemical Co. Antibodies to β-galactosidase were from Boehringer Mannheim Corp. Antibodies to the following proteins were also used in these studies under conditions described in the accompanying references: dynein heavy chain , DIC L5 , 74.1 , dynamitin , p150 glued , anti-p58 Golgi protein , and CENP-E . Fluorescein (FITC) and cyanine (cy3)-conjugated IgGs were obtained from Jackson ImmunoResearch Laboratories, Inc. HRP-conjugated IgGs were from Nycomed Amersham Inc. Antibodies were used alone or in combination as described in the text. Immunofluorescence microscopy was performed essentially as described . Unless otherwise stated, COS-7 cells expressing HA-Pc, β-galactosidase, pHAI, or mock transfected were fixed in 100% methanol at −20°C. Where indicated, cells were detergent-extracted to remove cytoplasmic protein before fixation (0.5% TX-100 in 80 mM Pipes, pH 6.8, 5 mM EGTA, 1 mM MgCl 2 , for 1 min). In most cases, monoclonal or polyclonal HA antibody was detected with FITC-labeled secondary antibody, and antibodies used in colabeling experiments were detected with cy3 secondary antibodies. In all cases, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect chromatin. Cells were observed using an Axiophot fluorescence microscope with a 100× objective (Carl Zeiss Inc.). Quantification of centrosomal staining in mitotic cells and DNA (DAPI) was performed as described . In brief, the total fluorescence from centrosomes and nuclei in individual cells was determined. Background values from three positions in the cytoplasm and camera noise (dark current) were subtracted (<10% of total). For centrosome staining, fluorescence signals were obtained from only one centrosome per mitotic cell, to avoid photobleaching. Cells with low, intermediate, and high expression levels were included in all analyses. For coexpression studies , HA-Pc and dynein, or dynactin cDNAs were cotransfected into COS-7 cells and processed 38–46 h later. Cells were washed in PBS, lysed in modified RIPA buffer at 4°C for 20 min (150 mM NaCl, 50 mM Tris, pH 8.0, 1 mM EGTA, 1% IGEPAL) with leupeptin, aprotinin, and AEBSF (Boehringer Mannheim Corp.), and precleared. Monoclonal anti-HA bound to protein G beads (Pharmacia Biotech) was added to lysates at 4°C for 12 h, and beads were collected and washed five times with modified RIPA buffer. Proteins were exposed to SDS-PAGE and transferred to PVDF membranes (Millipore Corp.). The presence of dynein/dynactin subunits was assayed by Western blot with anti-myc, anti-p50, and anti-p150 antibodies. COS-7 cells were transferred to methionine- and serum-deficient DME (GIBCO BRL) containing 50–100 uCi of [ 35 S]methionine (New England Nuclear). They were labeled for 4 or 18 h , washed in PBS, and lysed in 50 mM Tris, pH 7.5, 137 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 4 μg/ml aprotinin, 4 μg/ml leupeptin, 4 μg/ml antipain, 12.5 μg/ml chymostatin, 5 mM iodoacetamide, 130 μg/ml caproic acid, 12 μg/ml pepstatin, 200 μg/ml P-amino-benzamidine, and 1 mg/ml BSA. Protein G beads preblocked with COS-7 cell extract made from untransfected, unlabeled cells (4 h at 4°C), were added to precleared 35 S-labeled extracts with primary antibody, and immunoprecipitates were processed as described above. Dried gels or membranes were exposed to X-OMAT film (Kodak) for 24–48 h. COS-7 cells expressing HA-Pc or mock transfected were treated with nocodazole (10 μg/ml) for 1 h at 37°C to depolymerize microtubules. After removal of the drug, cells were incubated for 3 min to allow microtubules to regrow, then fixed in methanol and stained with α tubulin to reveal nucleated microtubules, as described previously . Previously, we demonstrated that functional abrogation of pericentrin disrupts centrosome and spindle organization in several systems . Based on these observations, we reasoned that an artificial elevation of pericentrin levels would provide additional information on protein function and interaction. To this end, we constructed and expressed an HA-Pc in COS-7 cells, and examined centrosome and spindle composition and function. As expected, HA-Pc had an electrophoretic mobility of ∼220 kD and was found in both Triton X-100 soluble and insoluble fractions . Immunofluorescence analysis demonstrated that the more abundant detergent soluble fraction was distributed throughout the cytoplasm , whereas the detergent insoluble fraction colocalized with γ tubulin at centrosomes . Centrosome localization of HA-Pc was unaltered when microtubules were depolymerized, suggesting that the protein was an integral component of centrosomes and not simply bound there by microtubules (data not shown). The organization of microtubules in interphase HA-Pc expressing cells was indistinguishable from control cells . Moreover, there was no detectable difference in microtubule nucleation from centrosomes . The most dramatic consequence of HA-Pc expression was disruption of mitotic spindle organization . A significant fraction of mitotic COS-7 cells at all expression levels exhibited spindle defects (75.7 ± 6.1%, n = 423), compared with nontransfected cells (2.5 ± 1.5%, n = 598) and vector DNA transfected cells (3.0 ± 1.0, n = 201). Three categories of spindle defects were observed. Spindles with structural defects were detected in 36.2% of transfected cells and included multipolar, monopolar, and distorted spindles . Mispositioned spindles were observed in 22.0% of the cells, and were often positioned far from the cell center . Spindles with misaligned, missegregated, and mono-oriented chromosomes were commonly observed . Spindle defects occurred alone or in combination. Despite the presence of improperly attached chromosomes, HA-Pc cells progressed through mitosis and were frequently observed in later stages of mitosis with missegregated chromosomes . The percentage of mitotic figures in the population of HA-Pc–expressing cells (3.1 ± 0.9%, n = 3,490) was not significantly different from control cells transfected with other constructs or mock transfected cells (2.9 ± 1.0 to 4.4 ± 2.1%, n = 5,002), and the cell viability and growth rate appeared unchanged. Nuclei exhibited a remarkably wide variation in DNA content. Values ranged from zero to five times those of controls , demonstrating that the cells were becoming aneuploid. From this analysis, we conclude that pericentrin overexpression causes multiple mitotic spindle defects leading to chromosome missegregation and aneuploidy. The spindle defects in pericentrin-overexpressing cells were similar to those previously observed in cells overexpressing the dynamitin subunit of dynactin . Dynactin is a protein complex which regulates the function of cytoplasmic dynein, a minus end microtubule motor protein involved in numerous physiological processes . Dynein and dynactin have been localized to prometaphase kinetochores, centrosomes, spindle poles, and the plasma membrane . Overexpression of dynamitin disrupts the dynactin complex, releases cytoplasmic dynein from mitotic kinetochores, disrupts mitosis, and alters the distribution of membranous organelles, including the Golgi complex . To test the possibility that cytoplasmic dynein or dynactin contributed to the pericentrin overexpression phenotype, we examined the distribution of these protein complexes in pericentrin-overexpressing cells. The level of cytoplasmic dynein immunoreactivity in mitotic cells was dramatically reduced at spindle poles . The motor appeared to be specifically displaced from spindle poles and not simply masked from antibody access for several reasons. First, diminished dynein staining was detected with two independent antibody preparations raised against the DIC (L5, polyclonal and 74.1, monoclonal). Second, control cells expressing β-galactosidase or untransfected cells had normal levels of dynein at their poles . Third, there was no detectable change in the distribution and abundance of several other centrosome and spindle pole components. The centrosome localization and levels of the dynactin subunits, dynamitin and p150 glued , did not appear to be altered, although there was some variability in p150 glued levels in prometaphase . There was no apparent change in the level of γ tubulin at individual spindle poles, even in cells with multiple poles . This suggests that multipolar spindles have normal centrosomes at their poles, each with the appropriate amount of γ tubulin (see Discussion). The spindle pole protein, NuMA, also appeared to be localized normally to poles of mitotic spindles . Cytoplasmic dynein was also dramatically reduced at kinetochores . In contrast, kinetochore localization and levels of dynactin and the kinesin-related protein, CENP-E , both appeared unchanged. Consistent with defects in the Golgi complex induced by overexpression of the dynamitin subunit of dynactin , HA-Pc overexpression caused dispersal of Golgi elements. This was observed by immunostaining with antibodies to the Golgi protein, p58 . In adjacent nontransfected control cells, Golgi complexes had the characteristic tightly focused appearance and were found in the perinuclear region of the cells . Disruption of the Golgi complex was also observed using a green fluorescent protein (GFP)-tagged N-acetylglucosamine transferase in cotransfection experiments with pericentrin (data not shown). Golgi complex dispersal did not appear to result from impaired microtubule integrity, as no detectable changes in the microtubule network were observed . The loss of cytoplasmic dynein from spindle poles and kinetochores, and the abrogation of cellular functions mediated by dynein (spindle positioning, Golgi complex organization) suggested that overexpressed pericentrin sequestered the motor in the cytoplasm. This was tested directly by coimmunoprecipitation assays. Antibodies to both DIC and DHC precipitated HA-Pc , whereas a control IgG preparation did not . Conversely, antibodies to HA, but not to control IgGs, precipitated DIC . Under the same conditions, antibodies to dynactin components (dynamitin and p150 glued ) did not precipitate detectable amounts of HA-Pc , although they immunoprecipitated other proteins of the dynactin complex . In cells metabolically labeled with [ 35 S]methionine, HA-Pc was specifically immunoprecipitated with antibodies to DHC, but not to preimmune sera . Moreover, despite very low levels of endogenous pericentrin in nontransfected control cells , we were able to specifically detect DHC after immunoprecipitation of pericentrin from lysates prepared from large numbers of cells . These results suggest that overexpressed pericentrin binds to and sequesters dynein in the cytoplasm, and prevents it from associating with its cellular targets. To determine whether the dynein–pericentrin interaction was direct or indirect, we cotransfected cells with HA-Pc and individual dynein and dynactin subunits, and performed a series of immunoprecipitation and immunoblot analyses. Immunoprecipitation of HA-pericentrin failed to pull down the DHCs and DICs or the dynactin subunits p150 Glued and dynamitin . However, a myc-tagged rat cytoplasmic dynein light intermediate chain (Hughes, S., A. Purohit, S. Doxsey, and R. Vallee, manuscript in preparation) and its COOH-terminal fragment N174 clearly coimmunoprecipitated with HA-pericentrin . When cells cotransfected with the LIC N174 fragment and HA-Pc were labeled with [ 35 S]methionine, the only bands specifically immunoprecipitated with anti-HA antibodies were HA-Pc and N174 . Dynactin did not appear to be required for the pericentrin–LIC interaction since overexpression of dynamitin had no effect on the ability of the proteins to coimmunoprecipitate (data not shown). These results provide strong evidence for a direct interaction between HA-Pc and the light intermediate chain of cytoplasmic dynein. We have found that pericentrin overexpression has profound effects on the organization, positioning, and function of mitotic spindles, and on the organization of the Golgi complex. Several studies show that cytoplasmic dynein is involved in processes affected by pericentrin overexpression . Consistent with a role for cytoplasmic dynein in mediating the pericentrin overexpression phenotype is the reduction of dynein staining intensity at the prometaphase kinetochore and the centrosome/spindle pole. Our immunoprecipitation data further support an interaction between pericentrin and cytoplasmic dynein. Our data indicate that the interaction is direct and specifically mediated by the light intermediate chains of the motor protein complex. Thus, this study provides the first evidence for a dynein–pericentrin interaction, and identifies the first functional role for LICs. The function of the light intermediate chains has been obscure. They have only been identified in cytoplasmic forms of dynein and contain well-conserved P-loop elements of unknown function near their NH 2 termini . Previous studies have implicated a different class of dynein subunit, the intermediate chains, in subcellular targeting. The intermediate chains reside at the base of the dynein complex and interact with the p150 Glued subunit of the dynactin complex . Dissociation of the dynactin complex by dynamitin overexpression was found to release dynein from prometaphase kinetochores. Together, these data supported a role for dynactin in anchoring dynein to at least one form of subcellular cargo through the intermediate chains . This mechanism has received further support from evidence that mutations in zw10, a dynactin-anchoring kinetochore component, also release dynein from the kinetochore . The current studies identify an additional and previously unsuspected mechanism for linking dynein to its cargo. The presence of cytoplasmic dynein, but not dynactin, in pericentrin immunoprecipitates, strongly suggests that dynactin is not necessary for the pericentrin/dynein interaction. Coexpression of recombinant dynein and dynactin subunits with pericentrin reveal a direct interaction with the light intermediate chains, further supporting a dynactin-independent mechanism. Thus, these results identify the light intermediate chains as an additional class of dynein-anchoring or -targeting subunit. Whether these polypeptides serve in a subset of dynein-mediated processes, such as interactions with soluble protein complexes versus membranous organelles or kinetochores, remains to be determined. Whether light intermediate chain-mediated dynein interactions are completely independent of dynactin also remains to be resolved. Examination of the behavior of GFP-pericentrin in living cells has revealed clear centripetal transport of pericentrin-containing particles to the centrosome (Young, A., R. Tuft, J. Dictenberg, A. Purohit, and S. Doxsey, manuscript submitted for publication). This behavior is correlated with a cell cycle-dependent accumulation of pericentrin and γ tubulin at the centrosome, which is strongly inhibited by nocodazole, antibody to cytoplasmic DIC, or overexpressed dynamitin. These data, together with the identification of a pericentrin–dynein interaction (this study), demonstrates that recruitment of pericentrin and γ tubulin to centrosomes involves dynein-mediated transport. Since pericentrin previously has been shown to interact with the γ tubulin complex , and more recently with protein kinase A (Diviani, D., L. Langeberg, A. Purohit, A. Young, S. Doxsey, and J. Scott, manuscript submitted for publication), we currently believe that pericentrin functions as a molecular scaffold that transports important activities to the centrosome and anchors them at this site. The ability of dynamitin overexpression to inhibit centrosome protein recruitment suggests a role for dynactin in pericentrin-mediated transport, despite the lack of evidence in the current study for a role for dynactin in the dynein–pericentrin interaction. It is conceivable that dynactin disruption affects pericentrin accumulation via a mechanism unrelated to direct pericentrin transport, such as the disruption of the microtubule cytoskeleton. Alternatively, dynactin could regulate dynein-mediated pericentrin motility independent of a role in linking pericentrin to dynein. Such a model contrasts with an obligatory role for dynactin in the attachment of dynein to kinetochores , but is consistent with our current evidence for an involvement of alternative dynein targeting mechanisms in different cellular processes. Finally, it is possible that pericentrin interacts with dynein by a bivalent mechanism involving both the light intermediate chains and dynactin, but that the latter interaction is poorly preserved in vitro. Our data support a cytoplasmic dynein sequestration model to explain the effects of pericentrin overexpression. Dynein is removed from at least two of the sites where it is normally found, the kinetochore and the spindle pole . The association of dynein with membranous structures is more difficult to assess because of the profusion of such structures in the cytoplasm, but the dispersal of the Golgi apparatus that we observe is strongly consistent with a loss of dynein from this organelle as well. Thus, we imagine that soluble pericentrin binds to the light intermediate chains and interferes with normal dynein targeting interactions. Interference of light intermediate chain localization or function by overexpressed pericentrin could result from competition with other light intermediate chain interactions in the cell. Alternatively, it could be due to steric interference by overexpressed pericentrin with the intermediate chain/dynactin interaction. Mapping studies have, in fact, shown the binding sites for the intermediate and light intermediate chains to be in close proximity within the DHCs (Tynan, S., and R. Vallee, unpublished results). Further work will be required to identify the full range of light intermediate chain functions. One distinction between the pericentrin and dynamitin overexpression effects is that there is no detectable change in the mitotic index of pericentrin-overexpressing cells. This result is puzzling in view of the similarity in mitotic defects observed in the two cases, including the production of multipolar mitotic spindles. The latter structures are suggestive of mitotic failure (i.e., cytokinesis failure) which typically occurs after a delay in mitosis. Although pericentrin-overexpressing cells do not exhibit a mitotic delay, they appear to grow and divide normally. This suggests a defect in the checkpoint that regulates the transition from metaphase to anaphase , an idea we are currently testing. Pericentrin previously has been shown to be part of a large protein complex that includes γ tubulin . Thus, it is possible that disruption of γ tubulin in pericentrin-overexpressing cells contributes to the spindle defects. However, we believe this is unlikely because recruitment of γ tubulin to spindle poles is not noticeably different than in control cells. Moreover, the ability of individual mitotic spindle poles to nucleate microtubules, a function thought to be mediated by γ tubulin, appears unchanged in pericentrin-overexpressing cells. Some pericentrin-overexpressing cells have multiple γ tubulin staining structures that seem to contribute to the formation of multipolar spindles. Since each of the multiple poles has approximately the same amount of γ tubulin as normal spindle poles, we believe that they represent bona fide centrosomes (with centrioles). We are currently investigating how these multiple foci of γ tubulin are generated and whether they contribute to aneuploidy in pericentrin-overexpressing cells. It is unclear why recruitment of γ tubulin and NuMA to spindle poles appear to be unaffected by the HA-Pc–induced dynein disruption since the evidence suggests that both proteins may also interact with, or be under the control of, cytoplasmic dynein. One possibility is that cell cycle variability in the localization and levels of these proteins , together with variability in the level of pericentrin overexpression, make it difficult to detect significant differences. Another possibility is that the proposed HA-Pc–induced sequestration of dynein may be less than complete, allowing some dynein-mediated transport to occur. This may be sufficient to localize the spindle pole proteins examined in this study, but insufficient to maintain Golgi complex organization or localize dynein to spindle poles and kinetochores. Alternatively, dynein may interact with many different cargoes (e.g. vesicles, protein complexes) whose localization is differentially affected by pericentrin overexpression. This could explain why NuMA and dynactin, which form a discrete complex with dynein in Xenopus extracts , appear to accumulate to normal levels at spindle poles. A final interesting feature of the pericentrin-overexpressing phenotype is the generation of aneuploid cells. In fact, pericentrin-overexpressing cells have chromatin levels both below and above diploid, suggesting that they undergo persistent chromosome missegregation as described . Since little is known about how aneuploid cells are generated, this cell system provides a powerful model to study this phenomenon. This system may also prove useful in understanding human tumorigenesis since pericentrin levels are elevated in most aneuploid tumors . For these reasons, it is important to determine the precise contributions of dynein and other pericentrin-interacting molecules in the generation of aneuploidy and spindle defects in pericentrin-overexpressing cells. | Study | biomedical | en | 0.999996 |
10545495 | Dictyostelium high speed supernatant (HSS) 1 was prepared as previously described . Wild-type cells [5 liters at a density of 4–8 × 10 6 cells/ml in HL-5 media containing 100 μg/ml streptomycin and 100 U/ml penicillin] were collected by centrifugation at 4,000 g for 15 min at 4°C and washed in 1 liter ice-cold Sorenson's phosphate buffer, pH 6.0 . Cells were resuspended in 1:1 wt/vol lysis buffer (LB: 30 mM Tris-HCl, pH 8.0, 4 mM EGTA, 3 mM DTT, 5 mM benzamidine, 10 μg/ml soybean trypsin inhibitor, 5 μg/ml TPCK/TAME, 10 μg/ml leupeptin, pepstatin A, and chymostatin, and 5 mM PMSF) containing 30% (wt/vol) sucrose. The suspension was split into thirds and each third was lysed by one passage through a nucleopore polycarbonate filter (5-μm pore size, 47-mm diameter; Costar Corp.) using a 10-ml syringe. The lysate was centrifuged at 2,000 g for 5 min at 4°C. The resulting post-nuclear supernatant was layered over a 1-ml cushion of LB/25% sucrose and centrifuged in a rotor (TLA 100.4; Beckman Instruments, Inc.) at 180,000 g for 15 min at 4°C to obtain an HSS. 5 liters of cells generated ∼20 ml of HSS. A microtubule–affinity-purified fraction (ATP releasate) was then prepared from the HSS as previously described . In brief, HSS was incubated with 15 U/ml hexokinase, 3 mM glucose, 4 mM AMP-PNP/MgCl 2 , 20 μM taxol, and 0.5 mg/ml taxol-stabilized microtubules for 20 min on ice. Microtubules and associated proteins were centrifuged through a 1-ml cushion of LB/25% sucrose containing 20 μM taxol at 85,000 g for 15 min (4°C). The microtubule pellet was resuspended in LB/5% sucrose containing 0.3 M KCl (1:12 vol of the original HSS volume) and immediately recentrifuged at 85,000 g for 15 min (4°C). The resulting supernatant (“salt wash”), containing predominantly minus-end–directed transport activity, was removed. The microtubule pellet was resuspended in phosphate buffer (10 mM potassium phosphate, pH 6.8, 1 mM EGTA, 3 mM DTT, 5 mM benzamidine, 10 μg/ml soybean trypsin inhibitor, 5 μg/ml TPCK/TAME, 10 μg/ml leupeptin, pepstatin A, and chymostatin, and 5 mM PMSF)/5% sucrose containing 5 mM ATP/MgCl 2 (1:20 vol of the original HSS volume) to elute the plus-end–directed transport activity. After incubation for 15 min on ice, microtubules were separated from the released proteins by centrifuging at 90,000 g for 15 min (4°C). The supernatant (ATP releasate) was collected and either assayed directly (see below) or frozen in liquid nitrogen. 1.5–2 ml of ATP releasate in phosphate buffer/5% sucrose was diluted to 2.26 ml in phosphate buffer/5% sucrose and CaCl 2 was added to 1.2 mM. 2.2 ml of the releasate was then loaded onto a 1.8-ml hydroxyapatite column (20 μm ceramic hydroxyapatite; American International Chemical) equilibrated in phosphate buffer/5% sucrose (for hydroxyapatite and Mono Q chromatography buffers, protease inhibitors included only 5 mM benzamidine, 5 μg/ml TPCK/TAME, and 1 mM PMSF). The column was run on the SMART chromatography system (Amersham Pharmacia Biotechnology). Sample was loaded at a flow rate of 250 μl/min and 600-μl fractions were eluted at 500 μl/min in two steps of 200 and 300 mM potassium phosphate (3.6 ml each). For motility assays, 45-μl aliquots of all fractions were dialyzed for 2 h against LB pH 8.0/5% sucrose using a microdialyzer system 100 (Pierce Chemical Co.), exchanging the buffer in the chamber once after 30 min of dialysis. For further chromatography, the 200-mM potassium phosphate peak fractions were pooled and dialyzed against LB pH 9.0/5% sucrose using a microdialyzer system 500 (Pierce Chemical Co.). The dialysate of the pooled hydroxyapatite fractions was diluted to 2.26 ml in LB pH 9.0/5% sucrose and 2.2 ml was loaded onto a 100-μl SMART system Mono Q column (Mono Q PC 1.6/5; Amersham Pharmacia Biotechnology) equilibrated in LB pH 9.0/5% sucrose. The column was loaded at 50 μl/min, and 100-μl fractions were eluted at 100 μl/min with a 115-mM NaCl step elution (500 μl), followed by a 170–300 mM NaCl gradient (1 ml). 45-μl aliquots of fractions were dialyzed against LB pH 8.0/5% sucrose as above for use in motility assays. Sea urchin sperm axoneme-nucleated microtubule structures were prepared in flow cells (10 μl) as described previously . KI vesicles were also prepared as described previously ; these vesicles, which are a mixed population isolated from the crude extract, were isolated by sedimentation through a sucrose cushion, treated with 0.3 M KI to dissociate weakly bound proteins, and then resedimented through a sucrose cushion. The flow cells were first washed with 10 μl of LB pH 8.0 (without PMSF)/15% sucrose to remove any free microtubules, followed by the introduction of 10 μl of assay mix consisting of, in the case of HSS, 5 μl of HSS, 3.5 μl LB/15% sucrose, 1 μl KI-washed vesicles, and 0.5 μl ATP-regenerating mix . Assay mixes for the ATP releasate consisted of 5 μl of ATP releasate, 3.0 μl LB/15% sucrose, 1 μl KI-washed vesicles, 0.5 μl of a 20-mg/ml casein stock, and 0.5 μl ATP-regenerating mix. Assay mixes for fractions from the hydroxyapatite and Mono Q columns (including the hydroxyapatite load) consisted of 8 μl of the fraction, 1 μl KI vesicles, 0.5 μl casein, and 0.5 μl ATP regenerating mix. The movement of organelles was observed using a microscope (Axioplan; Carl Zeiss, Inc.) equipped with differential interference contrast (DIC) optics, a 50- or 100-W mercury arc lamp, and a 63×, 1.4 NA Plan-Neofluor objective. Images were detected using a camera (Newvicon; Hamamatsu Photonics); contrast enhancement and background subtraction were performed with an image processor (Argus10; Hamamatsu Photonics), and recordings were made with a super VHS video tape recorder . Organelle motility was quantified by counting the number of movements in each direction on a single axoneme/microtubule structure. Only axonemes with clearly defined polarity were used. Recordings were performed on axonemes with between 6 and 12 microtubules (8–14 μm each in length) polymerized from the plus ends. If an organelle paused briefly and then continued in the same direction, it was scored as a single movement. Velocities of movements were measured using an NIH-IMAGE–based measuring program developed by J. Hartman; only vesicles that moved smoothly over a distance of at least 1.5–2 μm were scored. To observe linear organelle movements in live cells, 20 μl of null or control cells (grown to 3–7 × 10 6 cells/ml in HL-5 media containing 100 μg/ml streptomycin, 100 U/ml penicillin, and 5 μg/ml blasticidin (ICN Biomedicals Inc.) were introduced into flow cells (20 μl) made with coverslips that had been incubated overnight in 1 M HCl, and then washed extensively with water. The flow cells were inverted for 2 min, and then viewed by DIC optics as described above. Each cell was observed for 4 min, and the slide was discarded after 10 min of observation. Only linear and continuous movements >1 μm in length were scored. To observe the movement of mitochondria in live cells, 10 8 cells were collected by centrifugation at 800 g for 3 min at room temperature (25°C). Cells were resuspended in 10 ml HL5 containing P/S/blasticidin and 50 nM Mitotracker (Molecular Probes, Inc.). Cells were incubated for 15 min at 22°C while shaking at 180 rpm. The cells were repelleted and washed once in 10 ml Sorenson's phosphate buffer at room temperature (25°C) and resuspended in 5 ml HL5 plus P/S/blasticidin. Cells were incubated for 30 min at 22°C while shaking at 180 rpm. Cells were then diluted 1:5 in phosphate buffer and flowed into flow cells (20 μl) prepared with acid-washed coverslips as described above. The flow cell was inverted for 2 min, and then viewed by fluorescent optics using a 63×, 1.4 NA objective and a SIT camera (Hamamatsu Photonics). Each cell was viewed for 1 min and the slide was discarded after a maximum of 15 min of observation. Only linear and continuous movements >1 μm in length were scored. 200 μl ATP releasate in LB pH 8.0/5% sucrose/0.15 M NaCl was loaded onto a 2.2 ml 10–25% continuous sucrose gradient (in LB/0.15 M NaCl) and spun for 5 h at 200,000 g in a rotor (TLS-55; Beckman) (4°C). In parallel, a 200-μl mix of calibration standards (BSA, 4.3S; aldolase, 7.4S; catalase, 11.3S; ferritin, 17.6S; and thyroglobulin, 19.4S) at 1 mg/ml each in LB/5% sucrose/0.15 M NaCl was run on a separate gradient. 200 μl fractions were collected from each gradient. The sedimentation profiles of the calibration proteins were monitored by Coomassie staining, while the profile of DdUnc104 was monitored by immunoblotting with an affinity-purified DdUnc104 peptide antibody (see below). The S value of DdUnc104 was determined by comparing its position in the gradient to the positions of the standards plotted against their S values. 50 μl of the DdUnc104 peak from the sucrose gradient was loaded onto a 2.4-ml gel filtration column (SMART system: Superose 6 PC 3.2/30; Amersham Pharmacia Biotechnology) equilibrated in 30 mM Tris-HCl, pH 8.0, 4 mM EGTA, 3 mM DTT, 5 mM benzamidine, 5 μg/ml TPCK/TAME, and 0.15 M NaCl. The column was run at 30 μl/min and 50-μl fractions were collected. The elution profile of DdUnc104 was again monitored by immunoblotting. The elution profiles of calibration standards with known Stokes radii (3.1 nm ovalbumin, 4.8 nm aldolase, 5.2 nm catalase, 6.1 nm ferritin, and 8.5 nm thyroglobulin) prepared in the same buffer were assessed by Coomassie stain. Linear plots of the (−log K av ) 1/2 versus Stokes radius for each standard were used to determine the Stokes radius of DdUnc104 from its K av ( K av = V e − V o /V t − V o , where V e = elution volume, V o = void volume as determined by the elution volume of blue dextran, and V t = total column volume or the total accessible volume as assessed by the elution profile of 1 M NaCl). Partial specific volume and axial ratio calculations were made using the Sednterp program developed by John Philo. Coiled-coil predictions were made using the program COILS . Mono Q fractions containing the 245- and 170-kD polypeptides were pooled, TCA precipitated, and separated on 4–12% gradient polyacrylamide gels (Novex) under denaturing and reducing conditions followed by staining with Coomassie blue. After cutting out the protein bands, in-gel digestion with Endoproteinase Lys-C (Boehringer Mannheim Biochemicals) was carried out as described . Gel-extracted peptides were then fractionated using a Vydac microbore C8 column (The Separations Group) and individual peptides were subjected to Edman degradation with a protein sequencer (#492; Perkin-Elmer, Applied Biosystems Division). A peptide based on DdUnc104 amino acids 359–373 was synthesized with a COOH-terminal cysteine and used for immunization of a rabbit (QCB, Inc.). Peak bleeds were pooled, and the antibody was purified against the synthetic peptide coupled to a thiol-coupling resin using the QCB standard antibody purification protocol. The purified antibody was dialyzed into 80 mM Tris-HCl, pH 8.0, 4 mM EGTA for storage. For immunoblotting, samples were separated on 4–12% gradient polyacrylamide gels (Novex) and electroblotted to nitrocellulose membranes at 100 mA for 75 min. The blots were incubated with the DdUnc104 peptide antibody (1:500) overnight at 4°C, and then incubated in HRP-conjugated secondary antibody (1:2,000; Amersham Life Sciences) for 1 h at room temperature. Blots were developed using a chemiluminescence kit (NEN Life Sciences) and exposed to Hyperfilm (Amersham Life Sciences). One of the peptide sequences of the 245-kD protein was class-conserved among members of the Unc104/KIF1A subfamily. Therefore, fully degenerate oligonucleotides corresponding to the VVNEDAQ peptide (amino acids 360–366) obtained from peptide sequencing and to a sequence highly conserved in the Unc104/KIF1A family, KSYTMMG, were used to prime a PCR reaction using Dictyostelium oligo (dT)-primed first-strand cDNA as a template (Superscript Preamplification System; GIBCO BRL). The expected ∼780-bp PCR product was cloned and sequenced. Primers made to this PCR product were used to generate a 470-bp probe that was used to screen a Dictyostelium vegetative cDNA library (λZAP; Stratagene Inc.; kindly provided by Dr. Rick Firtel, University of California, San Diego, San Diego, CA). An ∼2-kb DdUnc104 cDNA clone containing the starting methionine and an upstream stop codon was obtained from the screen; however, this cDNA terminated prematurely. To obtain further downstream sequence, total RNA was extracted from vegetative cells (Trizol; GIBCO BRL), and poly A + RNA was further purified using an mRNA separator kit (Clontech). The poly A + RNA was used as a template to generate cDNA that was then used for a 3′ rapid amplification of cDNA ends (RACE) reaction (Marathon cDNA Amplification Kit; Clontech) using a primer based on the 3′ end of the λZAP fragment. This method yielded a 1.8-kb DdUnc104 fragment that was continuous with the λZAP fragment, but which still terminated prematurely. Primers based on the 3′ end of this new fragment were used in a new 3′ RACE reaction, using the same cDNA pool, to obtain a 3-kb fragment containing the remainder of the DdUnc104 gene. (Note: the DdUnc104 gene is also known as ksnA in Dictyostelium nomenclature.) To confirm the sequences obtained from the RACE products, two additional independent PCR reactions were performed for each segment and the resulting clones were sequenced. Percent identity between protein sequences was calculated using the worldwide web version of the program Blast available at http://www.ncbi.nlm.nih.gov/BLAST. Multiple sequence alignment was done with the program Pileup (Genetics Computer Group) and the output was shaded using MACBOXSHADE. A 1.4-kb cassette conferring blasticidin resistance was cloned between the adjacent BglII sites in the DdUnc104 sequence. The 3.2-kb construct was released from the plasmid by digestion with EcoRV and XbaI , and transformed into cells by electroporation as described . Transformants were selected on petri plates containing liquid DD-broth20 media supplemented with 5 μg/ml blasticidin (ICN Biomedicals Inc.). Isolated colonies were picked and transferred to 24-well plates. After further growth, the cells were collected for screening by PCR. Approximately 10 6 cells were resuspended in 50 μl lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.3% Tween-20, 60 μg/ml proteinase K) and incubated for 45 min at 56°C and 10 min at 94°C. 1 μl of the crude lysate was used as a template for 25-μl PCR reactions set up using standard protocols. Pairs of a primer specific to the blasticidin resistance cassette and a DdUnc104-specific primer flanking the disruption site were used for the PCR. Two independent null cell lines were obtained and were shown to behave identically. Genomic DNA was prepared from selected transformant strains (QIAGEN Inc.) and analyzed by Southern blotting as described previously using the EcoRV-XbaI fragment of the DdUnc104 cDNA as a probe. For Northern analysis, RNA was prepared using Trizol (GIBCO BRL) and the EcoRV-XbaI fragment was used as a probe. For Western analysis, 5 μl of ATP releasates prepared from null and control cells (see below) were immunoblotted using the affinity-purified DdUnc104 antibody. For analysis of in vitro motility, ATP releasates were prepared from null and control cells (500–800 ml at a density of 0.6–1 × 10 7 cells/ml in HL5 containing P/S/blasticidin) as described above, with two modifications. First, the 0.3-M KCl wash before ATP elution of the microtubule pellet was omitted to preserve minus-end motility in the ATP releasate. Second, the buffer for the ATP release was LB pH 8.0/5% sucrose containing 5 mM ATP/MgCl 2 . Previously, we developed a method of preparing extracts from D. discoideum that supported abundant bidirectional microtubule-based vesicle transport; the velocities and travel distances of moving organelles were similar to those displayed in vivo . We also showed that motion of KI-washed organelles, which did not move on their own, could be reconstituted by the addition of an HSS . Here, we wished to purify the factor(s) responsible for plus-end–directed vesicle transport using a biochemical fractionation scheme. Microtubules grown in a polarized manner from salt-extracted axonemes were used to determine the direction of vesicle movement in these experiments. Microtubule affinity was used as a first purification step since both plus- and minus-end vesicle transport activities cosediment with exogenously added microtubules in the absence of ATP and the presence of AMP-PNP and can be subsequently released from the microtubules by ATP . Such procedures are known to enrich for microtubule motors and associated proteins. Furthermore, the plus-end–directed organelle transport activity could be partially separated from the more abundant minus-end–directed activity by incubating the microtubules with 0.3 M KCl (to extract minus-end–directed activity selectively) before the ATP release step. This microtubule affinity procedure resulted in an 80-fold increase in specific activity for plus-end–directed organelle transport relative to the HSS ( Table ). To rule out the possibility that the vesicle transport observed was due to nonspecific interactions with membrane lipids, we tested whether treatment of KI vesicles with trypsin (100 μg/ml for 30 min on ice followed by soybean trypsin inhibitor, SBTI, at 4 mg/ml for 10 min) reduced their movement in the presence of the ATP release fraction. Plus-end–directed vesicle motility was decreased threefold by this treatment compared with a control in which trypsin was preblocked with SBTI before addition to the vesicles. Thus, proteins on the vesicle surface play an important role in motility. Since the ATP release fraction contained numerous polypeptides , we further purified the plus-end–directed vesicle transport activity using hydroxyapatite chromatography . The activity bound to the column and could be eluted by potassium phosphate. Three prominent polypeptides appeared in the activity-containing fractions: the ∼540-kD dynein heavy chain and two polypeptides of 245 and 170 kD. Even though dynein was prominent in the hydroxyapatite eluate, very little (less than two movements/axoneme) or no minus-end–directed transport was observed in several independent purifications. The peak fractions of the hydroxyapatite column were pooled, applied to a Mono Q column, and eluted with a shallow NaCl gradient, which proved necessary to separate the 245- and 170-kD polypeptides from one another . The organelle transport activity peaked consistently with the 245-kD polypeptide in six purification efforts, but activity was also detected in the later-eluting fractions that contained both the 170- and 245-kD polypeptides. In the Mono Q column, cytoplasmic dynein eluted earlier than the peak of plus-end–directed vesicle transport . Again, little or no minus-end transport activity was observed in the peak dynein fractions. The cofractionation of activity with the 245-kD polypeptide in the Mono Q column strongly implicated it as a factor that can stimulate plus-end motility. However, in the hydroxyapatite column, an early-eluting fraction containing the 170- but not the 245-kD polypeptide also stimulated transport. This finding raised the possibility that the 170-kD polypeptide was also capable of promoting plus-end–directed organelle movement. To address whether both polypeptides were truly independent transport-stimulating factors, we performed two additional experiments. First, we diluted a peak 245-kD Mono Q fraction (containing nominal 170-kD polypeptide) to a level of activity that was 25% of that in the 170-kD peak fraction. By silver staining, the quantity of the 245-kD polypeptide in this diluted fraction was greater than that found in an equal volume of the fourfold more active 170-kD peak fraction. This result indicates that organelle transport in the 170-kD peak fraction could not be stimulated solely by the 245-kD polypeptide contaminating this fraction. Next, we measured the velocities of plus-end–directed movements from column fractions that had minimal overlap in their content of 170- and 245-kD polypeptides . Movement velocities from the 245- or 170-kD–enriched fractions were 2.62 ± 0.50 ( n = 27) and 1.86 ± 0.74 ( n = 23) μm/s, respectively (mean ± SD; P < 0.001). The velocity histogram of movements generated by the 170-kD fraction appeared bimodal. The majority of movements (65%) was between 1 and 2 μm/s; by contrast, this velocity range represented only 11% of the movements stimulated by the 245-kD fraction. The 170-kD fraction also contained a subset of fast movements between 2 and 3.5 μm/s, which corresponded to the average velocity produced by the 245-kD fraction. Since the 170-kD fraction contained some contaminating 245-kD even in our purest fractions , it seems likely that these few fast movements were due to the 245-kD polypeptide. Thus, we conclude that the 245- and 170-kD polypeptides both stimulate plus-end–directed organelle transport, but at different velocities. Our hydroxyapatite and Mono Q fractionation experiments showed that the amount of organelle transport in our assay was always in quantitative agreement with the amount of the 245- and 170-kD polypeptides in the fraction. From examination of silver-stained gels from many columns, we could not identify any other polypeptide that clearly and consistently copurified with either the 245- or 170-kD polypeptides (though our gel system would not have clearly resolved polypeptides <25 kD). From this data, we believe that the 245- and 170-kD polypeptides can stimulate transport in the absence of associated polypeptides, although a low molecular weight factor or a catalytic activity present in low stoichiometry could have been present and not detected in this analysis. To establish the molecular identity of the 245- and 170-kD polypeptides, we obtained peptide sequence from tryptic fragments. Both polypeptides contained peptide sequences that implicated them as members of the kinesin superfamily. A peptide from the 170-kD protein contained the sequence LYLVDLAGSEK, which includes the highly conserved DLAGSE motif of the switch II region of the kinesin nucleotide binding site . The 245-kD polypeptide yielded a tryptic fragment containing the conserved VIxAL motif found in the α4 helix of kinesin motors. Since the 245-kD polypeptide was the more robust motor in our in vitro organelle transport assay, we chose this kinesin for further cloning and characterization. The 245-kD polypeptide was cloned using a combination of degenerate PCR, library screening, and RACE (see Materials and Methods). The complete open reading frame predicts a protein of 2,205 amino acids of 248 kD that contains all five peptides identified by direct sequencing . We are therefore confident that we cloned the correct polypeptide identified in our biochemical purification. Sequence alignments revealed a clear homology of the 245-kD polypeptide to the Unc104/KIF1A class of kinesin motors . Two founding members of this class, mouse (Mm) KIF1A (190 kD) and the C . elegans (Ce) ortholog Unc104 (174 kD), are monomeric kinesin motors that have been implicated in the transport of synaptic vesicle precursors in neurons . The Unc104/KIF1A class also includes motors implicated in other functions such as mitochondrial transport , Golgi-ER transport , and cytokinesis . The sequence of the 245-kD kinesin reveals the following domain structure. The first 353 residues show high amino acid identity to the conserved catalytic core that defines the kinesin superfamily . Within the kinesin superfamily, the catalytic core of the 245-kD kinesin is most similar to the Unc104/KIF1A class (40–60% identical residues). All members of the Unc104/KIF1A class also contain a highly conserved extension in loop 3 (L3), which is unique among kinesins (R. Case and R. Vale, unpublished observations). This L3 extension is also found in the 245-kD kinesin . Several, but not all, members of the Unc104/KIF1A class contain several lysine residues in the microtubule-binding loop (L12) . In the 245-kD kinesin, this loop contains only two lysines, and thus is much less charged. For ∼250 amino acids after the catalytic core, the 245-kD kinesin shows significant identity to all kinesins in the Unc104/KIF1A class . The latter half of this class-conserved region is homologous to a domain found in nonmotor proteins such as human AF-6 and Drosophila cno , and whose function is poorly understood. Beyond the AF-6/cno domain, the 245-kD kinesin continues to exhibit strong homology to CeUnc104 and MmKIF1A for an additional ∼370 amino acids; however, sequence identity to other Unc104/KIF1A class kinesins (e.g., KIF1B) does not continue far past the AF-6/cno domain . The sequence in the middle portion of the 245-kD kinesin is novel and contains repeats of glutamine or serine that are often found in Dictyostelium proteins . Residues 1523–1616 are predicted to form a pleckstrin homology (PH) domain whose greatest similarity in blast searches is to a COOH-terminal PH domain in MmKIF1A and CeUnc104 . This PH domain is not found in other motors belonging to this kinesin class . The sequences of MmKIF1A and CeUnc104 end shortly after the PH domain, but the 245-kD kinesin extends for another 589 amino acids. This region does not exhibit significant sequence identity to any other protein in the data base. In conclusion, the 245-kD kinesin shows more identity to the two ortholog motors CeUnc104 and MmKIF1A than to other members of the Unc104/KIF1A class. Since there is somewhat more nonmotor domain homology to CeUnc104 than to MmKIF1A , we term the 245-kD kinesin Dictyostelium (Dd) Unc104. We also examined DdUnc104 for possible coiled-coil domains using a sequence prediction program . This analysis revealed that both MmKIF1A and DdUnc104 contain two regions in the NH 2 -terminal half of the molecule (approximately amino acids 359–448 and 652–679 in DdUnc104; the second is also present in CeUnc104), which have a propensity towards coiled-coil formation . However, hydrodynamic analyses of MmKIF1A and CeUnc104 indicated that these regions do not cause dimerization. Interestingly, the majority of the unique ∼600–amino acid extension of DdUnc104 was also predicted to have a high probability of coiled-coil formation , raising the possibility that DdUnc104, in contrast to MmKIF1A and CeUnc104, is dimeric. To determine the quaternary structure of DdUnc104, we analyzed its hydrodynamic behavior using velocity sedimentation through sucrose density gradients and gel filtration (see Materials and Methods). In two separate experiments, the S value was 10.9 and 10.2 S (average 10.6). The sucrose gradient peak was then subjected to gel filtration, and a Stokes radius of 10.5 nm was measured in both experiments. Together with a partial specific volume of 0.73 cm 3 /g estimated from the DdUnc104 amino acid sequence, the native molecular weight was determined to be 480 kD. Given the polypeptide's molecular weight of 248 kD, this result indicates that DdUnc104 is a dimer and also argues against the presence of a stoichiometrically associated polypeptide of significant mass. These hydrodynamic data also suggest that DdUnc104 has an extended shape with an axial ratio of 15.1. Thus, DdUnc104 is the first dimeric motor described for the Unc104/KIF1A class of kinesin motors. To learn more about the function of DdUnc104 in vivo, the DdUnc104 gene was disrupted by homologous recombination. A gene-disruption construct was made by inserting a blasticidin resistance cassette into a DdUnc104 cDNA fragment. The linearized construct was transformed into wild-type cells by electroporation, and transformants were screened by PCR (see Materials and Methods). A cell line was identified that gave the PCR products expected from a gene disruption event. Disruption of the DdUnc104 gene was established by Southern analysis, and absence of the DdUnc104 RNA and protein was confirmed by Northern and Western blotting . A blasticidin-resistant cell line in which the construct had integrated elsewhere in the genome was used as a control . The DdUnc104 null cells grew normally and showed no gross morphological defects. In addition, after starvation, the null cells aggregated and differentiated normally and at a rate like that of wild-type cells. Thus, this motor is not essential for viability, cell division, or differentiation. The DdUnc104 knockout provided an opportunity to assess whether DdUnc104 was a bona fide organelle transport motor in vivo. To address this question, linear (>1 μm) movements of organelles in control and DdUnc104 null cells were observed by video DIC microscopy; earlier studies have shown that these movements are predominately microtubule based . Quantitative measurements revealed that the mutant cells exhibited a 62% reduction in overall movements . In Dictyostelium , organelle movement is not perfectly radial from the center to periphery, and hence it is very difficult to score the polarity of movement in vivo. Nevertheless, this result indicates that DdUnc104 drives organelle transport in vivo. Interestingly, when we examined the movement of mitochondria by labeling cells with a mitochondrial dye , no difference in the frequency of mitochondrial movements in null and control cells was observed . This result indicates that DdUnc104 is not required for the movement of mitochondria. Thus, the transport defect in the DdUnc104 null cells is not due to a generalized reduction in the movement of all organelles, but rather reflects a specific defect in the transport of an as yet unidentified organelle population(s). Since the polarity of organelle movement could not be determined in vivo, we analyzed whether the organelle motility defect in the DdUnc104 null cells was specific for the plus-end direction using our in vitro assay. An ATP release fraction prepared from either the control or null cells was incubated with KI-washed vesicles and scored for motility on our polarity-defined microtubule substrate. Minus-end–directed motility driven by dynein was nearly identical for the fractions obtained from control and null cells. However, dramatically, plus-end motility was decreased by 90% in the null cells . This result confirms that the DdUnc104 polypeptide is responsible for the majority of the plus-end–directed motor activity seen in our biochemical assay. Microtubule-based motor proteins have been purified using in vitro microtubule gliding activity or reactivity with pan-kinesin antibodies . However, these highly purified motors have not efficiently transported organelles. Here, we have approached the problem differently by directly purifying soluble factors needed for plus-end–directed movement of organelles. This strategy led to the discovery of two kinesin motors that drive organelle movement. Several findings argue that the purified motors are the ones that power plus-end–directed organelle transport in Dictyostelium . First, the specific activity of plus-end–directed organelle transport increased throughout the purification, indicating that the relevant activities from the starting extract were being enriched at each step. Second, the speed and quality of movement (continuous motion for several microns) produced by the two purified motors was virtually identical to that observed in extracts and in living cells . The role of the 245-kD DdUnc104 motor in organelle transport was further confirmed by a gene knockout. Although several other kinesin genes have been identified in Dictyostelium , the 245-kD DdUnc104 and 170-kD kinesin are likely to represent the major organelle transport motors, since the DdUnc104 knockout results in the loss of the vast majority of plus-end–directed organelle movement in vitro. It is likely that the remainder of the movement is produced by the 170-kD motor, and this can be established in the future by analyzing cells that have knockouts in both the DdUnc104 and 170-kD kinesin genes. In principle, our approach could have purified a soluble activator of an organelle-bound motor or a combination of an activator and a motor. Although these possibilities cannot be ruled out and further studies are required, our data does not favor these scenarios. First, there is no polypeptide that copurifies stoichiometrically with DdUnc104. This data, along with the hydrodynamic analysis, argues that this motor does not have an associated light chain, although it is possible that a small subunit (<25 kD) eluded detection in our SDS-PAGE analysis. Furthermore, based on many purification trials, we have not detected any substoichiometric polypeptide that consistently cofractionates with DdUnc104. We also believe that DdUnc104 is not activated by a protein that overlaps but does not copurify with the DdUnc104 fractions, since the relative amounts of organelle transport activity mirror the concentrations of DdUnc104. With regard to the membranes, all components required for transport are vesicle-associated after 0.3-M KI treatment, but are partially destroyed by trypsin. From these studies, we hypothesize that plus-end–directed organelle transport is driven by a direct interaction of DdUnc104 with a tightly associated, and possibly integral, membrane protein(s). Our results differ somewhat from in vitro studies of plus-end–directed organelle transport using other systems. A recombinant KIF1B motor was reported to transport mitochondria in vitro , although the average frequency and velocity of movements were not reported, and several examples of moving mitochondria shown in this paper reveal transport rates (<0.1 μm/s) slower than those seen in vivo. In contrast, vesicle movements produced by the two motors purified in our study occur over long distances and match in vivo velocities. For conventional kinesin, fractionation experiments by Schroer and Sheetz 1991 indicated that kinesin requires an activator for transporting organelles, although its identity has not been established through further biochemical purification. Interestingly, many of the plus-end–directed organelle movements in this activator fraction were faster than kinesin (>2 μm/s) and comparable in speed with the movements observed in our study. It is possible that the movement observed in this fraction was due not to a kinesin activator, but to an Unc104/KIF1A-type motor. The sequence of the 245-kD kinesin reveals that it belongs to the Unc104/KIF1A class of motors, since its amino acid identity extends well past the superfamily-conserved catalytic core. Based on phylogenetic trees of kinesin sequences from the database, the Unc104/KIF1A class encompasses ∼16 members from various organisms. Of these various members, DdUnc104 appears to be most similar to C . elegans Unc104 and mouse KIF1A. Most notably, its homology to CeUnc104/MmKIF1A uniquely extends well past the AF-6/cno domain found in many family members, and these three motors are also the only ones that contain a conserved PH domain at or near their COOH termini. However, DdUnc104 and CeUnc104/MmKIF1A must carry different types of cargo. MmKIF1A and CeUnc104 are expressed only in the nervous system, where they specifically transport synaptic vesicle precursors . Thus, a DdUnc104-like motor from unicellular organisms most likely evolved to acquire a more specialized function in the nervous system of higher eukaryotes. However, the conservation of nonmotor domains in DdUnc104 and CeUnc104/MmKIF1A suggests that the mechanisms by which they bind their cargo may be closely related. DdUnc104 is the first member of the Unc104/KIF1A kinesin subfamily identified and fully sequenced from a unicellular organism. A partial clone of a KIF1B homologue, TLKIF1, has recently been cloned from the thermophilic fungus Thermomyces lanuginosus , suggesting that members of this kinesin class may be common in lower eukaryotes. Since DdUnc104 is an evolutionarily distant member of the Unc104/KIF1A kinesin class, sequences that are uniquely conserved between it and other family members are likely to define regions that serve important functions for this class of motors. One such conserved motif is a unique extension in loop 3. Based on the location of this loop in the conventional kinesin and Ncd crystal structures, these extra amino acids would be expected to extend towards the nucleotide and hence may be involved in modulating enzyme kinetics. The AF-6/cno homology domain is also highly conserved between DdUnc104 and its higher eukaryotic relatives; its position adjacent to the catalytic core suggests that it may be involved in motor mechanics or regulation. In the COOH-terminal tail domain, the PH domains of DdUnc104, CeUnc104, and MmKIF1A are more similar to one another than to the PH domains from other proteins. PH domains are thought to be involved in membrane interactions , and the similar PH domains of these motors may reflect a conserved function in cargo binding. A surprising difference between DdUnc104 and other members of this class concerns their oligomeric state. KIF1A , KIF1B , and CeUnc104 (the NH 2 -terminal half) are monomeric, and sequence analysis of other members suggests that they also function using a single motor domain. Hence, this class has been referred to as the “monomeric kinesins” . However, the dimeric nature of DdUnc104 raises the question of whether other motors in this class can function as dimers under some circumstances. For DdUnc104, dimerization is constitutive due to the long coiled-coil COOH terminal to its PH domain. However, KIF1A and its higher eukaryotic relatives also contain sequences that are predicted to form coiled-coils , and perhaps binding of the motor tail to receptors on the membrane can trigger dimerization in a functionally equivalent manner. An alternative model is that higher eukaryotic Unc104/KIF1A family members have evolved to have efficient motor function as monomers. Consistent with this idea, some of the Unc104/KIF1A family members from higher eukaryotes have an insertion of several lysines in the microtubule binding loop. For MmKIF1A, these lysines have been shown to be important in enabling this monomeric motor to move processively along microtubules in vitro . In contrast, the equivalent loop in DdUnc104 has fewer lysines, perhaps because they are not needed for motility by this dimeric motor. Remarkably, although the knockout of the DdUnc104 motor dramatically decreases organelle transport, it has little effect on the morphology, division, and differentiation of these cells. This result suggests either that membrane trafficking can be achieved by vesicle diffusion, that DdUnc104 activity is redundant with another activity (perhaps with the 170-kD kinesin), or that other mechanisms act to compensate for the loss of this motor. However, with regard to this latter point, it is interesting that minus-end–directed motility is identical in extracts from the null and wild-type cells, indicating that the decrease in plus-end–directed motility does not elicit any compensatory change in the level of dynein-based movement. Our reconstituted assay system provides a starting point for identifying additional factors involved in organelle transport and for understanding their function. As documented in earlier work , we find that Dictyostelium cytoplasmic dynein loses its ability to transport organelles during purification, suggesting that it becomes separated from a required cytosolic factor. It will be interesting to determine if this factor is dynactin or whether another type of activator is used by Dictyostelium . In addition, this assay provides an opportunity for identifying the membrane receptors that interact with the DdUnc104 and 170-kD kinesin motors so that movement can eventually be reconstituted in a completely defined system. The molecular genetics offered by Dictyostelium also provides a testing ground for determining whether membrane proteins that bind kinesins in vitro indeed serve as bona fide receptors in vivo. | Study | biomedical | en | 0.999996 |
10545496 | Unless noted, all molecular biology experiments were done by standard methods . Fly husbandry and other related procedures were as described . All fly stocks, unless otherwise mentioned, are described in Lindsley and Zimm 1992 . A 150-bp PCR amplified KLP64D sequence was used as a probe to isolate several truncated cDNA clones from a Drosophila 0–4-h embryonic library cloned in pNB40 . A composite 2.5-kb KLP64D coding sequence was created from several cDNA clones. Nested deletions were created in the cloned cDNA fragments in either direction using an ExoIII kit (Promega Corp.) according to the protocol provided by the manufacturers. Both strands were sequenced using either the T7 or the T3 primers, and later the whole parental clones were sequenced using the internal primers generated from the primary sequences. Several overlapping genomic fragments were isolated from a Drosophila genomic library in Lambda DASH ® II using the KLP64D cDNA probe, and the coding sequences were further mapped to a 3.6-kb EcoRI/HindIII fragment, which was then cloned into pBluescript SK+ (Stratagene Inc.) and called pB11-4. Sequencing this genomic fragment revealed a single continuous open reading frame without any intron, and matched the KLP64D cDNA sequence. Several deletions in the KLP64D gene were isolated previously by imprecise excision of P-transposable elements inserted at the 3′ end of the gene . All mutant chromosomes were maintained over a TM3, Ser <y+> balancer in a yw background. We generated point mutations in the KLP64D gene by scoring failure of ethyl methanesulphonate (EMS)-induced lethal mutations to complement a deletion that removes the KLP64D gene, Df(3L)Klp64D A8 . n123 . In brief, mutagenesis followed standard methods in which 2-d-old adult isogenic w males were fed with 27 mM EMS in 2% sucrose solution for 16 h, put on fresh food for 1 d, and then groups of 10 males were mated to 40 w ; TM3, Sb Ser / TM6B, Tb Hu virgin females (60 matings). Individual male progeny that were TM3, Sb Ser /* or TM6, Tb Hu /* (where * indicates a mutagenized third chromosome) were then mated to four yw Df(3L)Klp64D A8 . n123 / TM3 , Ser <y+> virgins (9,000 matings). The progeny of these matings were screened for the absence of */ Df(3L)Klp64D A8 . n123 flies. TM3 , Ser /* males from appropriate lines were then mated to yw , TM3, Ser <y+>/ MKRS Sb virgins to create a stock. The mutagenized third chromosome was cleaned up and the lethal mutations mapped by recombination with a chromosome containing ru h th st cu sr e ca . We found eight recessive lethal mutants that failed to complement Df(3L)Klp64D A8 . n123 and that mapped to the KLP64D region. These lethal mutations fell into three complementation groups, one of which was found to identify the KLP64D gene. A 3.6-kb KLP64D genomic fragment in pB11-4 was cleaved out by NotI and XhoI endonuclease digest and ligated to the same sites of pUAST and named pUAS64D. This maintained proper orientation of the KLP64D coding sequences with respect to the UAS sites in pUAST. We recovered several stable transformant lines (marked by w + eye) in the X and in the second chromosome by injecting pUAS64D with a helper plasmid, containing the P-transposase gene, in yw embryos. These lines were then crossed to the Klp64D mutants. Rescue of lethality was scored both at the larval stages as well as in the adult, where the homozygous mutants are marked by yellow body color. For rescue experiments using the mouse KIF3A gene, an inducible transgene was created by inserting a full length KIF3A cDNA obtained by standard methods between the EcoRI and the NotI restriction sites of the pUAST vector . This arrangement placed the 5′ end of the KIF3A coding sequence near the GAL-UAS transcription activation sites. Stable transformant lines were then obtained by P-element–mediated transformation. Expression of the KIF3A transgene was induced in all neurons by Gal4 driven from the promoter of the elav gene inserted in the third chromosome . In situ hybridization was done according to Tautz and Pfeifle 1989 , with modification as described in Pesavento et al. 1994 and Perez and Steller 1996 . An antisense strand-specific ribo-probe was made from the 1.6-kb Spe I/Hind III fragment subcloned in pBluescript SK. An affinity column was prepared by cross-linking about 10 mg of truncated, bacterially expressed and purified glutathione S -transferase–KLP68D to 1 ml Affigel-10 (Bio-Rad Laboratories) beads according to the protocol supplied by the manufacturer. These were packed in a 15-ml plastic column (Bio-Rad Laboratories) and blocked thoroughly with 100 ml of 10% BSA (Sigma Chemical Co.) in 10 mM Tris-Cl, pH 7.0, 100 mM NaCl, and 5 mM KCl (TBS-BSA), then washed in succession with 15 ml of 100 mM glycine, pH 2.5, TBS-BSA, 100 mM triethanolamine, pH 11.5, and finally twice with TBS-BSA. It was then loaded with the antiserum, washed twice with 15 ml of TBS-BSA, and the bound antibody was extracted with 1 ml of 100 mM glycine, pH 2.5 . We tried all three methods of 1 M MgCl 2 , 100 mM glycine, and 100 mM triethylamine extraction and found glycine to be the most suitable. The purified antibody was dialyzed against TBS overnight and stored at 4°C with 0.2% NaN 2 . For Western blotting, ∼50 heads, thoraces, and abdomens from male and female adult flies were dissected and collected separately in plastic microcentrifuge tubes on ice. They were squashed in 500 μl sample loading buffer containing 0.1 M β-mercaptoethanol, 10% glycerol, and 0.1% bromophenol blue, and centrifuged at 14,000 g for 5 min at room temperature. The supernatant was boiled for 3 min and then separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane . The Western blots thus prepared were stained with several primary antisera and corresponding peroxidase-coupled secondary antisera (Jackson Laboratories, Inc.), and developed using the ECL kit (Amersham Pharmacia Biotech) according to the protocol supplied by the manufacturer. For coimmunoprecipitation experiments, methods were essentially as described in Yang and Goldstein 1998 . In brief, extracts of isolated Drosophila heads in RIPA buffer (50 mM Tris-Cl, pH 8.0, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl) were subjected to immunoprecipitation, run on SDS polyacrylamide gels, and then probed in Western blots by standard methods. Embryos were dechlorinated in 50% commercial bleach for 3 min and washed thoroughly in running distilled water, then fixed in heptane–fixative (2:1) interface as described in protocol 90 for 20 min at room temperature, and then devitelinized using methanol. The fixative contained 4% formaldehyde (EM grade) in 100 mM Pipes, pH 6.9, 1 mM EGTA, and 2 mM MgSO 4 . Larval tissues were dissected in Drosophila Ringers containing 130 mM NaCl, 4.7 mM KCl, 1.8 mM MgCl 2 , 0.7 mM KH 2 PO 4 , and 0.35 mM Na 2 HPO 4 and then fixed in the same fixative as mentioned above for 20 min at room temperature. Fixed tissue samples and embryos were washed with PBS and 0.1% Triton X-100 (PTX) and incubated with the primary antibody solution in PTX for 1 h. They were then washed with five changes of PTX and incubated with fluorescent secondary antibody (1:250 dilution in PTX; Jackson Laboratories, Inc.) for 1 h. Finally, after another wash with five changes of PTX, they were mounted with a drop of Vectashield™ mounting media (Vector Laboratories, Inc.). We used goat anti-HRP (1:1,000 in PTX; Cappel Inc.), purified rabbit anti-KLP68D , mAb anti-ChAT , rabbit anti-synaptotagmin (SYT) , and mAb anti–cysteine string protein (CSP) . The secondary antibodies alone do not stain anything in the control tissue. The stained tissue samples were observed using a Bio-Rad MRC1024 laser scanning confocal device which was fitted to a Nikon Diaphot 200, and the images were captured by the Laser-Sharp 3.0 software. Images were further processed in Adobe Photoshop 4.0 for presentation. Most of the pictures contain data from 3–10-μm thick regions in the tissue. KLP64D (previously named KLP4) was identified during a PCR screen for new Drosophila genes with homology to the highly conserved regions of the KHC motor domain . The gene was mapped to salivary gland chromosome band 64D. Northern analysis revealed a 2.6-kb mRNA enriched in the head . Sequence of the full-length KLP64D coding region was obtained from several cDNA clones as well as a genomic clone. Analysis of the sequence indicated a 2032-bp open reading frame (ORF), which encodes a 674–amino acids (aa) long polypeptide with a predicted molecular mass of 76.4 kD. Secondary structure prediction analysis suggested three distinct domains in the polypeptide, including a globular NH 2 -terminal domain of 385 aa, an alpha-helical coiled-coil central domain of 200 aa, and a globular COOH-terminal tail domain of ∼100 aa. The KLP64D sequence is most similar to the kinesin-II subunit KIF3A (94% similarity) of mouse and KRP85 (93% similarity) of sea urchin . Considerable similarity (66%) was found with another Drosophila kinesin-II polypeptide encoded by the KLP68D gene . Therefore, we placed KLP64D in the kinesin-II family with KIF3A and KRP85 as the likely homologues. This assignment is also supported by the finding that the lethality of KLP64D mutants can be rescued by expression of mouse KIF3A (see below). To obtain clues about the potential functions of kinesin-II in neurons, we examined the expression patterns of KLP64D and KLP68D in the developing Drosophila nervous system. In overview, we found that KLP64D and KLP68D are both expressed in a restricted set of neurons, which appear to correlate with the cholinergic subset. In situ hybridization to RNA revealed that KLP64D is expressed in neurons of both the central (CNS) and peripheral (PNS) nervous system. KLP64D RNA was first observed in the CNS at stage 12 when many CNS neurons begin extending their processes . Later in embryogenesis, at stage 16, KLP64D expression continues in the CNS, including the optic lobes . Prominent expression was also found in the PNS, where KLP64D RNA was observed in the cells of the dorsal and lateral chordotonal organs . Careful observation revealed that the staining in the abdominal lateral chordotonal (lch5) organs was restricted to the neuronal cells. In addition, we also found staining in the ventral and dorsal chordotonal neurons, the anterior sense organ and in the antenno-maxillary complex (data not shown). Expression in the CNS and PNS continues in the first instar larva and was still detectable in the third instar larval brain, although at a lower level . Expression in the third instar brain includes cells of the ventral ganglion and a few cells in the optic lobe . Antibody staining revealed preferential expression of KLP68D in a subset of neurons similar to those shown to express KLP64D, and shown previously to express KLP68D RNA . The antibody used was an affinity-purified antiserum raised against the 432-aa COOH-terminal domain of KLP68D . In Western blots of adult head lysate, the purified serum stained a single band of 90 kD, which is close to the expected molecular mass of full-length KLP68D. In stage 17 embryos, the purified KLP68D antibody strongly stained a subset of PNS neurons, including chordotonal neurons, cells in the antenno-maxillary complex , bolwig organ cells , and other sensory neuron cells in the epidermis. To visualize all neurons, the same embryos were also stained with anti-HRP antibody , which, as expected, labeled the cytoplasm and membranes of all neuronal cell bodies and processes . This double staining experiment helped establish the identity of the KLP68D-positive sensory neurons in the epidermis , and revealed that KLP68D may be specific to a subset of PNS sensory neurons, whose common thread may be that they are all cholinergic (see below). KLP68D was also detectable in the CNS starting at stage 16 in the embryo, where it was most pronounced in the first instar larva stage in the ventral ganglion as well as in the optic lobe. Unlike the sensory neurons, the expression in the CNS was not distinguishable in specific cell types, owing to the small size and high density of cell bodies in this region. Some cells in the optic lobe were strongly stained early in the first instar stage, but the staining was not seen at a later stage. In the ventral ganglion, uniform staining appeared in the neuropil and cell cortex. No specific staining was detectable in CNS at the third instar stage. A comparison of the KLP68D and KLP64D expression patterns suggests that both KLP64D and KLP68D are expressed in similar tissue subsets and most likely in the same set of cells. However, the overlap could be determined with certainty only in the neurons of the chordotonal organs , bolwig organ cells, and in the antenno-maxillary complex. We noted that the KLP64D and KLP68D distribution was similar to that of acetyl cholinesterase RNA expression and ChAT RNA and lacZ reporter expression in the PNS , which are good markers for cholinergic presynaptic neurons in Drosophila and other insects. The neurons innervating the chemosensory, chordotonal, and larval bolwig organs are known to be cholinergic. In addition, many interneurons of the CNS also express ChAT and acetyl cholinesterase . Unlike vertebrates, motor neurons in Drosophila generally use glutamate instead of acetylcholine as their neurotransmitter . Similar to what others have found, our efforts to localize ChAT antigen itself in the neuronal cell bodies of the PNS and CNS using ChAT antiserum were not successful, and so we could not determine the overlap of ChAT and KLP68D in the cell bodies with precision. However, since all of the cholinergic synapses are located in the CNS, ChAT staining was detected in the synaptic bulbs of the CNS neuropil , which provided an assay for ChAT transport in KLP64D mutants (see below). To test further whether KLP68D and ChAT are coexpressed in cholinergic neurons, we used kinesin light chain ( Klc − ) mutant larvae. Homozygous Klc mutant larvae die at the late second instar and early third instar stages with progressive distal paralysis. The axons of these larvae develop focal swellings along their length and contain accumulated cargos of various types that appear to block single axons in axon bundles . We observed numerous KLP68D and ChAT immunoreactive clogs in the axons of these animals , which probably result from the blockage of ChAT and KLP68D transport in cholinergic neurons. Quantitation in several nerve bundles revealed strong coincidence between ChAT immunoreactivity and that of KLP68D in the axons ( Table ). This observation confirms the suggestion that both ChAT and KLP68D are expressed in the same cells and transported in the same axons. These findings are significantly different than observed for ChAT and a ubiquitous axonal cargo, SYT . Although there are a large number of SYT-positive clogs in the axon, only a small percentage (37.4%) are also ChAT-positive . These data suggest that not all axons and axonal clogs contain ChAT and therefore, that the observed ChAT and KLP68D association is not fortuitous. Since other members of the kinesin-II family are often associated in heterodimeric complexes, we conducted coimmunoprecipitation experiments to probe the association of KLP68D and KLP64D. These experiments were complicated by the absence of an antibody specific for KLP64D and the lack of cross-reactivity in Western blot experiments of antibodies reactive with the KIF3A and KRP85 homologous proteins (data not shown). However, it was possible to express functional mouse KIF3A in Drosophila using the GAL4-UAS system , and to demonstrate that KIF3A rescues KLP64D mutants (see below). Flies expressing KIF3A could then be used in coimmunoprecipitation experiments. Immunoprecipitations using the K2.4 mAb raised against sea urchin KRP85 precipitated both KIF3A and KLP68D in flies expressing KIF3A, but not in wild-type . Complementary immunoprecipitations using anti-KLP68D also precipitated both KIF3A and KLP68D in flies expressing KIF3A, but not in wild-type . Control immunoprecipitations precipitated no immunoreactive material in any genotype. Thus, we propose that KLP64D, which is the sequence and functional homologue of KIF3A, also associates with KLP68D in vivo. Previous work led to the generation and characterization of a set of deletions in and around the KLP64D gene . These deletions were obtained by imprecise excision of several P-transposable elements inserted near the 3′ end of the KLP64D gene . One such deletion, Df(3L)Klp64D A8 . n123 , has a left breakpoint that extends to the extreme 5′ end of the KLP64D protein coding sequence. To generate KLP64D mutations, we used EMS mutagenesis and screened for lethal mutants that failed to complement Df(3L)Klp64D A8 . n123 . We isolated eight mutants that fell into three different complementation groups. Meiotic recombination mapping with respect to roughoid and hairy placed the lethality in all of these chromosomes near the KLP64D gene. One of the complementation groups containing three of the EMS alleles, Klp64D k1 , Klp64D k5 , and Klp64D k33 , identifies the KLP64D gene, since these mutants show changes in the KLP64D coding sequence. Specifically, Klp64D k1 has a change from Gln (CAA) to a stop (UAA) at codon 13, Klp64D k5 has a change from a highly conserved Glu (GAG) to Lys (AAG) at codon 551, and Klp64D k33 has a change from T to G in the 3′ untranslated region 4 bp after the predicted normal stop codon. We presume that Klp64D k33 causes mRNA instability, since its phenotype is similar to that of Klp64D k1 , which is likely to be a null-mutant since it has a stop codon early in the gene. Heteroallelic combinations among these mutants give some complementation and a few very uncoordinated and near-paralyzed adults. All three KLP64D mutants are homozygous lethal. In a typical crowded culture, these mutants die at or before the early third instar larval stage. If the mutants are raised in conditions of low density and without competition from wild-type larvae, a few mutant animals will survive to be pharate pupae and occasionally eclose as fully formed adults. All of these mutant adults exhibited an acutely uncoordinated walk, are unable to stand, and died in a few hours. Mutant third instar larvae exhibit slight to severe sluggishness, and roll abnormally from side to side during crawling. These observations suggest a neuronal function of the KLP64D gene. To confirm that mutations in the KLP64D gene were responsible for the lethality and other behavioral defects, we used genetic rescue experiments. Although large KLP64D genomic constructs were unstable in bacteria, we were able to construct pUAS64D, which contained an ∼3.6 kb EcoR1/HindIII genomic fragment with a full-length KLP64D coding sequence, 447 bp of 5′ upstream sequences, including the TATA box, and about 0.8 kb of 3′ sequences in pUAST . We reasoned that expression of KLP64D from this construct would be initiated from its own promoter if the necessary enhancer element is present within the limited 5′ upstream region. Alternatively, the GAL4-UAS at the 5′ end can be used to drive KLP64D expression through the tissue-specific expression of specific promoter–Gal4 fusion genes . We obtained several independent stable transformants and crossed them to various Klp64D alleles to test whether they can rescue the lethality and other abnormalities. The pUAS64D construct by itself rescued the lethality and uncoordinated behavior of Klp64D k1 and Klp64D k5 , either in homozygous condition or hemizygous with the Df(3L)Klp64D A8 . n123 chromosome. These results confirm that the lesions in the KLP64D gene caused the lethality and the uncoordinated behavior. We also used genetic rescue experiments to establish further that KLP64D is a true functional homologue of KIF3A and KRP85. Thus, a complete KIF3A cDNA was placed under the control of GAL4-UAS sequences and introduced into the Drosophila genome by P-element transformation. Then we combined this transgene with a transgene encoding GAL4 driven by the neural elav promoter . This combination of elements gave complete rescue of Klp64D k1 heterozygous with a Df(3L)Klp64D A8 . n123 chromosome. Thus, when yw/Y ; UAS-Kif3A<w+>/+; Df(3L)Klp64D A8 . n123 elav-Gal4 <w+> /TM3, Ser e <y+> was crossed to yw/yw; +/+; Klp64D k1 /TM3 Ser e <y+>, 92 progeny carrying UAS-Kif3A<w+>/+; Df(3L)Klp64D A8 . n123 elav-Gal4 <w+>/ Klp64D k1 were recovered, whereas no progeny carrying +/+; Df(3L)Klp64D A8 . n123 elav-Gal4 <w+>/ Klp64D k1 were recovered out of a total of 414 progeny, which is well within expected proportions. The expression pattern and the behavioral defect in Klp64D mutant animals led to the hypothesis that the KLP64D motor complex might be involved in transporting the components of the acetylcholine-producing machinery. We tested this view by staining Klp64D mutant animals with ChAT antiserum. In wild-type third instar larvae, we found ChAT in abundance at many synapses in the neuropil of the ventral nerve chord and in the optic lobes of the larval brain , whereas very little was observed in the cell bodies of the brain cortex. Counter staining with rhodamine-labeled α-bungarotoxin, which binds to the nicotinic acetylcholine receptors (nAChR) , showed that the ChAT antigen is distributed in the neuropil similar to nAChR . No ChAT immunoreactivity was observed in the axons of wild-type larvae. This staining pattern is consistent with previous reports . In Klp64D k1 and Klp64D k5 homozygous third instar larvae, we observed accumulation of ChAT immunoreactivity in the cell cortex of the ventral ganglion. Closer examination of these mutant samples revealed considerable ChAT immunogen in the nerve roots and in the cell bodies . In some samples, the ChAT immunoreactivity in the neuropil was reduced strikingly (data not shown) relative to the wild-type control. The abnormal staining pattern suggests that loss of KLP64D affects ChAT transport to the synapse, since no such ChAT staining is observed in the wild-type axons . The α-bungarotoxin staining of nAChR on the postsynaptic membrane in the Klp64D k1 homozygous mutants showed no detectable difference from wild-type control. We tested whether the loss of ChAT immunoreactivity at the synapse and the abnormal accumulation in the axons are a secondary effect of the lethality or other neural defects in the mutant animals lacking KLP64D. We examined Khc − and Klc − larvae, which die at the second instar and third instar larva stage, respectively, with significant axonal transport defects . We found that the intensity of ChAT staining in late second instar Khc − as well as in late third instar Klc − brains was not significantly reduced or altered as compared with the wild-type. Hence, it is unlikely that reduction of ChAT immunoreactivity is a secondary effect of lethality or general axonal transport defects. To test whether ChAT accumulation in Klp64D k1 axons is a specific effect of KLP64D malfunction or a result of general axonal clogging, we stained Klp64D k1 homozygous larvae at the third instar stage with antiactin , anti-SYT (data not shown) , and anti-CSP . We used Klp64D k1 /TM3 larvae as the wild-type control . Since both SYT and CSP are integral parts of synaptic vesicles, they are transported to all chemical synapses and are likely to accumulate in general axonal clogs as seen in Khc and Klc mutants . We found no accumulation of SYT and CSP in the axons of the Klp64D mutant larvae . In addition, we also stained the larvae with anti-HRP, antitubulin, and antiactin and found no detectable accumulation in the axon. Finally, we also noted that ChAT accumulation in Klp64D mutant axons is distinct from the clogs found in Klc or Khc mutants , since these accumulations do not cause obvious swelling of the axons. In fact, EM observation suggests that general CNS neuronal and axonal morphology is normal in Klp64D mutants (data not shown). Therefore, mutations in the Klp64D gene appear to cause selective accumulation of ChAT as opposed to general clogging. In fact, these results suggest that there are probably no clogs in these axons, which is dissimilar to Klc and Khc mutants. In this paper we report two principal findings. First, we found that KLP64D and KLP68D, which appear to be mutually associated motor polypeptides of the kinesin-II family, are expressed preferentially in cholinergic neurons in Drosophila . Second, we found that mutations in the KLP64D gene cause accumulations of ChAT in neuronal cell bodies and axons. Since other apparently generic axonal cargos do not appear to accumulate in these mutants, and since the overall neural morphology of mutant animals is relatively normal, the data suggest that these kinesin motor proteins are required for transport of ChAT, and perhaps a small number of other cargos in axons of cholinergic neurons. There are several important implications of these findings. A striking conclusion that can be drawn from the preferential expression data and the relative selectivity of the axonal transport defect in KLP64D mutants is that KLP64D and KLP68D in Drosophila are required for axonal transport of a subset of axonal cargos, one of which is ChAT. This conclusion suggests that a common assumption about the general organization of the axonal transport machinery may be incorrect. That is, axonal transport is usually thought of as a neuronal process that uses a general machinery of neuronal motors, adapters, and carriers for transport of both specialized and ubiquitous axonal components . Consistent with this view, there is good evidence for the existence of a general axonal transport machinery that uses ubiquitous neuronal kinesins such as unc104/KIF1A, KIF1B, and various forms of true kinesin to transport cargos used in all neurons such as mitochondria, synaptic vesicle components, and channels . However, our data suggest that there also may be specialized transport pathways activated or enhanced in specific classes of neurons. These neuronal classes may be defined by neurotransmitter or other phenotype, and may activate specific motors, adapters, and/or carriers for axonal transport. While kinesin-II in Drosophila (KLP64D and KLP68D) may provide the first recognizable example of such a cell type–specific axonal transport system, there may be others awaiting recognition or discovery. Although the reported expression patterns of kinesin motor proteins in vertebrate systems do not provide a clear test of the hypothesis that some motors are harnessed in cell type–specific axonal transport pathways, there are a few relevant anecdotal observations in the literature. In particular, KIF3B, KIF3C, and neuronal KHC have been reported to have expression patterns that are not uniform among neurons . KIF3B was reported to be expressed in a subset of Purkinje cells , whereas neuronal KHC exhibits greatly elevated expression in a subset of neurons . However, at present, in the absence of double staining experiments with good markers of neuronal phenotype, it is difficult to gauge whether some kinesins have strict cell type specificity in vertebrate neurons. Nonetheless, it is clear that even ubiquitous neuronal motor proteins, such as neuronal KHC, may not be expressed equivalently in all neuronal types. Although some of this nonuniformity in expression might be a consequence of overall transport requirements in large neurons, some of the heterogeneity might result from the need for cell type–specific axonal transport pathways in phenotypically differentiated neurons. There is considerable information regarding the function of kinesin-II family members in several organisms. In particular, a variety of data suggest that motors of the kinesin-II family are specialized for the transport of cytosolic, nonmembrane enclosed constituents such as dyneins or central pair components of cilia and flagella. In Caenorhabditis , kinesin-II motors are needed for the function of sensory cilia as demonstrated by the phenotype of osm-3 mutants . In sea urchins, antibody microinjection experiments demonstrate the requirement for these motors in transport of ciliary components, perhaps those of the central pair microtubules . In vertebrates, motors of this family have been found in the immotile dendritic sensory cilium of the rod photoreceptor, where they could move ciliary or sensory components and are required to form early embryonic cilia of the node . Finally, in Chlamydomonas , there is good evidence to support the view that kinesin-II motors are needed to drive the movement of intraflagellar particles or rafts of axonemal components such as inner arm dyneins . Together, these data have suggested a requirement for kinesin-II in transport in ciliated cells or neurons with degenerate dendritic sensory cilia. What has been unclear is the possible role of this family of motor proteins in axons of sensory and nonsensory neurons. Our work in Drosophila suggests that a unifying theme for kinesin-II motors may be that they are specialized to move cytosolic proteins in motile cilia and flagella, modified immotile dendritic sensory cilia, and in certain classes of axons. Since ChAT is generally thought to be a cytosolic protein, its dependence upon kinesin-II in Drosophila for transport may reflect this specialization. There is also evidence that kinesin-II motors may be associated with membranous components . Thus, kinesin-II motors could, in principle, move both complexes of cytosolic protein components in axons and cilia as well as interact with membranous vesicles. Alternatively, the complexes of cytosolic proteins could interact intermittently with membranes to give the observed associations, similar to the suggestion that has been made for bead movements on the surface of Chlamydomonas flagellar membranes . An intriguing possibility in this regard is that the combinatorial interactions of kinesin-II polypeptides in obligate heterodimers might allow some forms to associate with soluble complexes and some to associate with membranous vesicles . In this context, it remains formally possible that ChAT in Drosophila is not moved as a soluble protein complex, but instead as a “hitch-hiker” on membranous vesicles. Further work will be necessary to fully resolve these issues. The transport of soluble proteins such as ChAT is normally attributed to the slow axonal transport machinery, which is driven by an unknown mechanism . However, past work is not conclusive on the question of whether ChAT is transported in the fast or slow axonal transport compartment , and at present there is no evidence as to whether Drosophila has a slow axonal transport pathway. An economical but speculative hypothesis is that although kinesin-II motors are generally thought of as fast axonal transport motors based on their speed of movement in vitro and on the velocity of particle transport in flagella, they could, in principle, also drive some components of the slow transport system. For example, if ChAT-containing particles spent only a small fraction of time interacting with kinesin-II motors, this could lead to slow transport of ChAT. Alternatively, if the overall balance of anterograde versus retrograde transport of ChAT-containing particles is small, then net transport rates of ChAT would be slow. Finally, we note that if either of these parameters can be regulated by the axon, then the actual rate of ChAT transport might also vary in different cell types or circumstances, thus accounting for the diversity in rates reported. In this context, recent work demonstrating that kinesin-I may have a role in movement of intermediate filament subunits in nonneural cells is consistent with this general view . At present the question of how slow transport is driven remains open, but the possibility that kinesin-II motors might play a role warrants further investigation. | Other | biomedical | en | 0.999994 |
10545497 | Cosmids spanning the region where the osm-1 mutation has been previously mapped were analyzed for ORFs that may encode the 20–amino acid peptide previously reported to be part of the OSM-1 protein . Overlapping cosmids were identified using AceDB software, and their sequences were obtained through NCBI GenBank. All predicted ORFs on cosmids T25D1, T27B1, K08B5, F59C12, and C06G1 (provided by Dr. Alan Coulson, Sanger Center, Cambridge, UK) were analyzed for their inclusion of the 20–amino acid peptide sequence that is conserved in both C . elegans and Chlamydomonas homologues of OSM-1 . The peptide was localized to ORF T27B1.1, but the cosmid was found to contain a deletion upon restriction enzyme analysis. The overlapping cosmid C15H5 was used instead for subsequent subcloning. The predicted OSM-1 polypeptide sequence was analyzed for sequence motifs and predicted domains using various algorithms provided on the ExPASy homepage, proteomics tools (http://www.expasy.ch). The osm-1 gene and upstream regulatory sequences were subcloned into the pPD95.77 GFP genomic transformation vector provided by Dr. Andrew Fire (Carnegie Institute of Washington, Baltimore, MD) using standard molecular biology protocols . Specifically, a 3.1-kb osm-1 gene fragment representing the most 3′ end of the osm-1 gene was PCR amplified from cosmid C15H5 using the primers 5′-ttggatcctctgcgtctcttatccc-3′ and 5′-caat cccggg gacaacgaaatagccaacgg-3′ (SmaI site underlined; added to facilitate the in-frame ligation of osm-1 to GFP). This 3.1-kb fragment was shuttled through PCR-Blunt (Invitrogen Corp.) and ligated into pPD95.77 using BamHI and SmaI sites. Then, an SphI-BamHI 5.2-kb fragment representing the 5′ end of the osm-1 gene plus 2.5 kb of upstream sequence was excised from cosmid C15H5 and added to the aforementioned pPD95.77 vector containing the 3.1-kb 3′ end fragment. Finally, a BamHI-BglII 3.8-kb genomic fragment representing the middle of the osm-1 gene was added into the pPD95.77 vector containing the 5.2- and 3.1-kb fragments, fully reconstituting the entire osm-1 gene (9.7 kb) plus 2.5 kb of upstream promoter sequence. Heritable lines of transgenic worms carrying extrachromosomal arrays of OSM-1::GFP–encoding constructs were created by coinjection of the aforementioned osm-1 /pPD95.77, and plasmid pRF4 containing the semidominant marker mutation rol-6 , into homozygous osm-1 (p808) hermaphrodites by methods previously described . Heritable roller lines were selected, and rescue of the osm-1 mutant phenotype was assayed by dye filling as previously described . The uptake of DiI into amphid and phasmid chemosensory neurons was monitored by epifluorescence microscopy. Heritable lines carrying GFP-tagged kinesin-II accessory polypeptide (KAP::GFP, strain 12.5) were made by similar methods and described previously . OSM-6::GFP worms were provided by Drs. Jocelyn Shaw and Robert K. Hermann (University of Minnesota, St. Paul, MN). Males expressing OSM-6::GFP or KAP::GFP were crossed with homozygous mutant che-3 or osm-3 hermaphrodites, and the F1 heterozygotes were selfed to obtain OSM-6::GFP; che-3 , OSM-6::GFP; osm-3 ; and KAP::GFP; che-3 progeny . Che-3 and osm-3 homozygosity was verified by dye filling. We were unable to create OSM-1::GFP; che-3 progeny because of the low male mating efficiency characteristic of roller mutants. The progeny of these crosses were scored for fusion protein expression patterns by laser scanning confocal microscopy and in vivo transport by methods described below. The che-3 , osm-1 (p808) , and osm-3 (p802) strains were obtained through the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN). For transport assays, one strain expressing OSM-6::GFP was used , one strain expressing KAP::GFP was used (12.5), and two strains expressing OSM-1::GFP were used (SL16 and SL17). Adult transgenic worms expressing translational fusions of OSM-1::GFP, OSM-6::GFP, and KAP::GFP were assayed for in vivo transport by time-lapse fluorescence microscopy. Specifically, GFP-expressing worms were anesthetized with 10 mM Levamisole and mounted onto poly- l -lysine–coated slides. Paralyzed, nontwitching worms were selected using bright field microscopy at 10×, and transport was visualized by collecting fluorescent images at 100× at 1 frame per second or 1 frame per 0.5 s intervals using a Nikon Eclipse E600 microscope equipped with a Uniblitz automated shutter driver and Metamorph Imaging System software (Universal Imaging Corp.). Images were collected for a minimum of 1 min, and movies were created from stacked images using Metamorph Imaging software. Still images were taken from individual frames of time-lapse collections. We routinely used 10 mM Levamisole (rather than the 1 mM concentration that is often used with C . elegans) to completely inhibit twitching and, thus, to facilitate microscopic observations of transport. In control experiments, we found that rates of transport were identical in 1 and 10 mM Levamisole (e.g., in 1 mM Levamisole, OSM-6::GFP particles were observed to move at 0.65 ± 0.06 μm/s anterogradely [10 animals, 52 GFP particles], and 1.08 ± 0.07 μm/s retrogradely [10 animals, 30 GFP particles]). Moreover, we found that the paralyzing effects of the anesthetic are reversible at both concentrations; worms treated with 1 or 10 mM Levamisole for periods of time corresponding to the length of typical transport assays completely recover movement within 24 h of being transferred to anesthetic-free buffer. Velocities of transport were determined by analyzing time-lapse transport movies for moving dots of fluorescence. The relative positions of individual fluorescent particles were monitored over multiple frames (≥4) and velocities were calculated by determining total distance moved per given time period using an objective micrometer. Anterograde IFT movement was designated as the movement of fluorescent particles from the transition zone of sensory cilia toward the ciliary endings at the tip/nose of the worm; retrograde transport was designated as movement of fluorescent particles from the tip back toward the transition zone . Bidirectional movement of GFP particles was also analyzed in associated dendrites of amphid and phasmid chemosensory neurons, with anterograde intradendritic transport corresponding to the movement of GFP particles from the cell body toward the transition zone, and retrograde intradendritic transport as the movement back toward the cell body . We previously used a time-lapse fluorescence microscope–based transport assay to visualize the anterograde movement of kinesin-II::GFP (labeled on the KAP subunit) and OSM-6::GFP within sensory cilia (but not dendrites) of amphid chemosensory neurons in living transgenic C . elegans . However, in that study, we were unable to visualize the retrograde transport of these IFT motors and raft particles, which left unanswered the question of whether these proteins are moved back along the cilia and dendrites by a retrograde retrieval pathway , or if instead they are degraded and rendered nonfluorescent at the distal tip of the cilia. Here, we show that kinesin-II and its cargo molecules do in fact move in the retrograde direction as well as the anterograde direction in both dendrites and cilia of amphid and phasmid chemosensory neurons . Moreover, we have extended our analysis to a second presumptive cargo molecule, OSM-1, which required the production of OSM-1::GFP–expressing transgenic lines. Previous work showed that the osm-1 gene is essential for the formation and/or maintenance of chemosensory cilia that detect chemosensory cues, and that osm-1 mutants display defects in ciliary structure resulting in defects in fluorescent dye-filling ability and osmotic avoidance behavior . The OSM-1 protein is encoded by ORF T27B1.1 on genomic cosmid T27B1 . To study the transport of the OSM-1 protein within dendrites and cilia of chemosensory neurons, we constructed transgenic lines carrying extrachromosomal arrays of constructs driving the expression of an OSM-1::GFP fusion protein under the control of the endogenous osm-1 promoter. Specifically, a 12.1-kb genomic fragment that included the osm-1 gene and 2.5 kb of upstream sequences were placed in-frame with the GFP gene in a C . elegans transformation vector and injected into the osm-1 homozygous mutant background. In the osm-1 mutant, defects in sensory ciliary structure give rise to corresponding defects in the uptake of fluorescent dyes such as DiI into six pairs of amphid channel neurons in the head and two pairs of phasmid channel neurons in the tail . The OSM-1::GFP fusion protein appears to be functional as it rescues the osm-1 mutant phenotype as demonstrated by the restoration of the dye-filling ability . Initially, it was only possible to clearly visualize anterograde IFT in sensory cilia , but we are now able to readily visualize retrograde transport as well. Thus, we observed the bidirectional transport of kinesin-II and its presumptive cargo in vivo within the sensory ciliary regions indicated in steps 2 and 3 of Fig. 1 c, by time-lapse fluorescence microscopy of adult transgenic worms expressing KAP::GFP, OSM-1::GFP, and OSM-6::GFP fusion proteins. All three GFP fusion proteins are expressed in amphid and phasmid chemosensory neurons, where they appear to concentrate at the base of the transition zones, which correspond to the basal bodies of motile and sensory cilia. These fusion proteins are seen to emerge from the transition zones as small fluorescent particles that move in a bidirectional fashion along the sensory cilia, displaying anterograde movement from the transition zone toward the tip of the ciliary axoneme and retrograde movement from the cilium tip back toward the transition zone . The average velocities of intraflagellar transport of OSM-1::GFP, OSM-6::GFP, and KAP::GFP, were very similar in a given direction ( Table ), although the average rates differed between anterograde and retrograde IFT ( Table ). The average rates of anterograde transport were ∼0.7 μm/s for these three proteins, which agree with the rates reported previously for OSM-6::GFP and KAP::GFP in sensory cilia . The retrograde transport velocities of OSM-1::GFP, OSM-6::GFP, and KAP::GFP within sensory cilia were also similar ( Table ); average rates of retrograde transport were ∼1.10 μm/sec, which is consistent with the hypothesis that the retrograde IFT transport pathway is shared between these molecules. We observed the bidirectional transport of KAP::GFP, OSM-1::GFP, and OSM-6::GFP in dendritic segments of amphid and phasmid chemosensory neurons . The rates of anterograde movement (corresponding to the movement of GFP particles from the cell body along the dendrite toward the transition zone of sensory cilia) and retrograde transport (corresponding to the movement of GFP particles from the transition zone along the dendrite back toward the cell body) were very similar for all three GFP fusion proteins . Specifically, KAP::GFP, OSM-1::GFP, and OSM-6::GFP showed average rates of anterograde dendritic transport of ∼0.7 μm/sec and average retrograde transport rates of ∼1.0 μm/sec. These velocities are essentially the same as those measured in sensory cilia. In all transgenic worms examined, nonmoving GFP particles were seen along the dendrites. Because these fluorescent spots were of various sizes and immotile, they are likely to be GFP aggregates that have formed within the cell. To investigate whether the CHE-3 cytoplasmic dynein drives the retrograde transport of kinesin-II and its presumptive cargo protein, OSM-6, within sensory cilia, we expressed the KAP::GFP and OSM-6::GFP fusion proteins in a che-3 homozygous mutant background and assayed for transport by time-lapse fluorescence microscopy. The che-3 reference allele, which we used here, has been sequenced and found to contain a premature ochre stop codon approximately halfway through the predicted protein sequence . This truncates the heavy chain at the first amino acid after the third P-loop, which is conserved in all dyneins, eliminating much of the catalytic domain and rendering che-3 a strong candidate null or severe loss-of-function mutation (Wicks, S., C. deVries, H. Van Luenen, and R. Plasterk, manuscript submitted for publication). This conclusion is supported by the observation that che-3 over three separate deficiencies is phenotypically the same as che-3 homozygotes . Genomic cosmid F18C12 encodes the DHC1b orthologue, CHE-3, and this completely rescues the che-3 mutant phenotype based on microscopy observations of ciliary morphology and assays for dye filling and chemotaxis behavior (Wicks, S., C. deVries, H. Van Luenen, and R. Plasterk, manuscript submitted for publication; Grant, W., personal communication). The che-3 ORF, F18C12.1 , predicts a 4,131–amino acid DHC1b polypeptide that, when compared to other proteins in the GenBank sequence database including multiple dynein heavy chains, shares highest homology (44% identity, 54% similarity in the region corresponding to amino acids 898–2,071 of CHE-3) with partial cDNA sequence encoding DHC1b in Chlamydomonas . Strikingly, over many hours of observation, we never saw retrograde IFT in cilia of che-3 mutants, in contrast to the robust transport that we consistently observed in wild-type worms. This inhibition was specific for retrograde IFT since we observed that anterograde IFT continued unabated in the shortened cilium of che-3 mutants. Accordingly, we observed an accumulation of both KAP::GFP and OSM-6::GFP at the distal tips of the slightly truncated cilia in che-3 mutants, compared with the normal enrichment of these fusion proteins in the transition zones of wild-type worms , presumably because of the continuous anterograde movement of OSM-6::GFP and kinesin-II to the distal tip of cilia, but defective retrieval. This accrual of fluorescence at cilia tips was obvious in the amphid sensory cilia of the head and phasmid sensory cilia of the tail . The fact that anterograde IFT persists in che-3 mutant animals suggests that the inhibition of retrograde transport does not reflect a nonspecific disruption of transport in general. To further address whether the inhibition of retrograde transport seen in the sensory cilia was an indirect, nonspecific effect of having an abnormal, truncated axoneme, we monitored OSM-6::GFP expression and transport in the osm-3 mutant background. The che-3 and osm-3 mutants have similar axonemal defects; both are missing the distal segments of the axoneme, but retain their middle and proximal segments . che-3 differs from osm-3 in that the middle segments of its cilia have swollen tips . Using confocal microscopy, we compared the ciliary defects of osm-3 and che-3 and estimated that both are missing the distal 2.5 μm of their axoneme, with che-3 missing an additional 0.8 μm from its middle segment. Thus, the che-3 dynein mutant retains ∼4.2 μm of the middle and proximal segments of their axoneme, of which 1.6 μm is bulbous in nature and the other 2.6 μm appears normal and visibly supports anterograde transport. Bidirectional transport of OSM-6::GFP was normal in the shortened cilia of the osm-3 mutant (0.66 ± 0.07 μm/s anterogradely, 1.09 ± 0.09 μm/s retrogradely), and the OSM-6::GFP fusion protein demonstrated normal localization to the transition zones and cilia . These results argue that the type of inhibition of retrograde transport seen in che-3 mutants is not seen in all structurally abnormal cilia, which is consistent with it being a specific effect. To determine if CHE-3 cytoplasmic dynein was also responsible for the retrograde intradendritic transport of kinesin-II and its presumptive cargoes from the sensory cilia back to the cell body, we analyzed the transport of the KAP::GFP and OSM-6::GFP fusion proteins in sensory neuronal dendrites in the che-3 homozygous mutant background . Although these fusion proteins show dramatic accumulation at the tips of both amphid and phasmid sensory cilia because of defects in retrograde IFT along the ciliary axoneme, the retrograde transport of these proteins from the transition zone back toward the cell body occurs normally in the corresponding dendrite . The retrograde transport of KAP::GFP and OSM-6::GFP in che-3 mutant amphid dendrites occurred at an average velocity of ∼1.0 μm/s in both cases ( Table ), which is similar to their retrograde transport velocities in wild-type backgrounds. Thus, CHE-3 dynein appears to be involved specifically in retrograde IFT . This paper, together with our previous study , documents the use of a fluorescence microscope–based transport assay to observe the bidirectional movement of a motor, heterotrimeric kinesin-II, and two proteins that are essential for sensory ciliary function, OSM-1 and OSM-6, along cilia and dendrites within chemosensory neurons of living C. elegans . Previously, we observed anterograde movement of these molecules along cilia , but retrograde transport was not observed. Improvements in the assay have allowed us to show in the current study that these molecules are indeed moved along cilia and dendrites in a retrograde direction at the same rate, which may reflect the operation of a retrieval pathway for IFT motors and IFT rafts. Moreover, through the use of a che-3 dynein motor mutant, we obtained results that are consistent with the hypothesis that CHE-3 dynein participates in the early phase of retrieval of the anterograde motor, heterotrimeric kinesin-II, and its presumptive cargo molecules by moving these proteins from the distal tip of the ciliary axoneme back to the transition zone . However, the later phases of retrograde transport of these IFT proteins appear to depend on a different motor that drives retrograde intradendritic transport. It is plausible that the transport system that we are characterizing serves to transport key neuronal components from their site of synthesis in the cell body, out along the dendrite, to the distal tip of the sensory cilium. Such components could include chemosensory receptors and other sensory signaling molecules that are concentrated in sensory cilia as well as structural components of ciliary axonemes. The observation that mutations in two components of this transport pathway, OSM-1 and OSM-6, cause defects in both sensory ciliary structure and chemosensory behavior supports this hypothesis. OSM-1 and OSM-6 proteins are members of a set of ∼25 genes that are required for sensory ciliary structure and function in C . elegans . . Both the osm-1 and osm-6 mutants were identified in a screen for osmotic avoidance defective behavior . Both are dye filling defective and have structural abnormalities in axonemes of their chemosensory cilia that cause defects in other behaviors such as chemotaxis and thermotaxis . The osm-6 mutant was cloned and characterized by Collet et al. 1998 and the osm-1 mutant was cloned by Jocelyn Shaw and Stephen Stone (personal communication). The predicted ORF for OSM-1 (T27B1.1) suggests that OSM-1 is a novel, large molecular weight protein with no obvious motifs or transmembrane domains, which is consistent with the hypothesis that OSM-1 is a nonmembrane-bound component of IFT raft particles . The OSM-1 and OSM-6 polypeptides are proposed to be components of C . elegans IFT raft particles that convey structural components such as axonemal precursors that are required for ciliary assembly and/or maintenance . The precise functions of the OSM-1 and OSM-6 proteins are unclear, but they may act as carriers of cargo, or may play a more direct role in axonemal assembly, such as chaperones or structural components of axonemes. The severely shortened axonemes and ectopic microtubule assembly characteristic of osm-1 and osm-6 mutants would be consistent with such roles in ciliary assembly. Alternatively, OSM-1 and OSM-6 may represent signaling molecules that are localized to sensory cilia, and are involved in the reception/integration pathway(s) of environmental stimuli, similar to receptor molecules that are transported from the cell body to their site of action in sensory cilia . Indeed, the OSM-6 protein shows some homology with the 40-kD mammalian protein NGD5, which was identified as an mRNA that is downregulated with prolonged exposure to an opioid receptor agonist and is thus involved in opioid receptor signaling . Both OSM-6 and NDG5 contain potential PxxP motifs that may modulate their interaction with other signaling molecules containing SH3 domains . The transport of OSM-1::GFP, OSM-6::GFP, and KAP::GFP particles is likely to be physiologically relevant because they move very consistently at the same rate and share similar transport velocities, and because both the OSM-1::GFP and the OSM-6::GFP fusion proteins rescue the cilia defects of osm-1 and osm-6 mutants, respectively. It is likely that the IFT rafts containing these proteins function to convey cargo by moving bidirectionally along the ciliary axoneme, picking up cargo in the cell body or at the transition zone, and delivering it to its site of activity or incorporation at the distal tip of the axoneme. The retrieval of kinesin-II and IFT raft particles to the transition zone or to the cell body by retrograde transport could then serve to regenerate the available pool of anterograde IFT motor and raft components. The similar rates of anterograde transport of OSM-1::GFP, OSM-6::GFP, and KAP::GFP in both IFT transport along sensory cilia and dendritic transport along corresponding amphid neuronal dendrites, suggests, but does not prove, that these molecules share a common transport pathway from the cell bodies to the tip of the sensory cilium. Based solely on their similar transport rates and localization, it is plausible to suggest that OSM-1 and OSM-6 are transported by kinesin-II in both sensory cilia and sensory neuron dendrites. This is consistent with the suggestion that OSM-1 and OSM-6 polypeptides represent C . elegans homologues of proteins that are components of the IFT rafts that are transported by heteromeric FLA-10 kinesin in Chlamydomonas . In contrast, the diacetyl receptor, Odr-10, moves along dendrites at a faster rate and, thus, is thought to be transported by a kinesin-II–independent pathway. In addition to heterotrimeric kinesin-II, C . elegans chemosensory neurons contain another heteromeric kinesin complex, Osm-3–kinesin. The Osm-3–kinesin motor does not appear to be responsible for the anterograde transport of IFT raft components as the OSM-6::GFP fusion protein localizes normally and demonstrates normal anterograde IFT and dendritic transport in the osm-3 homozygous mutant background. To directly test the hypothesis that kinesin-II drives the anterograde transport of OSM-1 and OSM-6 will require a method for specifically inactivating this motor protein and assaying for a corresponding inhibition of the transport of OSM-1 and OSM-6 proteins. At the current time, the absence of a kinesin-II mutant makes this difficult and for this reason, we are currently undertaking a reverse genetic approach with the goal of identifying mutations in genes encoding kinesin-II subunits. The availability of mutants in the che-3 gene allowed us to assess the role of the cytoplasmic dynein CHE-3 in retrograde transport. Like anterograde transport, the retrograde IFT and dendritic transport of OSM-1, OSM-6, and KAP occur at similar rates, suggesting that they all may be transported retrogradely toward the transition zone (IFT) or toward the cell body (dendritic transport) by a common mechanism in wild-type worms. We have investigated if the minus end–directed microtubule motor, CHE-3 dynein, was responsible for the retrograde transport within sensory cilia and sensory neuron dendrites. We observed a specific inhibition of retrograde transport in cilia, but not in dendrites of che-3 mutants, indicating a distinct subcellular transport role for the CHE-3 dynein in retrograde IFT. CHE-3 is a divergent form of DHC that shares homology with DHC1b, the dynein isoform responsible for retrograde IFT transport in Chlamydomonas flagella . The C . elegans che-3 mutant has sensory cilia that lack the distal portions of their axoneme, but have normal looking axonemes lying adjacent to normal transition zones . Like osm-1 and osm-6 , che-3 mutants show ectopic assembly of microtubules below the transition zones; but, in contrast to osm-1 and osm-6 which have normal diameter axonemes, che-3 mutant cilia have enlarged, bulb-shaped tips filled with dark ground material . The dark material that accrues at the tips of che-3 mutant sensory cilia is likely because of the accumulation of IFT rafts, representing proteins such as OSM-1, OSM-6, and kinesin-II. In support of this idea, we did observe an accumulation of OSM-6::GFP and KAP::GFP at the tips of sensory cilia and a loss of normal enrichment at the transition zone in che-3 mutant worms, indicating defects in the retrograde transport of these proteins from the distal tip of the cilium back toward the transition zone. Although a mislocalization of OSM-6::GFP in che-3 mutants has been seen before, it was reported as an accumulation in two large bilateral patches near the region of amphid neuron endings . By coupling high resolution confocal microscopy with the analysis of OSM-6::GFP and KAP::GFP transport in che-3 mutants, we were able to directly visualize the anterograde IFT that leads to the accumulation of these proteins, to resolve the sites of accumulation at the distal tip of the cilia, and to document the absence of retrograde transport specifically in cilia (but not the corresponding dendrites). Thus, CHE-3 cytoplasmic dynein is required for the retrograde IFT of kinesin-II and IFT raft particles along sensory cilia. We are proposing that CHE-3 dynein functions as a motor that drives the retrograde transport of kinesin-II and IFT raft particles along axonemal microtubules in sensory cilia. However, we cannot formally exclude the possibility that the inhibition of retrograde IFT in che-3 worms is an indirect, nonspecific consequence of the aberrant axonemes of che-3 mutant cilia. Two key pieces of data seem to argue against this: (1) the ciliary defects would have to specifically affect retrograde transport since we see normal anterograde IFT along the shortened che-3 axoneme; and (2) both anterograde and retrograde transport occur normally in the osm-3 mutant background, which has similarly shortened axonemes. Although we cannot rule out the possibility that retrograde transport may require axonemal structures that are missing or aberrant in che-3 mutants, such structures must be present in the osm-3 mutant where retrograde transport occurs normally. Thus, the defective transport we see is specific for the retrograde direction, and is not a general consequence of ciliary structural deformity. Although retrograde IFT is abolished in sensory cilia in the che-3 mutant background, bidirectional transport occurs normally in the corresponding dendrite, indicating that CHE-3 dynein is not the retrograde motor for these molecules in sensory neuron dendrites. The che-3 mutation is thought to be a null or severe loss-of-function allele (Wicks, S., C. deVries, H. Van Luenen, and R. Plasterk, manuscript submitted for publication; Grant, W., personal publication), so we cannot rule out the possibility that some residual CHE-3 activity may be sufficient to drive retrograde transport in dendrites but not in cilia. However, it is plausible that a different cytoplasmic dynein isoform or another minus end–directed motor protein may be responsible for the retrograde traffic of these and other proteins from the transition zone back to the cell body. Although abundant evidence exists implicating conventional kinesin, monomeric kinesins, and cytoplasmic dynein in bidirectional axonal transport , intradendritic transport has not been well characterized. However, since cytoplasmic dynein isoforms are thought to be responsible for fast retrograde axonal transport, it is plausible to propose they are also responsible for the retrograde transport of kinesin-II and associated cargo in C . elegans chemosensory dendrites. There are at least two cytoplasmic DHC genes in C. elegans : one that is encoded by ORF T21E12.4 on genomic cosmid and more homologous to DHCs found in other organisms that are involved in various forms of intracellular transport ; and CHE-3, which is encoded by the F18C12.1 ORF on cosmid F18C12 . The former was previously purified from whole worm extracts and may be responsible for the retrograde dendritic transport of kinesin-II and cargo molecules. Alternatively, there are kinesin-related motors that have carboxy-terminal motor domains and are thought to move toward the minus ends of microtubules . In adult mouse neurons there is a carboxy-terminal motor, KifC2, which localizes to dendrites, axons, and cell bodies of neurons in the central and peripheral nervous systems . KifC2 may be involved in retrograde axonal transport and/or the transport of multivesicular bodylike organelles in dendrites . The C . elegans genome sequencing project has identified at least six ORFs that may encode carboxy-terminal motor proteins and, therefore, potential retrograde transport motors, but a true homologue of KifC2 has not been described. However, it should be noted that the microtubule polarity in amphid and phasmid neuron dendrites has not yet been determined. A requirement for a minus end–directed motor would only be true if the microtubules in sensory dendrites are uniform, with plus ends distal to the cell body, similar to the uniform microtubule arrangement characteristic of vertebrate axons. If these dendritic microtubules are actually of mixed polarity, as seen in the dendrites of some vertebrate neurons , then in theory either plus or minus end motors could facilitate retrograde dendritic traffic. The time-lapse fluorescence microscope–based assay described here allows the direct visualization of the in vivo transport of GFP-tagged motor and cargo molecules in both wild-type and mutant backgrounds, and enables us to assess the role of intradendritic and intraflagellar transport in chemosensory neuronal function and behavior in this simple animal. This approach has already revealed that the presumptive retrograde motor, CHE-3 dynein, participates in the retrograde intraflagellar transport of key ciliary components. Moreover, the transport pathway that we are studying appears to play a critical role in nervous system function as mutations in the che-3 , osm-1 and osm-6 genes display severe defects in sensory cilia. Furthermore, because all known chemical attractants, repellents, and pheromones are sensed through ciliated receptors in C . elegans , these mutants are defective in many essential behaviors. The use of this transport assay in concert with the multiple mutants that exist in sensory ciliary structure and function should allow a detailed elucidation of the role of motor and cargo molecules in the pathway of sensory ciliary assembly and function, and in other basic neuronal processes, many of which are likely to be conserved in higher organisms. | Study | biomedical | en | 0.999997 |
10545499 | SPP, dihydroSPP, sphingosine, N,N -dimethylsphingosine, and C2-ceramide were from Biomol Research Laboratory Inc. [γ- 32 P]ATP (3,000 Ci/mmol) was purchased from Amersham Corp. Alkaline phosphatase (bovine intestinal mucosa, type VII-NT) and pertussis toxin were from Sigma Chemical Co. Serum, medium, and G418 were obtained from Biofluids, Inc. Restriction enzymes were from New England Biolabs Inc. Monoclonal antibodies against c-myc were from Zymed, and anti–mouse Texas red dye–conjugated goat antibody was from Jackson ImmunoResearch Laboratories, Inc. The Anti-Fade kit was from Molecular Probes. The bromodeoxyuridine incorporation detection kit and anti–mouse FITC-conjugated IgG were obtained from Boehringer Mannheim Biochemicals. Bisbenzimide hydrochloride was from Calbiochem Corp. Acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) was from Bachem. Anti-Fas IgM (clone CH-11) was from Upstate Biotechnology Inc. NIH 3T3 fibroblasts and human embryonic kidney cells were grown in high glucose DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l -glutamine supplemented with 10% calf serum and fetal bovine serum, respectively . Jurkat T leukemia cells were cultured in RPMI 1640 containing 10% fetal bovine serum. Before addition of exogenous stimuli, cells were resuspended at a density of 0.75–1 × 10 6 cells/ml in medium containing 5 μg/ml transferrin and 5 μg/ml insulin in place of serum . SPHK1a was subcloned into a modified pcDNA3 vector (Invitrogen Corp.) to express proteins with an NH 2 -terminal c-myc epitope tag (a gift from Dr. Peter Burbelo, Georgetown University, Washington, DC) by PCR using a 5′ primer with a BamH1 restriction site (5′-GAG GGATCC GAACCAGAATGCCCTCGAGGA-3′), and as the 3′ primer, the last 21 nucleotides of the SPHK1a sequence with an EcoRI overhang (5′-GAG GAATTC TTATGGTTCTTCTGGAGGTGG-3′). For transient expression, plasmids were transfected into cells using Lipofectamine Plus (Life Technologies, Inc.) according to the manufacturer's instructions at a 5:1 ratio with pCEFL-GFP, which encodes green fluorescent protein (GFP; a generous gift of Dr. Silvio Gutkind, NIH, Bethesda, MD). Transfection efficiencies were typically 30 and 40% for NIH 3T3 and HEK293 cells, respectively. Jurkat T cells were transfected by electroporation at a cell density of 10 6 cells/ml with 15 μg of DNA using a Gene Pulser apparatus (Bio-Rad Laboratories) at 400 V and 960 μFa. Stable transfectants containing pcDNA3 plasmids were selected in medium containing 1 mM sodium pyruvate and 0.5 g/liter G418 (NIH 3T3 fibroblasts) or 1 g/liter G418 (HEK293 and Jurkat cells). For all experiments, nonclonal pools of stably transfected cells were used to avoid clonal variability. Cells were incubated in low serum or serum-free media for at least 24 h. The media was then removed, cells were washed with PBS and scraped in 1 ml 25 mM HCl/methanol, and SPP levels were measured essentially as described . In brief, lipids from the cells were then extracted with 5 ml of chloroform/methanol/1 M NaCl (2:1:2, vol/vol) containing 100 μl 3 M NaOH. SPP is water soluble at alkaline pH, and partitions into the aqueous phase. SPP in the aqueous phase was dephosphorylated with alkaline phosphatase (25–50 U) in buffer containing 1.2 M glycine buffer pH 9.0 and 75 mM MgCl 2 . After 1 h at 37°C, 40 μl concentration. HCl was added, and sphingosine was extracted and quantitated by phosphorylation with sphingosine kinase and [ 32 P]ATP . Total phospholipids in cellular lipid extracts were quantified by a colorimetric reaction with malachite green exactly as previously described . HEK293 cells were grown to subconfluency in 100-mm dishes in 10% FBS media, washed and incubated in serum-free media containing 40 μCi/ml [ 32 P]orthophosphate for 24 h to label the phospholipid pools to isotopic equilibrium. In some experiments, sphingosine (5 μM) was added to the media 10 min before termination of the 24-h incubation period. The media was then collected and the cells scraped from the dishes. Cellular and secreted [ 32 P]SPP were extracted in alkaline conditions as described above, followed by acidic extraction with chloroform/methanol/concentration. HCl (100:100:1, vol/vol), to partition [ 32 P]SPP into the organic phase. [ 32 P]SPP was resolved on TLC with 1-butanol/ethanol/acetic acid/water (80:20:10:20, vol/vol), visualized and quantified as described . Cells were incubated in low serum or serum-free media for at least 24 h, and then harvested and lysed by freeze-thawing in buffer containing 20 mM Tris, pH 7.4, 20% glycerol, 1 mM β-mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 40 mM β-glycero-phosphate, 15 mM NaF, 10 μg/ml leupeptin, aprotinin and soybean trypsin inhibitor, 1 mM PMSF, and 0.5 mM 4-deoxypyridoxine. Cell lysates were fractionated into cytosol and membrane fractions by centrifugation at 100,000 g for 60 min 4°C. Sphingosine kinase activity was determined in the presence of 50 μM sphingosine, dissolved in 5% Triton X-100 (final concentration 0.25%), and [ 32 P]ATP (10 μCi, 1 mM) containing MgCl 2 (10 mM) as previously described , and specific activity was expressed as picomoles of SPP formed per minute per milligram protein. Cells grown on glass coverslips coated with collagen I were incubated overnight in DMEM supplemented with 2 μg/ml insulin, 2 μg/ml transferrin, and 20 μg/ml BSA. Cells were washed with PBS and fixed in 3.7% formalin and 0.1% Triton X-100 for 20 min. After washing with PBS, cells were permeabilized for 10 min with 0.5% Triton X-100 in PBS, washed again, and incubated with anti–myc antibody for 20 min at room temperature. After washing, cells were incubated with anti–mouse antibody conjugated with fluorescein or Texas red for 20 min. After washing three times with PBS, coverslips were mounted on slides using an Anti-Fade kit and cells were photographed using an inverted fluorescence microscope (Eclipse TE200; Nikon Inc.) connected to a digital camera . 24 h after transfection, NIH 3T3 cells were serum starved in DMEM supplemented with 2 μg/ml insulin, 2 μg/ml transferrin, and 20 μg/ml BSA, and then stimulated with various agents. After 16 h, cells were incubated for 3 h with bromodeoxyuridine (BrdU, 10 μM), and then fixed in 4% paraformaldehyde containing 5% sucrose, pH 7.0, for 20 min at room temperature. After washing with PBS, cells were incubated in permeabilization buffer (0.5% Triton/PBS, pH 7.4, containing 10 mg/ml BSA) for 20 min at room temperature, and then incubated for 1 h at room temperature with monoclonal anti–BrdU antibody in the presence of DNAse (1,000 U/ml) . After washing with PBS, cells were stained with Texas red–conjugated anti–mouse antibody in 5% BSA/PBS for 1 h, washed with PBS, and then photographed using an inverted fluorescence microscope connected to a digital camera. Cells expressing GFP and cells with positive BrdU staining were counted. At least three different fields were scored with a minimum of 100 cells scored per field. Stably transfected NIH 3T3 fibroblasts were plated in 24-well clusters at a density of 5 × 10 3 cells/well in DMEM containing 10% calf serum. After 24 h, cells were washed with DMEM containing 0.5% calf serum and incubated in same media. The media was replaced every 2–3 d. At the indicated times, cultures were pulsed with 1 μCi of [ 3 H]thymidine for 6 h and radioactivity incorporated into trichloroacetic acid–insoluble material measured as previously described . Values are the means of triplicate determinations and standard deviations were routinely <10% of the mean. Stably transfected NIH 3T3 fibroblasts were trypsinized and counted. Aliquots containing 2 × 10 6 cells were centrifuged, washed twice with PBS, and resuspended in 40 mM citrate buffer, pH 7.6, containing 250 mM sucrose and 5% DMSO. After propidium iodide staining of cellular DNA, cell cycle analysis was performed with a FACStar plus® flow cytofluorometer (Becton Dickinson & Co.) . Stably transfected NIH 3T3 fibroblasts (1,000 cells) were plated in 24-well plates in DMEM containing 10% calf serum. After 24 h, cells were washed twice with DMEM and then grown in DMEM containing 0.5 or 10% calf serum. At the indicated times, cells were washed with PBS, fixed with 70% ethanol for 10 min, and stained with crystal violet. Incorporated dye was dissolved in 100 μl of 0.1 M sodium citrate in 50% ethanol, pH 4.2, and the absorbance was measured at 540 nm . In some experiments, cells were trypsinized and counted in a hemocytometer. Apoptosis was assessed by staining cells with 8 μg/ml Hoechst in 30% glycerol/PBS for 10 min at room temperature as previously described . Cells expressing GFP were examined with an inverted fluorescence microscope. Apoptotic cells were distinguished by condensed, fragmented nuclear regions. The percentage of intact and apoptotic nuclei in cells expressing GFP fluorescence was determined . A minimum of 500 cells were scored in a double-blinded manner to minimize subjective interpretations. In some experiments, viable cells were determined by trypan blue exclusion. Enzyme reactions were performed in 96-well plates with 20 μg of cytosolic proteins and a final concentration of 20 μM Ac-DEVD-AMC substrate as previously described . Fluorescent AMC product formation was measured over a 30-min period at excitation and emission wavelengths of 360 and 460 nm using a Cytofluor II fluorometer plate reader (PerSeptive Biosystems). Cytosolic fractions (10–25 μg) were boiled in Laemmli sample buffer, separated on 10 or 15% SDS-PAGE, and blotted to nitrocellulose. The membranes were blocked with nonfat dry milk in 0.1% Tween-20–PBS for 2 h, and incubated overnight with anti–c-myc monoclonal antibody (1 μg/ml) or 2 h with rabbit antiserum specific for the p17 subunit of caspase-3 (kindly provided by Dr. Donald Nicholson, Merck), in the same buffer containing 1% nonfat dry milk. Immunocomplexes were detected by enhanced chemiluminescence as described previously . Similar to our previous results with transiently transfected cells , sphingosine kinase activity in cell lysates from NIH 3T3 and HEK293 cells stably expressing c-myc–tagged SPHK1a was dramatically increased . Western blot analysis of cytosolic fractions using anti–c-myc antibody revealed a specific protein band with an apparent molecular weight consistent with the predicted size of c-myc–sphingosine kinase that was absent in vector-transfected cells . SPP levels were also elevated in cells expressing sphingosine kinase by four- to eightfold , a level that did not correlate with the large fold increase in sphingosine kinase activity measured in vitro. One possible explanation for this discrepancy is that availability of cellular sphingosine might limit the production of SPP. In agreement with our previous results , when cells were acutely treated with exogenous sphingosine, which is readily taken up , levels of SPP were further increased three- to sixfold, suggesting that although availability of sphingosine may be important for regulating SPP levels, it is probably not the only critical factor influencing levels of SPP in cells overexpressing sphingosine kinase. Since activated platelets can release SPP , and the EDG family of G protein–coupled SPP receptors have recently been identified , it was important to determine whether sphingosine kinase–transfected cells, which have notable increases in SPP levels, secrete SPP into the medium. No significant release of SPP into the extracellular media could be detected by our mass measurements, even after addition of sphingosine to both NIH and HEK293 sphingosine kinase–transfected cells. To increase the sensitivity of detection of secreted SPP, we labeled HEK 293 cells to isotopic equilibrium with [ 32 P]P i and analyzed the labeled SPP in cells as well as in the medium . Despite the large increases in [ 32 P]SPP detected in cells overexpressing sphingosine kinase, there was no detectable labeled SPP released into the medium. Similar results were also found with NIH3T3 fibroblasts. However, in agreement with previous studies , we could readily detect secretion by human platelets after thrombin treatment. Both methods to measure SPP levels gave identical increases in intracellular SPP in transfected and sphingosine-treated cells. Based on the sensitivity of these methods (1–2 pmol SPP/sample), it is estimated that the concentration of SPP in the extracellular media must be <0.4 nM, a concentration well below the K d for binding of SPP to its receptors (8–25 nM) . Both membrane-associated and cytosolic sphingosine kinase activities have been described in mammalian tissues and cell lines . However, the amino acid sequence of murine SPHK1a suggests that it should be a cytosolic protein . In agreement with our previous study on transiently transfected cells , most of the sphingosine kinase activity in cells stably expressing c-myc–tagged sphingosine kinase was cytosolic and the relative amounts of sphingosine kinase activity in cytoplasmic versus membrane fractions were similar in vector- and sphingosine kinase–transfected cells ( Table ), suggesting that the small c-myc tag does not affect localization of sphingosine kinase. Immunohistochemistry with antibodies against c-myc revealed that sphingosine kinase has a diffuse distribution in the cytosol and somewhat denser expression in perinuclear sites in both NIH 3T3 and HEK 293 cells . Previously, we and others have shown that PDGF stimulates sphingosine kinase activity in various cell types . PDGF stimulated cytosolic c-myc–sphingosine kinase activity in transfected NIH 3T3 fibroblasts to a similar extent as its effect on endogenous sphingosine kinase ( Table ), indicating that c-myc–sphingosine kinase activity is regulated by the signaling pathways triggered by growth factors in the same manner as the native enzyme. Unlike activation of protein kinase C, activation of sphingosine kinase by PDGF does not induce significant translocation to the membrane fraction. Collectively, these data suggest that cells overexpressing sphingosine kinase are a useful tool to study intracellular actions of SPP. Interestingly, endogenous SPP was present in both the cytoplasm and membrane fractions and overexpression of sphingosine kinase markedly increased both by nearly the same extent ( Table ). In contrast to most phospholipids, glycosphingolipids, and other sphingolipid metabolites, such as ceramide and sphingosine, which are found mainly in membranes, 20% of the total cellular SPP is in the cytosol ( Table ). Studies with sphingosine kinase inhibitors suggested that the mitogenic effect of SPP might be due to intracellular actions . However, others have suggested that the mitogenic effect is mediated by binding of SPP to the EDG family of G protein–coupled receptors . In view of this controversy, it was of interest to examine the proliferation of cells whose intra- rather than extracellular levels of SPP are increased after transfection with sphingosine kinase. Transient expression of c-myc–sphingosine-1-phosphate (SPHK) in NIH 3T3 cells not only increased intracellular levels of SPP, it also increased the proportion of cells in the S phase of the cell cycle measured as incorporation of BrdU into nascent DNA . The growth promoting effects of sphingosine kinase were further enhanced by suboptimal concentrations of PDGF (1 ng/ml) and serum (0.1%) , which are known to stimulate sphingosine kinase . However, addition of exogenous SPP increased BrdU incorporation in empty vector-transfected cells twofold, but had no effect on sphingosine kinase–transfected cells, suggesting that these cells are already maximally stimulated by their intracellular SPP. It should be pointed out that these experiments were carried out in the presence of 2 μg/ml insulin, which is a known survival factor for these cells and was used to prevent cell death caused by serum deprivation. Since transient expression of sphingosine kinase increased the proportion of cells in the S phase, it was of interest to determine whether there was a corresponding increase as a result of stable transfection. Nonclonal pools of stably transfected cells were studied to avoid potential phenotypic changes due to selection and propagation of clones derived from single individual cells. Stable expression of sphingosine kinase had a dramatic effect on [ 3 H]thymidine incorporation into DNA in cells cultured in low serum media, while DNA synthesis was low in empty vector-transfected cells over the course of 8 d in 0.5% serum . DNA flow cytometry was used to characterize the distribution of cells in the cell cycle. FACS ® analysis revealed that after 2 d in 0.5% serum, >95% of the vector-transfected NIH 3T3 fibroblasts were in G 0 /G 1 phase and only a small fraction were in S and G 2 /M phases ( Table ). Overexpression of sphingosine kinase reduced the fraction of cells in G 0 /G 1 and, in agreement with the DNA synthesis data, increased the proportion in the S phase and, to a lesser extent, in the G 2 /M phase. Even in the presence of 10% serum, which markedly increased the proportion of cells in the S phase and G 2 /M phase, sphingosine kinase transfection further increased the proportion of cells in the S phase ( Table ). This data suggests that either a greater proportion of sphingosine kinase–transfected cells are cycling and/or that the duration of the G 1 phase is shortened compared with vector-transfected cells. Growth curves in low serum of vector and sphingosine kinase–transfected 3T3 fibroblasts diverged after 2 d and growth of the sphingosine kinase–transfected cells was markedly enhanced thereafter, demonstrating that the alteration in cell-cycle distribution resulted in increased growth rate . Moreover, even in the presence of 10% serum, sphingosine kinase expression was still able to significantly increase proliferation , and to increase the saturation density (not shown). Analyses of the growth curves during the exponential growth phase revealed that sphingosine kinase expression decreased the doubling time in 0.5% calf serum from 34 ± 1.4 to 27 ± 1.8 h, although no differences were observed in the doubling times in full serum (vector, 17 ± 2.2 h; SPHK, 16 ± 1.2 h). Furthermore, computation of the parameters of the cell cycle during exponential growth revealed that sphingosine kinase markedly shortened the duration of G 1 phase of the cell cycle by 31 ± 2% in low serum, suggesting that sphingosine kinase is important for nontransformed cells to progress through the G 1 -S boundary. However, in cells growing in full serum, although overexpression of sphingosine kinase increases the proportion of cells in S phase with a corresponding decrease in G 0 /G 1 ( Table ), there were no significant changes in the duration of either of these phases of the cell cycle. To further investigate the possibility that the EDG family of SPP receptors might be involved in the proliferative response induced by overexpression of sphingosine kinase, stably transfected 3T3 fibroblasts were treated with pertussis toxin, as these receptors are known to act through pertussis toxin-sensitive G proteins . In contrast to many other biological responses, including the mitogenic effect of exogenous SPP, which are inhibited by pertussis toxin , it did not abrogate the increase in DNA synthesis induced by expression of sphingosine kinase in the presence of PDGF, suggesting that sphingosine kinase acts independently of G i proteins . In contrast, treatment of cells with a specific inhibitor of sphingosine kinase, N,N -dimethylsphingosine (DMS), at a concentration that inhibits sphingosine kinase and decreases SPP levels, blocked the effects of sphingosine kinase overexpression . Similar results were obtained in cells transiently expressing sphingosine kinase (data not shown). Moreover, although 2 μM SPP stimulated proliferation in vector and sphingosine kinase–transfected cells , 10 μM SPP markedly stimulated DNA synthesis of vector-transfected cells while inhibiting proliferation of sphingosine kinase–transfected cells. Interestingly, 10 μM dihydro-SPP, which lacks the trans double bond present in SPP and binds and signals through Edg-1, -3, and -5 , only slightly stimulated DNA synthesis in both vector- and kinase-transfected cells , suggesting that SPP receptors are equally responsive in vector as well as sphingosine kinase–transfected cells. Exogenous SPP has been shown to suppress apoptosis induced by cytokines, such as TNF and Fas ligand , serum deprivation , and to be important in the survival effects of NGF , vitamin D3 , and cAMP . In contrast, more recently it has been suggested that SPP protected human T lymphoblastoma cells from apoptosis induced by antibodies to Fas, CD2, and CD3/CD28, or C6-ceramide by binding to EDG-3 and EDG-5 GPCRs . However, if the intracellular level of SPP is a critical factor that determines cell survival, then it is expected that overexpression of sphingosine kinase should suppress apoptosis. In agreement with previous studies , prolonged serum deprivation induced apoptosis in NIH 3T3 fibroblasts , where shrinkage and condensation of nuclei were clearly evident . Transient and stable expression of sphingosine kinase in NIH 3T3 fibroblasts suppressed the appearance of apoptotic nuclei induced by serum starvation . In contrast, sphingosine kinase expression had almost no effect on apoptosis resulting from treatment with staurosporine , a broad spectrum protein kinase inhibitor that is known to induce apoptosis in normal and neoplastic cells . Moreover, DMS, but not pertussis toxin, inhibited the cytoprotective effect of sphingosine kinase overexpression in stably as well as transiently transfected NIH 3T3 fibroblasts. It should be pointed out that serum withdrawal markedly increases ceramide levels in many cell types, and it has been proposed that ceramide mediates, at least in part, serum deprivation–induced cell death . Thus, it was of interest to examine the effect of sphingosine kinase expression on apoptosis of other types of cells. Expression of sphingosine kinase in HEK293 cells also markedly inhibited apoptosis induced by serum deprivation and by the cell permeable ceramide analogue, C2-ceramide, although to a lesser extent . These cytoprotective effects of sphingosine kinase overexpression were also not inhibited by pertussis toxin treatment. In contrast, apoptosis induced by staurosporine, a well recognized inducer of apoptosis in HEK293 cells , was not suppressed by expression of sphingosine kinase . Caspases, a family of aspartate-specific cysteine proteases, play a critical role in the execution phase of apoptotic cell death by cleavage of a specific set of cytosolic and nuclear proteins leading to disassembly of the cell . Fig. 6 B demonstrates that serum withdrawal induced activation of caspase-3, as shown by the appearance of the p20 and p17 subunits, which correlated with the onset of apoptosis. Overexpression of sphingosine kinase reduced processing of caspase-3 . Human leukemic Jurkat T cells are a well-characterized model system that readily undergo apoptosis and are sensitive to C2-ceramide–induced apoptosis . Moreover, exogenous SPP suppressed Fas- and ceramide-mediated apoptosis in these cells . Serum deprivation or brief treatment of Jurkat T cells (3 h) with Fas monoclonal antibody or C2-ceramide caused extensive cell death, as measured by the appearance of nuclear fragmentation, which was reduced by expression of sphingosine kinase (data not shown). The fluorogenic substrate Ac-DEVD-AMC, which corresponds to the cleavage site found in executioner caspases targets, was used to measure the activation of these caspases. Indeed, serum deprivation, Fas ligation, and C2-ceramide treatment of Jurkat cells resulted in a time-dependent increase in DEVDase proteolytic activity , which preceded the appearance of fragmented nuclei. Overexpression of sphingosine kinase blocked the increase in DEVDase activity induced by serum deprivation and C2-ceramide and had a lesser yet still significant effect on Fas ligation–induced DEVDase activation. In agreement, sphingosine kinase effectively prevented activation of caspase-3, as measured by the decrease of its processing to p20 and p17 forms following serum deprivation and C2-ceramide addition, but had a smaller effect on Fas-induced activation . Apparently contradictory reports describe intra- and extracellular actions of SPP in diverse cell types . While much evidence has accumulated recently indicating that SPP functions as a ligand for the EDG family of GPCRs, which are linked to diverse biological responses , less is known of the role of SPP as a second messenger. SPP has been proposed as an intracellular mediator of cell growth , mainly based on the use of exogenously added SPP in the high micromolar range, stimulation of sphingosine kinase by growth factors, as well as sphingosine kinase inhibitors. For example, in Swiss 3T3 cells, competitive inhibitors of sphingosine kinase block some of the proliferative signals elicited by PDGF , including activation of mitogen-activated protein kinase, cyclin-dependent kinases (Cdc2 and Cdk2 kinases), and activation of the transcription factor AP1 . However, the proliferative response to exogenously added SPP is partially sensitive to pertussis toxin, suggesting the potential involvement of G i -coupled cell surface receptors . Thus, it was important to develop another approach to unequivocally determine the role of intracellularly generated SPP. To this end, we transiently and stably transfected cells with sphingosine kinase, which markedly increased intracellular levels of SPP. In contrast to activated platelets that release stored SPP in a PKC-dependent manner , transfected NIH 3T3 fibroblasts and HEK293 cells did not secrete any detectable SPP, suggesting that only certain cell types are capable of secreting SPP and that these transfected cells should be useful to study the intracellular roles of SPP. Interestingly, SPP levels were increased to the same extent by sphingosine kinase expression in both the cytosol and in membrane fractions, whereas other sphingolipid metabolites, ceramide and sphingosine, are predominantly located in membranes. This observation is very intriguing since it might suggest that, similar to another second messenger, inositol trisphosphate, it can move between intracellular compartments. BrdU incorporation, cell cycle analysis, and growth curves indicate that sphingosine kinase transfection can significantly increase the proliferative rate of nontransformed NIH 3T3 fibroblasts. Enforced expression of sphingosine kinase not only resulted in higher levels of SPP, it also increased the proportion of cells in the S phase of the cell cycle, expedited the G 1 /S transition, and reduced the doubling time, especially in low serum conditions, indicating that intracellular SPP is an important regulator of cell growth. In support of this notion, microinjection of SPP or intracellular generation of SPP by photolysis of incorporated caged SPP , stimulated DNA synthesis of Swiss 3T3 fibroblasts. Moreover, in contrast to the sphingosine kinase inhibitor, pertussis toxin did not suppress proliferation induced by sphingosine kinase overexpression. However, pertussis toxin markedly inhibited SPP-induced proliferation of endothelial cells . It is possible that there is a complex interplay between cell surface receptor signaling and intracellular targets for SPP, which can contribute to its mitogenic response in certain cell types. Moreover, the discovery of G protein–coupled receptors in the nucleus and the presence of intracellular EDG-1 in distinct perinuclear location raises the possibility that intracellular SPP might signal through receptors located inside cells. Sequence analyses identified homologues of sphingosine kinase in numerous widely disparate organisms, including yeast , demonstrating that sphingosine kinase is a member of a novel but highly conserved gene family and is distinct from almost all other known lipid kinases. Interestingly, of all the lipid kinases, sphingosine kinase shares the highest degree of homology to diacylglycerol kinase ζ (DGK-ζ) . However, overexpression of DGK-ζ, which regulated diacylglycerol levels in the nucleus , had opposite effects to sphingosine kinase on the cell cycle, increasing the doubling time and the fraction of cells in the G 1 /G 0 phase of the cell cycle . The effects of SPP on cell growth appear to be evolutionarily conserved, as phosphorylated long-chain sphingoid bases also regulate proliferation and survival of yeast . Spontaneous mutants of Saccharomyces cerevisiae with diminished sphingosine kinase activity had reduced growth rates and failed to pass through the diauxic shift and successfully initiate respiratory growth , suggesting that yeast sphingosine kinase might play a role in mediating growth responses to changes in nutrients. Because exogenously added dihydro-SPP was unable to affect yeast growth , these results further substantiate a role for intracellular SPP in cell growth regulation. In agreement, deletion of SPP lyase, which results in accumulation of phosphorylated sphingoid bases in yeast, causes unregulated proliferation on approach to the stationary phase . Moreover, expression of G 1 cyclin, whose downregulation may be directly involved in mediating the growth arrest induced by starvation, was maintained at a higher level throughout the post-diauxic phase in these lyase-deficient yeast . This growth advantage was associated with a failure to arrest cells in G 1 and a marked increase in stationary phase DNA content per cell. In agreement, overexpression of LBP1 and LBP2, which are specific SPP phosphatases , reduced levels of long-chain phosphorylated sphingoid bases and arrested cells prematurely at the G 1 phase of the cell cycle . These effects on the yeast cell cycle are mirror images of the effects of overexpression of sphingosine kinase and increased SPP levels in mammalian cells that result in shortening of the G 1 phase of the cell cycle and accumulation of cells in the S phase when deprived of growth factors. Sphingosine kinase activity and SPP levels can be regulated by growth and survival factors . Interestingly, the levels of SPP in 3T3 fibroblasts overexpressing sphingosine kinase, despite the enormous increases in in vitro activity, were maximally increased only four- to eightfold. This level was similar to the level of SPP produced in the same cells after stimulation by PDGF and serum, addition of sphingosine, or even SPP itself . These observations suggest that cells tightly regulate their levels of SPP, consistent with its role as a second messenger. In addition to sphingosine kinase, rapid degradation by SPP lyase and/or SPP phosphatase may also play an important role in determining the steady state levels of SPP. Another possible explanation for the lack of correlation of sphingosine kinase activity and SPP levels is that the substrate for the overexpressed sphingosine kinase, sphingosine, may be located in a different subcellular compartment. Exogenous SPP (2 μM) stimulated growth of vector-transfected and sphingosine kinase overexpressing cells; however, high concentrations of SPP (10 μM) inhibit, rather than enhance, growth of sphingosine kinase–transfected cells. In agreement, while increases in intracellular levels of long-chain phosphorylated sphingoid bases enhance growth of yeast , substantially higher increases reduce their growth rate . Paradoxically, although the processes of cell growth and cell death appear to be opposing and mutually contradictory, substantial evidence now suggests that the pathways of apoptosis and mitosis may be mechanistically related or tightly coupled . Mammalian cells overexpressing sphingosine kinase not only had higher growth rates, but also exhibited enhanced survival in serum-free conditions. Previously, it was shown that exogenous SPP can antagonize apoptosis mediated by a number of stress stimuli, including serum withdrawal, TNF-α, Fas ligation, and ceramide elevation . However, the importance of intracellular SPP has been challenged by the recent suggestion that protection from apoptosis may be mediated by binding of SPP to EDG-3 and EDG-5 GPCRs . In contrast, our results indicate that increased cellular SPP levels in various cell types due to overexpression of sphingosine kinase mimics the protective effect of exogenous SPP against apoptosis, especially in response to growth factor withdrawal, supporting the concept that the cytoprotective actions of SPP are mediated intracellularly. In agreement, we previously found that dihydroSPP, which binds to and signals through the EDG family of GPCRs , unlike SPP, did not prevent apoptosis and conversely, the nonhydrolyzable SPP-phosphonate, which does not bind to these receptors, mimicked the effect of SPP on proliferation and survival . Because the sphingolipid metabolite ceramide is a mediator of stress responses , both in mammalian cells and in yeast , we proposed that the relative intracellular levels of these two sphingolipid metabolites (ceramide and SPP) is an important factor that determines whether cells will survive or die . Consistent with this hypothesis, we found that transient expression of sphingosine kinase modified the sphingolipid metabolite balance, resulting in higher levels of SPP, with concomitant decreases in the levels of sphingosine and ceramide . Our present results show that expression of sphingosine kinase in 3T3 fibroblasts, HEK 293, and Jurkat T cells suppressed apoptosis induced by serum deprivation, known to increase ceramide levels, or by the ceramide analogue, C2-ceramide, with concomitant inhibition of activation of the executionary caspase-3. Similarly, transient expression of sphingosine kinase in U937 cells reduced apoptosis induced by both serum starvation and TNF-α treatment, as determined by DNA fragmentation and DEVDase activity assays (Cuvillier, O., and S. Spiegel, unpublished observations), suggesting that the protective effects of intracellularly generated SPP are not restricted to certain cell types. Because the proximal molecular targets by which ceramide activates caspase-3 and related CED-3 subfamily caspases remain largely unidentified, further work is necessary before the target(s) for SPP can be more precisely identified. This survival function of SPP might also be part of an ancient stress response since, in S . cerevisiae , nutrient deprivation activates sphingosine kinase and yeast mutants, with decreased sphingosine kinase activity also displayed an increased sensitivity to heat stress . Furthermore, accumulation of phosphorylated long chain sphingoid bases in S . cerevisiae either due to the deletion of LBP1 and LBP2 genes , or deletion of DPL1 , a gene encoding long-chain–base phosphate lyase , correlates with increased survival at an elevated temperature , suggesting that long-chain–base phosphates may play a physiological role in heat stress resistance in yeast. In summary, this study substantiates a role for sphingosine kinase–derived SPP as a second messenger in cell proliferation and survival. Sphingosine kinase belongs to a new class of lipid kinases, different in structure and biochemical properties than other lipid kinases, such as the phosphatidylinositol 3-kinase family, but similar in the broad spectrum of signals and in vital and versatile cell functions that they regulate. | Other | biomedical | en | 0.999996 |
10545500 | Sodium orthovanadate, β-glycerophosphate, aprotinin, and leupeptin were obtained from Sigma Chemical Co. Histone 2B was purchased from Boehringer Mannheim Corp. γ[ 32 P]ATP was from ICN Biomedicals. Protein A–Sepharose CL-4B was obtained from Pharmacia Biotech Inc. Purified myosin II was a generous gift from Dr. Tom Egelhoff (Case Western Reserve University, Cleveland, OH). To clone PAKa, a pair of degenerate primers was made based on the amino acid sequences conserved among the kinase domains of yeast Ste20 and human PAK65 (the two primers corresponded to the amino acid sequences VAIKK and DFGLAR). A full-length cDNA clone (3.6 kb) was obtained by screening a 12–16 h developmental λZAP library with a probe amplified by PCR. PAKa was disrupted in the thymidine auxotroph JH10 by inserting the Thy gene cassette into codon 791, and transformants were selected. For overexpression experiments, PAKa and PAKa mutants were subcloned into the DIP-J expression vector . PAKa was tagged at the COOH terminus with the FLAG epitope by using primer 5′-TTACTTGTCATCGTCGTCCTTGTATTGTGCAGTTAAATTTTGTAATTG-3′. The HA epitope tag was added to the NH 2 terminus of PAKa by using primer 5′-AAAATGACCTACCCATA-CGATGTTCCAGATTACGCTGAAGAGAAACCAAAAAGTACA-ACTCCACCA-3′. myr PAKa was obtained by PCR using primer 5′-ATGGGTTCATCAAAATCAAAACCAAAAGATCCATCACAAC-GTCGTCGT-3′, a sequence coding for the first 16 amino acids of chicken c-Src, composed of the myristoylation signal and the basic amino acid cluster sufficient for stable membrane association. To select for strains expressing a high level of PAKa, transformants were selected at 40 μg/ml of G418, which in our hands produces clones that express encoded proteins at high levels. To select clones that express at a low level, we selected and maintained cells at 15 μg/ml of G418. Clones were screened to identify those exhibiting a low level of exogenous PAKa expression and exhibiting wild-type phenotypes in actin staining. For immunofluorescence staining, cells were starved in 12 mM sodium phosphate buffer (pH 6.2) for >5 h and fixed with 4% formaldehyde for 5 min. Cells were permeabilized with 0.5% Triton X-100, washed, and incubated with 1.4 μg/ml anti-FLAG mAb or rabbit anti-HA antibody (1/200 dilution) in PBS containing 0.5% BSA and 0.05% Tween-20 for 1 h. Cells were washed in 0.5% BSA containing PBS and incubated with FITC-labeled anti-mouse or anti-rabbit antibodies for 1 h. After washing, cells were observed with a 60× oil immersion lens on a Nikon Microphot-FX microscope. Images were captured with a Photometrics Sensys camera and IPLAB Spectrum software. Anti-HA antibody was purchased from Santa Cruz Biotechnology. Monoclonal anti-FLAG antibody was obtained from Kodak. F-actin was stained with FITC-labeled phalloidin (Sigma Chemical Co.). Rabbit antimyosin II antibody was a generous gift from Dr. James A. Spudich (Stanford University, Stanford, California). Log-phase vegetative cells were washed and resuspended at a density of 2–3 × 10 6 cells/ml in Na/K phosphate buffer and pulsed for 5 h with 30 nM cAMP every 10 min. The cells were collected by centrifugation and resuspended at a density of 2–3 × 10 7 cells/ml. The cAMP receptor activity was downregulated by bubbling air through the cell suspension for 10 min . The 200-μl samples were lysed by mixing with an equal volume of 2× lysis buffer (50 mM Tris, pH 7.6, at room temperature, 200 mM NaCl, 20 mM NaF, 2 mM vanadate, 50 mM β-glycerophosphate, 6 mM sodium pyrophosphate, 2 mM EDTA, 2 mM EGTA, 4 μg/ml leupeptin, 4 μg/ml aprotinin, 2% NP-40, 20% glycerol, 2 mM DTT). 1 μl of antibody was added to 200 μl supernatant and incubated on ice for 1 h. The formed immune complexes were collected with 50 μl of a 1:1 slurry of protein A beads in lysis buffer by incubation under agitation for 1 h at 4°C. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM MOPS, pH 7.4, at room temperature, 25 mM β-glycerophosphate, 20 mM magnesium chloride, 1 mM DTT, 30 mM KCl for myosin II assay). PAKa activity was measured in an immunocomplex kinase assay after immunoprecipitation with the anti-FLAG antibody. The beads were incubated with 75 μl kinase buffer containing 5 μCi γ[ 32 P]ATP, 5 μM cold ATP, and 5 μg histone 2B or myosin II as a substrate. The reaction was stopped by the addition of 25 μl 4× sample buffer and boiling for 5 min. The samples were separated by SDS-PAGE (12.5%), blotted onto a PVDF membrane (Millipore Corp.), and exposed to film. Cells were pulsed with 30 nM cAMP at 6-min intervals for 5 h, and cells were washed and resuspended in Na/KPO 4 buffer containing 200 μM CaCl 2 and MgCl 2 . A small volume of cells was plated on glass-bottomed microwell plates (MatTek, Inc.) and allowed to adhere to the surface for ∼20 min. A micropipette filled with 150 μM cAMP was positioned to stimulate cells by using a micromanipulator (Eppendorf-Netheler-Hinz GmbH), and the response and movement of cells was recorded by using a time-lapse video recorder and NIH Image software (1 image every 6 s). The movement of cells and changes of cell shape were analyzed with the DIAS program . Cytoskeletal proteins were isolated as proteins insoluble in detergent NP-40. Cells were harvested by centrifugation and resuspended at 10 8 cells/ml. cAMP (100 μM) was added to cells and 500 μl of cells were taken at each time point and immediately added to 500 μl of 2× lysis buffer as described for the kinase assay. After vortexing a few times, the tubes were placed on ice for 10 min, then allowed to warm to room temperature for 10 min. The samples were spun for 4 min at 11,000 g and the supernatant was discarded. After washing with 1× lysis buffer, the protein pellet was dissolved by boiling in 2× SDS-PAGE sample buffer. Samples were run in 8% gel, and protein bands were stained with Coomassie blue. Protein bands were scanned, and changes in myosin II content in cytoskeleton were measured by using IPLAB software. Rac1B, RacE, RasG, and human Cdc42 were amplified by PCR and cloned into yeast two-hybrid fish vector pG4-5 as described previously . The clones were sequenced to confirm the reading frame and the absence of mutations. Point mutations corresponding to Q61L in Ras were made with the Transformer Site Directed Mutagenesis Kit from CLONTECH Laboratories, Inc. Constructs containing site-directed mutants were sequenced to confirm the nucleotide substitutions and the absence of other mutations. Rac1B was a generous gift from Su Dharmawardhane (University of Texas, Austin, TX), RacE was from Arturo De Lozanne (Duke University, Durham, NC), RasG was from Robert Insall (University of Birmingham, UK), and HsCdc42 was from Michael Karin (University of California, San Diego, CA). For overlay assays, Rac1B and HsCdc42 were cloned into GST fusion constructs. The constructs were sequenced to confirm the reading frame and the absence of errors in the cloning/amplification. GST fusion proteins were produced in Escherichia coli and overlay assays done according to the methods of Manser et al. 1994 . We identified PAKa in a PCR screen for PAK/Ste20 family kinases using degenerate primers based on conserved sequences in the kinase domains of Ste20 and mammalian PAK. Sequenced PCR products carrying part of a potential PAK family member were used to screen a cDNA library and several family members were identified, one being PAKa. One ∼3.5-kb PAKa cDNA clone contained the complete PAKa open reading frame . The domain structure of PAKa is similar to that of mammalian PAK1 . PAKa has a predicted Cdc42/Rac, a small G protein binding domain (CRIB), a COOH-terminal kinase domain, and a long NH 2 -terminal putative regulatory domain containing a highly acidic region and a potential SH3-interacting polyproline stretch. In addition, the NH 2 -terminal domain contains an Akt/PKB and multiple cAMP-dependent kinase (PKA) consensus phosphorylation sites. Comparisons of the kinase and CRIB domains of PAKa and other members of this family are shown in Fig. 2A and Fig. B , respectively. The PAKa kinase domain has the highest sequence identity to the kinase domain of Dictyostelium MIHCK (59% identity) and human PAK1, mouse PAK3, and Saccharomyces pombe PAK1 (∼48% identity each). The PAKa CRIB domain shares strong homologies with the CRIB domain of Dictyostelium MIHCK and PAKs from other species . To examine the specificity of the PAKa CRIB domain for small G proteins, we used the yeast two-hybrid system to assay the interactions of the PAKa CRIB domain with activated forms of Dictyostelium Rac1B, human Cdc42 (a Dictyostelium homologue has not yet been identified), Dictyostelium RacE, and Dictyostelium RasG, the latter two of which are required for cytokinesis in Dictyostelium . As we show in Fig. 2 C, the PAKa CRIB domain preferentially interacts with DdRac1B and HsCdc42. We confirmed the interaction of DdRac1B and HsCdc42 with the PAKa CRIB using an overlay assay . We observed strong interaction of a GST-CRIB/PAK fusion protein with DdRac1B GTPγS and Cdc42 GTPγS (data not shown). These results are consistent with, but do not prove, that PAKa is a member of the PAK family of Rac/Cdc42-regulated protein kinases. Northern blot analysis indicates that PAKa encodes a single developmentally regulated transcript (3.7 kb) that is expressed in vegetative and early developing cells, and is upregulated during aggregation and multicellular stages . No hybridization signal was detected when the probe was used on a blot carrying RNA isolated from paka null cells (data not shown). PAKa was disrupted by homologous recombination (see Materials and Methods). Random transformants were selected and screened for disruption of PAKa by Southern and RNA blot analysis. All clones that showed disruption of PAKa did not exhibit an mRNA signal on RNA blots, and had the same developmental and growth phenotype. As depicted in Fig. 3 A, paka null cells exhibit a defect in completing cytokinesis when grown in suspension. Under these conditions, the increase in cell number indicates paka null cells grow more slowly than wild-type cells because the cells become multinucleate, as revealed by an increase in number of nuclei in a cell . When grown in suspension for five days, paka null cells are considerably larger than wild-type cells, and DAPI (4′, 6-diamidino-2-phenylindole) staining shows an average of eight nuclei per cell . However, when paka null cells are grown on plastic, they grow predominantly as mono- and dinucleate cells (data not shown), dividing by traction-mediated cytofission, as previously described for myoII null cells . Our data suggest that paka null cells might have difficulties in regulating cytoskeletal organization required for cytokinesis, but karyokinesis is normal. The cytokinesis and developmental defects of paka null cells can be complemented by overexpression of PAKa from the Actin 15 ( Act15 ) promoter (data not shown). To examine the effect of disrupting PAKa on multicellular development, we plated paka null and wild-type cells on nonnutrient agar and followed development. Compared with wild-type cells, development of paka null cells is very delayed . Tight aggregates do not form until 16–17 h, and some mounds arrest at this stage. For those that proceed further, the first fingers are not formed until ∼24 h. Mature fruiting bodies form at ∼36 h, containing sori that are smaller than those of wild-type cells (data not shown). To examine the role of PAKa further, we created a series of PAKa mutant constructs: a kinase dead, putative dominant negative mutant of PAKa made by changing the Lys residue in the ATP binding site in the kinase domain to Ala (PAKa K394A , designated PAKa DN ); a putative constitutively active PAKa (PAKa CA ) containing only the kinase domain ; a PAKa mutant containing the CRIB and kinase domains (PAKa G+K ); and a PAKa carrying the NH 2 -terminal myristoylation signal from Src ( myr PAKa), which has been demonstrated to constitutively target mammalian and Dictyostelium proteins uniformly to the plasma membrane . The structures of these constructs are shown in Fig. 1 B. We overexpressed the mutant PAKs and wild-type PAKa from the constitutive Act15 promoter. For these experiments, the overexpression mutants were selected at high G418 concentrations to yield clones expressing a high level of the specific PAKa protein (see Materials and Methods). Cells expressing PAKa DN have a phenotype similar to, but more severe than, that of paka null cells . Aggregation is very delayed and most mounds arrest and do not develop further. The more severe phenotype of PAKa DN -expressing cells compared with paka null cells may be due to the inhibition of another PAK-like kinase by PAKa DN . Cells overexpressing PAKa CA exhibit severe aggregation defects and never form mounds. We targeted PAKa to the plasma membrane by expressing myr PAKa. Indirect immunofluorescence indicates that myr PAKa, also tagged at the COOH terminus with the FLAG epitope, localizes uniformly to the plasma membrane around cells . These cells exhibit very delayed aggregation and development, suggesting that proper subcellular localization of PAKa might be important for regulating the in vivo function of PAKa during aggregation . Cells overexpressing wild-type PAKa or PAKa G+K exhibit a wild-type pattern of aggregation and multicellular development, and form normal-looking fruiting bodies (data not shown). To examine whether overexpression of PAKa mutants has an impact on the actin cytoskeleton, we performed phalloidin staining of aggregation-competent cells. Wild-type cells are usually polarized and show localized F-actin assembly at lamellipodia of the leading edge and sometimes, to a lesser degree, at the posterior cortical region of the retracting cell body . paka null cells also appear elongated, but are not as polarized in the distribution of the actin cytoskeleton as wild-type cells. paka null cells have multiple, randomly localized F-actin–rich pseudopodia-like protrusions . Cells overexpressing wild-type PAKa (PAKa OE ), although lacking a developmental phenotype, exhibit changes in F-actin organization. PAKa OE cells have multiple F-actin–enriched crowns along the periphery of cells . This pattern is very similar to that of racgap1 null cells and cells overexpressing constitutively active DdRac1B (DdRac1B Q61L ; Chung, C.Y., S. Lee, C. Briscoe, and R.A. Firtel, manuscript submitted for publication), suggesting that the activity of PAKa might be regulated via Rac1B. Despite this aberrant F-actin organization, PAKa OE cells are elongated. Cells expressing PAKa DN lack prominent actin-rich lamellipodia and actin staining is very diffuse and scattered around the cell periphery . Some of the cells show microspikes , but these microspikes do not appear to be enriched in F-actin. In contrast, cells expressing PAKa CA accumulate assembled F-actin in multiple membrane ruffles over the entire cell. Some of these cells are multinucleate, although not to the extent of paka null cells, suggesting that a putative nonregulated form of PAKa, as well as a lack of PAKa, can result in cytoskeletal defects that prevent proper cytokinesis. Moreover, although these cells elongate, they fail to show a polarization of the actin cytoskeleton, consistent with their inability to aggregate properly. These results suggest that PAKa can mediate rearrangements in the actin cytoskeleton. Cells expressing PAKa G+K do not exhibit a prominent change in F-actin distribution (data not shown) compared with wild-type cells and in contrast to cells expressing PAKa CA , suggesting that the CRIB domain might negatively regulate kinase function, consistent with work on human PAK1 . As wild-type PAKa has the CRIB domain, it is possible that the upstream, NH 2 -terminal sequences deleted in PAKa G+K are involved in regulating the function of the CRIB domain in controlling PAKa activation. Cells expressing myr PAKa exhibit domains of F-actin along the entire membrane cortex, consistent with a uniform distribution of myr PAKa along the cortex of the cells . These cells fail to elongate or polarize . We expect that the upregulated assembly of F-actin results from the activity of the membrane-localized PAKa. To examine whether the changes in the cytoskeletal organization described above alter chemoattractant-induced cell migration, we compared the chemotactic movement of paka null cells to that of wild-type cells by examining their movement toward the chemoattractant using the DIAS image analysis software . Cells competent to chemotax toward cAMP (aggregation-competent cells) were obtained by pulsing cells in suspension for 5 h with 30 nM cAMP, conditions that mimic the cAMP signaling during the phase leading up to aggregation in vivo . This protocol maximally induces the expression of aggregation stage genes required for aggregation, including the cAMP receptor, cAR1, and the coupled G protein α subunit, Gα2. Wild-type cells are usually well-polarized and move quickly and linearly toward the cAMP source. As we show in Fig. 6 A, wild-type cells produce pseudopodia almost exclusively at the edge in the direction of the micropipette, and produce very few random lateral or rear pseudopodia (an average of 1.5 lateral pseudopodia per 10 min). Wild-type cells make few changes in the direction of movement, as shown in Fig. 6 A. In sharp contrast, paka null cells produce more random pseudopodia in various directions. In addition, paka null cells exhibit random changes in the direction of movement, which might result from the protrusion of lateral pseudopodia, leading to an inefficient chemotaxis toward the cAMP source. The movement of the four paka null cells shown in Fig. 6 B illustrates these phenotypes. Cells protrude multiple pseudopodia and, in cell c, two pseudopodia in the same cell become dominant (rather than one), resulting in the formation of two independent leading edges and the independent movement of the two halves that remain connected. In addition, paka null cells do not retract their rear cell body as efficiently as wild-type cells. The posterior of wild-type cells contracts and lifts from the substratum after the leading-edge pseudopod has been extended ; paka null cells appear defective in this process . As illustrated by Fig. 6 B, cells a and c, the anterior of the paka null cell continues to extend its cell body toward the cAMP source until the cell reaches a certain length and then the posterior retracts very rapidly. Probably due to the combination of random changes of the direction of movement and the difficulty in retracting the rear cell body, the speed of movement of paka null cells (4.95 μm/min) is about half that of wild-type cells (8.63 μm/min). PAKa DN cells produce more random pseudopodia than wild-type cells and have an increased frequency of turning, similar to paka null cells . PAKa DN cells exhibit difficulty in retracting the rear cell body as depicted in Fig. 6C , Fig. c . As the leading edge of a cell moves forward, the rear cell body elongates, due to the lack of retraction, and then retracts very rapidly. PAKa CA cells, as expected from the upregulated F-actin assembly over the entire cell, are flattened and appear adherent and stationary. The cell–substratum contact area of these cells is significantly larger than that of wild-type cells . Upon stimulation by cAMP, PAKa CA cells do not polarize and produce few dominant pseudopodia. These cells are not capable of changing their shape; hence, their migration is very slow. Our results suggest that PAKa plays an important role in the regulation of cytoskeletal organization, which is presumably required for maintaining cellular polarity and preventing the formation of random, lateral pseudopodia. The inhibition of the formation of random pseudopodia might prevent cells from making random changes of direction when moving up a chemoattractant gradient. To examine where PAKa is localized in polarized, aggregation-competent cells, PAKa was tagged with FLAG at the COOH terminus or HA at the NH 2 terminus, and expressed in wild-type cells. The transformants carrying a construct of tagged PAKa were selected and maintained at a low concentration of G418 to select strains that expressed PAKa at a low level. Strains were selected for those that did not exhibit the altered actin cytoskeletal defects shown in the high overexpression strains used in Fig. 4 and Fig. 5 . The localization of tagged PAKa was determined in aggregation-competent cells by indirect immunofluorescence. FLAG-tagged PAKa mostly localizes to the posterior cortical region of the cell body and is not found in the leading edge where F-actin is concentrated . The staining pattern of FLAG-PAKa in the posterior cortex overlaps with the phalloidin staining of F-actin in that region. Similarly, HA-tagged PAKa localizes to the posterior cortex . Knowing that paka null cells have a cytokinesis defect, we examined the localization of HA-tagged PAKa in wild-type cells undergoing cytokinesis. As depicted in Fig. 7F and Fig. G , HA-PAKa localizes to the cortex of the cleavage furrows of dividing cells. These results are consistent with involvement of PAKa in cytokinesis. The subcellular localization of PAKa during chemotaxis and cytokinesis is very similar to that of myosin II. This raises the possibility that PAKa might regulate myosin II and, in turn, control the cytoskeletal organization in the posterior cell body. To test this possibility, we examined the distribution of a myosin II–green fluorescent protein (GFP) fusion protein in wild-type cells and paka null aggregation stage cells. Myosin II–GFP mainly localizes at the cortex of the rear cell body in wild-type cells as has been described previously . However, in paka null cells, we observe a much more diffuse staining pattern of myosin II–GFP throughout the cells and no posterior cortical cap. We examined the localization of myosin II by indirect immunofluorescence staining using the antimyosin II antibody . Myosin II is concentrated in the posterior cell body in aggregation-competent wild-type cells as shown by the myosin II–GFP fusion, whereas the staining of myosin II in paka null cells does not exhibit a strong subcellular localization. In contrast, we found strong myosin II staining along the membrane cortex of cells expressing myr PAKa, suggesting that anchoring PAKa to the plasma membrane promotes assembly of myosin II filaments at the membrane cortex . This increased myosin II assembly at the membrane cortex is not found in cells expressing myr PAKa DN , reflecting that myosin II assembly at the membrane cortex is due to the PAKa activity. However, cells expressing myr PAKa DN do not have a posterior that is enriched in myosin II. We expect this is because myr PAKa DN functions (like non–myr-tagged PAKa DN ) as a dominant negative protein and inhibits intrinsic PAKa activity. Like myr PAKa, myr PAKa DN predominantly localizes to the membrane cortex as determined by immunostaining . Cells expressing PAKa CA have regions highly enriched in myosin II throughout the cell. These highly enriched myosin II-containing regions probably contain highly assembled myosin II. This result strongly suggests that PAKa might be involved in the regulation of myosin II assembly into the cytoskeleton. To test this idea, we determined the amount of myosin II in cytoskeleton fraction in wild-type cells, paka null cells, and cells expressing PAK CA in both vegetative growth and aggregation stages. In vegetatively growing cells, the level of myosin II assembled into the cytoskeleton in both wild-type and paka null cells is low, compared with cells at the aggregation stage . However, cells expressing PAK CA display a higher level (approximately four times higher than wild-type cells) of myosin II assembly in the cytoskeleton. This result is consistent with PAKa activity being involved in regulating myosin II assembly. The level of myosin II in the cytoskeleton in aggregation-competent, wild-type cells is significantly higher (seven to eight times higher) than that in growth-stage, vegetative cells . This difference is probably due to myosin II assembly in the rear cell body upon cell polarization in aggregation-competent cells. In paka null cells, the level of assembled myosin II is ∼65% of that of wild-type cells, suggesting that PAKa is required to control the level of myosin II assembly. Interestingly, there is no striking difference of myosin II level between wild-type cells and cells expressing PAK CA at the aggregation stage (probably because the assembly of myosin II is already activated in aggregation-competent cells). We examined whether PAKa function is important in controlling changes in myosin II assembly into the cytoskeleton upon cAMP stimulation . As shown in Fig. 9 B, in response to cAMP stimulation, the level of myosin II heavy chain in the cytoskeletal fraction in wild-type cells rises to a peak at 20–30 s and returns to the basal level by 60–80 s, consistent with previous observations . However, this peak of myosin II assembly upon cAMP stimulation into the cytoskeleton is absent in paka null cells. These results suggest that PAKa might be involved in regulating myosin II assembly via either direct phosphorylation of myosin II or regulation of kinases controlling myosin II assembly. To determine whether Dictyostelium PAKa activity is stimulated in response to the chemoattractant, aggregation stage cells expressing FLAG-tagged PAKa were stimulated by cAMP, lysed at various times after stimulation, and PAKa–FLAG was immunoprecipitated from the detergent-soluble fraction (see Materials and Methods). Fig. 10 A shows that the kinase activity of immunoprecipitated PAKa towards a nonspecific substrate, H2B, rapidly increases three- to fourfold in response to cAMP. Maximal H2B phosphorylation is obtained within 30 s, after which the activity decreases slowly with kinetics similar to those of the adaptation of cAMP receptor-stimulated ERK2 kinase activity . The kinetics of PAKa activation are very similar to those of PAK activation in human neutrophil stimulated by fMet-Leu-Phe . In a process of verifying that there is an equal amount of PAKa–FLAG in each immunoprecipitate, we observed that the amount of PAKa–FLAG in the detergent-soluble fraction is rapidly decreased in response to cAMP. This decrease of PAKa–FLAG in the detergent-soluble fraction is due to the incorporation of PAKa–FLAG into a detergent-insoluble cytoskeleton fraction . The kinetics of PAKa relocalization into the cytoskeleton are very similar to the kinetics of myosin II assembly into the cytoskeleton upon cAMP stimulation. This result suggests that PAKa is involved in the regulation of myosin II assembly. When PAKa kinase activity is normalized for the amount of PAKa protein, activity increases approximately ninefold. We tested whether PAKa might directly regulate myosin II through its phosphorylation in the COOH-terminal tail fragment . As shown in Fig. 10 A, we do not observe myosin II phosphorylation by PAKa, suggesting that PAKa indirectly regulates the assembly of myosin II, probably via the regulation of MHCKs. As expected, PAK CA immunoprecipitated from aggregation-competent cells has three to four times higher basal activity against H2B , but the activity is not regulated in response to cAMP stimulation, probably because the activity is already maximal. To access the localization of PAKa in live, migrating cells, we fused the NH 2 -terminal regulatory domain of PAKa (excluding the CRIB and kinase domains) to GFP. Cells expressing N-PAKa–GFP chemotax normally (data not shown). As we demonstrate in Fig. 11 A, N-PAKa–GFP fusion protein localizes to the cortex at the cell's posterior, as we observed with FLAG- or HA-tagged PAKa, suggesting the NH 2 -terminal domain used in this fusion is sufficient for proper subcellular localization of PAKa. As cells migrate, the N-PAKa–GFP fusion protein remains at the rear cortical region , suggesting that PAKa activity is localized in the posterior and thus, may be important to maintain the polarity of cells during chemotaxis and retract the posterior cell body. To determine whether the localization of PAKa can be altered by disrupting cell polarity, we examined the distribution of N-PAKa–GFP by overlaying the cells with a receptor-saturating cAMP solution . As cells lose their polarity and round up, N-PAKa–GFP remains associated with the cortex, but diffuses away from its previous location to be distributed along the membrane cortex over the entire cell. Fig. 11 C shows the quantification of the stimulus-induced relocalization of N-PAK–GFP at the plasma membrane. The differences in fluorescence intensities before and after the addition of cAMP over the cell were measured along a thin line through the central portion of cells. In the polarized cell (frame 1), the fluorescence intensity in the rear cell body is tenfold higher than the intensity in the leading edge. However, the fluorescence intensity in the rear cell body is remarkably reduced by 50 s after cAMP stimulation. The intensity in the leading edge is increased ∼2.5 times. The kinetics of the translocation of N-PAK–GFP from the rear cell body to leading edge are presented in Fig. 11 D as a measure of the fluorescence intensity along the thin line through the anterior part of the cell. The translocation of N-PAK–GFP reached maximum ∼30–40 s after stimulation, which is similar to the kinetics of PAK kinase activity upon the cAMP stimulation. To examine whether the localization of holo-PAKa shows similar changes upon cAMP stimulation, cells expressing PAKa–FLAG were pulsed and plated on coverslips. Cells were bathed with 100 μM cAMP and fixed at 0, 25, and 50 s after cAMP stimulation and the localization of PAKa–FLAG was determined by indirect immunofluorescence staining. PAKa was localized in the rear cell body of polarized, unstimulated cells and relocalized along the membrane cortex 25 s after cAMP stimulation, which is very similar to the relocalization of N-PAK–GFP. In contrast to the distribution of N-PAK–GFP, PAKa staining in cells fixed at 50 s was absent in the cortex where new pseudopodia were formed, suggesting PAKa might be excluded in newly formed pseudopodia. The difference in the behavior between PAKa and N-PAK–GFP might result from the lack of a CRIB domain in N-PAK–GFP. We have identified a gene encoding a serine/threonine kinase, PAKa, that is a putative member of the PAK family of protein kinases. We find that PAKa has a dynamic subcellular localization. PAKa localizes to the cleavage furrow of cells undergoing cytokinesis and the posterior cortex of polarized cells and colocalizes in the same region of the cell with myosin II. Using a GFP fusion, we demonstrate that the NH 2 -terminal region lacking the CRIB domain is sufficient for the proper subcellular localization of PAKa in live, chemotaxing cells. If cells are overlaid with a receptor-saturating concentration of cAMP, the N-PAKa–GFP fusion proteins remain associated with the cell cortex, but delocalize along the cortex of the cell as it loses its polarized shape. Our data suggest that the NH 2 -terminal domain associates with a dynamically localized component of the cell cortex that is found in the posterior of polarized cells. Moreover, we suggest that the establishment of polarity in the cortical region responsible for this localization and the subsequent rearrangements of the cortical region are controlled through the chemoattractant receptors. We suggest that the localization of PAKa to a specific subdomain of the cell is a mechanism for restricting its activity to a specific site in the cell. This might be particularly important if its substrate is more uniformly distributed. Expression of PAKa CA , which is randomly localized in the cells, or myr PAKa, which is uniformly membrane-localized, upregulates the actin and myosin cytoskeletons and the cells' lack of cell polarity. These results are in agreement with our view that PAKa's tethering to the posterior of the cell is an important aspect of regulating PAKa function and may be important in maintaining the polarity of chemotaxing cells. paka null cells exhibit two major phenotypes associated with an inability to properly regulate the cytoskeleton: the cells fail to complete cytokinesis when grown in suspension and the cells exhibit defects in chemotaxis. Our results provide the first identification of an essential role of a putative PAK in cytokinesis. Dictyostelium cells and mammalian cells undergo cytokinesis via the formation and constriction of a myosin II-rich contractile ring in the cleavage furrow. myoII null cells do not divide in suspension due to the inability to perform cytokinesis, but undergo cytokinesis on an adhesive surface by traction-mediated cytofission in which dividing daughter cells are pulled apart . Several small G proteins and GAPs are required for proper cytokinesis in Dictyostelium . These genes include: RasG ; two IQGAP-related genes, Ddrasgap1 and GapA ; and a novel Rac protein, RacE . RasG might function via the activation of the PI3 kinase because deletions in two PI3 kinase genes (PI3K1 and PI3K2) lose the ability to grow in suspension and RasG interacts with the Ras binding domain of PI3K1 and PI3K2 (Reddy, T.B.K., and R.A. Firtel, unpublished observations). One of the IQGAP-related proteins, Ddrasgap1, interacts with activated DdRac1B and is thus a potential effector of Rac1B , which interacts with PAKa. It is not known if Rac1B interacts with Ddrasgap1 in vivo and regulates its role in cytokinesis. racE null cells fail to divide in suspension and become multinucleated . RacE could be an upstream regulator of PAKa. However, we only observe a strong interaction with activated Rac1B in yeast two-hybrid system and no interactions with activated RacE. paka null cells exhibit defects in chemotaxis and morphogenesis during multicellular development. The chemotaxis phenotype is exemplified by inability of paka null cells or wild-type cells expressing PAKa DN to maintain polarity, regulate the formation of pseudopodia, and control the contraction of the rear cell body during cell migration. In chemotaxing cells, myosin II aligns along the posterior of the cell, forming a C-shaped cap . This myosin cap contracts, causing the posterior of the cell to lift and allowing it to retract towards the leading edge . In myoII and paka null cells, the retraction of the posterior of the cell during chemotaxis is defective . We find that this C-shaped cap of myosin II in wild-type cells is much less visible or absent in paka null cells. Moreover, we found that paka null cells are defective in the assembly of myosin II into the cytoskeleton upon cAMP stimulation, suggesting that PAKa is involved in the regulation of myosin II assembly. In addition, cells expressing PAKa CA have a much higher level of myosin II assembly in the cytoskeleton in vegetatively growing cells. These data are consistent with PAKa playing an important role in the regulation of myosin II assembly into the cytoskeleton. Consistent with this, PAKa, as previously demonstrated for myosin II , is important for suppressing lateral pseudopod formation. Both myoII and paka null cells form more lateral pseudopodia than do wild-type cells . The inability to inhibit lateral pseudopod formation in myoII null cells may be associated with a reduction in cortical tension of these cells . The higher frequency of protrusion of lateral pseudopodia in paka null cells probably results from the reduction in cortical tension due to the lack of myosin II filament assembly at the posterior cortex. These cytoskeletal defects might also be a major cause of the morphological defects during multicellular development of paka null cells and myoII null cells . We demonstrate that PAKa kinase activity against H2B is stimulated in response to cAMP; however, our data indicate that myosin II is not phosphorylated by PAKa. This observation is consistent with a requirement of PAKa for the maintenance, but not the disassembly, of myosin II fibers. The regulation of myosin II filament assembly occurs through receptor-mediated phosphorylation of myosin II heavy chain tail and myosin II light chain . Phosphorylations at threonine residues in the myosin II tail by two kinases, MHCKA and MHC-PKC, lead to the disassembly of myosin filaments . MHCKA is expressed during growth and development . The mhckA null cells are viable, but display partial defects in myosin localization . Cells overexpressing MHCKA display reduced myosin II assembly in the cytoskeleton and become large and multinucleated in suspension, which is very similar to phenotypes of myoII null and paka null cells. In this context, PAKa is likely to regulate myosin II assembly via inhibition of MHCKA. MHC-PKC is particularly interesting, since it translocates from the cytosol to the plasma membrane/cortex and is activated upon cAMP stimulation, leading to the disassembly of myosin II fibers . Disruption of the gene encoding MHC-PKC leads to an upregulation of the myosin II cytoskeleton and chemotaxis defects . Our data are consistent with a model in which PAKa phosphorylates and thereby inhibits the activity of MHC-PKC and possibly MHCKA. According to such a model, this posterior localization of PAKa would allow MHCK-mediated disassembly of myosin II fibers at the leading edge while inhibiting the disassembly of myosin II fibers at the posterior. Myosin II light chain kinases are not likely to be substrates of PAKa, since light chain phosphorylation controls myosin II motor activity and not its assembly . Our results are consistent with the finding that cells expressing a myosin II mutant in which the three mapped MHC-PKC phosphorylation sites are converted to Ala do not maintain their shape and are unable to suppress the formation of lateral pseudopodia . Thus, the ability to sequentially assemble and disassemble myosin II, possibly in response to the activation and adaptation of PAKa kinase activity, appears to be critical for cell movement. Dictyostelium PAKa has high homology to Dictyostelium MIHCK. In Acanthamoeba and Dictyostelium , PAK homologues phosphorylate myosin I heavy chain and stimulate the Mg 2+ -dependent ATPase activity . Immunohistochemical studies demonstrate that three classic myosin Is, myoI-B, myoI-C, and myoI-D, are concentrated at the leading edge of chemotaxing cells , and are not found in the cleavage furrow, suggesting that myosin Is are not PAKa substrates. PAKs have been implicated in the morphological changes resulting from changes in the actin cytoskeleton associated with Rac and Cdc42 . Microinjection of activated PAK1 protein into quiescent Swiss 3T3 cells induces the rapid formation of lamellipodia, filopodia, and membrane ruffles , which is very similar to the effect of microinjection of activated Rac and Cdc42. The interaction between the proline-rich domain of PAK1 and a protein containing an SH3 domain appears to be important for regulating PAK1 activity. Nck, an adapter protein containing SH2 and SH3 domains, binds PAK1 and induces PAK1 relocalization to the membrane, leading to its activation . Other studies demonstrate that localization of PAK to membranes stimulates the kinase activity of PAK and our data with myr PAKa are consistent with this model. Although we did not examine whether the proline-rich domain of PAKa interacts with proteins containing an SH3 domain such as Nck, PAKa probably associates with an SH3 domain via the PPxP sequence, resulting in the localization of PAKa to the cortex. We note that PAK CA leads to a hyperpolymerization of actin. We do not know if this is a direct effect of PAKa on regulators of the actin cytoskeleton or is indirect through the control of myosin II. It is also possible that the PAKa kinase domain, when expressed by itself, may not show the same level of substrate specificity as the whole protein. Our observation that the expression of myr PAKa results in a hyperpolymerization of myosin and actin along the periphery of the cell, reinforces the model that the subcellular localization of PAKa to the posterior of the cell may be essential for its proper function. We suggest that this overexpression along the entire membrane or the localization by the myristoylation leads to a constitutive activation of the kinase. Although we have not demonstrated that PAKa is directly regulated by Rac1 and/or Cdc42, the GTP-bound, but not GDP-bound, forms of Rac1B and HsCdc42 bind to the PAKa CRIB domain. These data are consistent with, but do not prove, that PAKa is a bone fide PAK. In contrast to the kinase domain alone, overexpression of the entire PAKa protein or the kinase and CRIB domains (PAKm G+K ) does not cause a major change in the actin or myosin cytoskeleton. This finding is consistent with observations of mammalian PAKs in which the CRIB domain and an adjacent domain negatively regulate PAK kinase activity . We expect that binding of activated Rac or Cdc42 to the CRIB domain of PAK regulates the activation of the kinase, whereas the more NH 2 -terminal domain regulates PAKa's localization. Cells overexpressing PAKa exhibit an upregulated assembly of F-actin, resulting in multiple actin-enriched crowns, which is very similar to the F-actin organization of cells expressing Rac1B Q61L or of null cells of the Rac1 GAP, DdRacGAP1 (Chung, C.Y., S. Lee, C. Briscoe, and R.A. Firtel, manuscript submitted for publication). These similarities of F-actin organization suggest PAKa may be important in regulating F-actin organization . A Dictyostelium Ste20 family kinase that phosphorylates severin, a Ca 2+ -dependent F-actin fragmenting protein, was purified and cloned recently , indicating a direct signal transduction from the plasma membrane to the cytoskeleton by phosphorylating actin-binding proteins. PAKa might control F-actin organization by phosphorylation of actin binding or actin-bundling proteins. The identification of the downstream substrates of PAKa and the determination of the role of phosphorylation by PAKa in regulating the function of the substrate should resolve these issues. | Other | biomedical | en | 0.999997 |
10545501 | The procedures of Nelson et al. 1975 were adapted for our experiments, as described previously , with modifications as outlined in Results. Neutrophils were isolated using standard techniques . For each cell tracking experiment, assays were set up and allowed to incubate at 37°C for 75 min, at which time they were moved to a warmed (37°C) microscope stage, and cell movements were recorded for 15 min. Agarose gels contained endotoxin-tested RPMI-1640 (Sigma Chemical Co.), 10% heat-inactivated bovine calf serum (serum inactivated at 57°C for 25 min), 1.2% agarose (GIBCO BRL), and 10 mM Hepes (GIBCO BRL), pH 7.2. For standard assays, five 3.5-mm-diam holes were cut 2- or 1.5-mm apart in a linear array. For experiments with three equidistant wells in an equilateral triangle, a template-guided steel punch was used to form three 3-mm-diam wells 3.3-mm apart. Cells and agonists were suspended in RPMI-1640, 10% heat-inactivated bovine calf serum, and 10 mM Hepes, pH 7.2. Chemoattractants used were Leukotriene B 4 (Sigma Chemical Co.) and recombinant human interleukin-8 (a gift from Antal Rot, Sandoz-Forschungsinstitut, Vienna, Austria). Images were captured every 30 s on an inverted microscope (captured area = 780 μm orthogonal to the axis of the wells × 584 μm along axis of wells) or every 15 s on a laser scanning confocal microscope using Lasersharp software version 2.1 (Bio-Rad; captured area = 800 × 800 μm). For confocal microscopy experiments, neutrophils were labeled with 100 nM (final concentration/10 7 cells) of either SYTO-13 (green) or SYTO-15 (orange; Molecular Probes Inc.) for 25 min at room temperature in migration medium, washed, resuspended, and stored on ice until the start of the assay. To create a uniform field of chemoattractant, 1 nM LTB4 was incorporated into the agarose gel. Two wells, 7.5 mm apart, were cut in the gel. At t = 0, each well was filled with 10 5 cells in 20 μl of medium containing 1 nM LTB4. At t = 60 min (15 min before filming), well contents were overlaid with agarose containing 1 nM LTB4. Each videotape (or series of confocal images) was converted to an NIH Image time-lapse movie, with one image every 30 (or 15) s. Cells moved on average 1–1.5 cell lengths/min, so cells moved less than a cell length between frames and could be identified from one image to the next. (The length of migrating cells was ∼20 μm as cells spread out significantly under the agarose.) An analysis region was defined based on the position of the filmed cells relative to the chemoattractant-containing wells, and only cells that were the desired distance from each well at the beginning of the movie were tracked. The analysis region always excluded cells within 100 μm of the field edge because they could not be reliably tracked for the entire 11.5-min analysis period. Usually, 90% of the cells within the analysis region could be followed for the entire 11.5-min analysis period (always = 87.5%). Once the analysis region was defined, each cell was numbered and its x, y coordinates were measured on the first image and on every subsequent image in the image stack, with the x-axis parallel to the edge of the cell starting well and the y-axis orthogonal to the well edge (parallel to a line connecting the starting well and the distant well). The (x, y) data for each cell was exported to a Microsoft EXCEL spreadsheet and used for the subsequent calculations of chemotactic bias. The filmed field was aligned so that the axis of the experimental wells on the plate passed vertically through the center of the field, with the forward direction defined as directly away from the cell starting well. For cells migrating in a uniform field, the direction of a cell's motion during a given time interval was calculated trigonometrically using the cell's (x, y) coordinates. To determine an average direction at each time point, the cell's angle relative to the forward direction (0°) was calculated over three overlapping time intervals; for example, the cell's initial direction was calculated as the average of the cell's angle from 0 to 1 min, 0.5 to 1.5 min, and 1 to 2 min of observation. A cell was considered to be persistent until it achieved an angle >90° from its starting angle. To assure accurate assessment of the starting angle, the few cells that were not traveling at least 1 cell length/min (20 μm/min) during the initial 2-min period were excluded from the analysis. The analysis was begun within 3 min of the start of the videotape, at the first time at which the cell was traveling at least 20 μm/min. Each cell's (x, y) coordinates were used to calculate the cell's efficiency of forward migration, or forward migration index (FMI), during an initial 2-min analysis period, and its efficiency of forward migration during a subsequent test period of 4–9.5 min (see Results). The FMI was calculated as follows: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{FMI}}=\frac{{\mathrm{Forward\;Progress}}}{{\mathrm{Total\;Path\;Length}}}=\frac{{\mathrm{{\Sigma}{\Delta}y}}}{{\mathrm{{\Sigma}}} \left \left( \left \left({\mathrm{{\Delta}x}}^{2}+{\mathrm{{\Delta}}}y^{2}\right) \right ^{-{1}/{2}}\right) \right }\end{equation*}\end{document} with Δx and Δy assessed for each 30-s interval throughout the analysis period. For most experiments, the FMI was only calculated for the initial 2 min of observation and the subsequent 9.5 min; 11.5 min was the longest period for which nearly all cells selected could be followed without interruption (without leaving the field), and thus, an initial 2-min and subsequent 9.5-min observation provides the most reliable measure of the cells' time-averaged behavior. For each experimental condition, we plotted the initial versus subsequent FMI for all tracked cells and fitted a line through these data points by linear regression. The y-intercept of this line indicates the cells' chemotactic bias (see Results). We used standard statistical techniques to weigh and pool data acquired on different days. Intercepts of regression lines were compared using a t test. To understand the behavior of neutrophils migrating in overlapping chemoattractant gradients, we needed to develop a quantitative method to evaluate chemotaxis (directed cell motility) and to distinguish its contribution from the other factors influencing cell migration in our model. We first characterized cell migration in the presence of a uniform field of chemoattractant: chemoattractants stimulate random cell motility (chemokinesis) when present at a uniform concentration . We incorporated LTB4 directly into an agarose gel, at an optimal concentration for stimulation of cell movement (1 nM). Neutrophils (in medium containing the same concentration of LTB4) were added to a well in the gel and allowed to migrate from their starting position for 75 min, videotaped, and analyzed over the next 15-min period. To assess the initial orientation of the cells within the field of view, we determined the average angle of cell migration during the first 2 min of the 15-min observation period. As shown in Fig. 1 a, the distribution of cell orientations was not random, but rather a majority of videotaped cells pointed away from the starting well, or forward (27/43 or 63%), in spite of the absence of a gradient. We postulated that this initial forward orientation might be the result of the following: (a) a mass action effect, in which most cells enter the field of observation from the direction of the starting well coupled with (b) a directional persistence, the well-described tendency of neutrophils to continue in their recent direction, rarely making sharp turns, even in the absence of an orienting gradient . Together, these two phenomena could result in an initial forward bias in cell orientation. The forward bias could also result from real chemotaxis because of, for example, cellular chemoattractant destruction. To distinguish between these possibilities, we analyzed cell behavior in more detail. Consistent with previous reports, cells migrating in a uniform field in our assay displayed considerable directional persistence: after 5 min, only ∼50% of cells had turned >90° from their starting direction; by 10 min, ∼90% of cells had turned . To the degree that cells persist in their initial directions, an initial forward bias in cell orientation would be expected to lead to a forward bias in subsequent cell movements. Therefore, any analysis of the cells' chemotactic behavior must be corrected for the cells' initial orientation. To assess the relationship between the initial and subsequent direction of cell movements, we calculated each cell's FMI during an initial 2-min observation period, and then during subsequent 4, 7, or 9.5-min observation periods. The FMI is the ratio of the net distance the cell progressed in the forward direction (away from the starting well) to the total distance the cell traveled as it wandered through the videotaped field (see Materials and Methods). The FMI measures the efficiency of a cell's forward migration during a given time interval. . Fig. 1c Fig. e , show the relationship between the initial and subsequent forward migration indices for cells migrating in a uniform field of LTB4. Fig. 1 c shows the subsequent FMI, calculated over a 4-min period (from t = 2–6 min), plotted against the initial index, calculated over the first 2-min period of observation (t = 0–2 min). The regression line through these data has a positive slope (+0.55), indicating that cells that start out moving forward tend to continue to progress forward during the subsequent 4-min period, and that cells that initially move backward tend to continue to progress backward. The correlation between the initial and subsequent FMI is statistically significant . The slope of the linear regression line of the initial versus subsequent FMI is an indicator of the degree to which cells persist in their initial direction; therefore, it can be considered a persistence index. Fig. 1d and Fig. e , plots the cells' forward migration indices, as calculated over longer intervals (of 7- and 9.5-min duration), against their initial migration indices determined in the first 2 min of observation. Again, there is a significant positive correlation between the cells' initial and subsequent movements, but it becomes less prominent as cells are tracked for longer intervals (when the subsequent migration index is determined over 7 min, from t = 2–9 min, the slope = 0.33, and r = 0.41, P = 0.01; when determined over a 9.5-min period, from t = 2–11.5 min, the slope = 0.32, and r = 0.42). The correlation between the initial and subsequent forward migration indices, and the reduction of this correlation over time, is consistent with the effect of directional persistence on cell movement, and with the decay of this effect as cells are followed for a longer time interval. Cells displayed similar behavior when migrating in a uniform field containing both IL-8 and LTB4 (not shown). The plots comparing the cells' initial and subsequent movement describe, in essence, neutrophils' turning behavior. To understand the data presented in Fig. 1 e more fully, it is useful to consider several example cells. Cell A pointed forward during the initial 2-min analysis period, as indicated by its positive initial FMI (+0.92), and it continued to progress forward during the subsequent 9.5-min analysis period. Cell B, like A, pointed forward during the initial 2-min analysis period, but did not maintain this course throughout the subsequent period. Cell B has a negative FMI during the 9.5-min tracking period (−0.46), indicating that, overall, this cell went backward during that time, and therefore, must have turned from its initial direction. Cell C pointed backward during the initial period, but turned during the subsequent period because, overall, it progressed forward during the subsequent 9.5-min analysis period (subsequent FMI = +0.26), whereas cell D also initially pointed backward, as evidenced by its negative initial forward migration index (−0.79), and it continued to progress backward during the subsequent period. Once the relationship between the cells' starting directions and their subsequent progress is known, it can be used to assess whether cells display a directional turning bias. To do so, we asked to what degree an average cell progresses forward if it starts out pointing neither forward nor backward, that is, pointing to the left or right, with an initial FMI of zero. This value is equivalent to the y-intercept of the regression line that fits the initial versus subsequent migration index data. Note that the y-intercept of the plot for cells migrating in a uniform field of chemoattractant, in our system, is always close to zero , so that a cell starting out with no bias forward or backward, on average, makes no net progress forward or backward. In fact, this is the behavior expected for cells migrating in the absence of a chemotactic gradient. Thus, by comparing cells' initial direction to their subsequent movement, we have arrived at a measurement of directed cell movement normalized for cells' starting orientations. This measurement, the y-intercept, describes the turning bias associated with true chemotaxis and is hereafter termed the chemotactic bias. The regression line through the cells' initial versus subsequent forward migration indices is therefore: subsequent FMI = chemotactic bias + (persistence index) × (initial FMI). Next, we assessed the behavior of cells migrating in an LTB4 gradient. In this experiment, we videotaped cells migrating from their starting well towards an LTB4-containing well 2 mm away. The initial and subsequent migration indices were determined for each cell as above. In this and the following experiments, the subsequent FMI was determined over a 9.5-min period only. The plot comparing initial and subsequent movement reveals several noteworthy features of the cells' behavior . First, like cells migrating in a uniform field, cells that start out migrating forward tend to progress forward during the subsequent analysis period (i.e., then tend to have positive subsequent forward migration indices). However, in contrast to cells migrating in a uniform field, even cells that start out pointing backward, or towards the cell starting well, tend to make net progress forward (up the LTB4 gradient) during the subsequent 9.5 min. Consequently, the y-intercept of the regression line through these data, or the chemotactic bias, is positive (0.34, with a 95% confidence interval of 0.24–0.43). This indicates that, in this setting, an average cell that started out pointing orthogonal to the LTB4 gradient makes net progress forward, toward the LTB4 source, during the subsequent 9.5 min. Based on these data, we can conclude that cells exposed to this LTB4 gradient display a significant turning bias, and tend to progress towards the gradient source, independent of initial orientation and of directional persistence. The minimal residual effect of directional persistence is apparent in the slight positive slope of the regression line through these data. The experiments that follow were analyzed in the same way, and in each case the y-intercept of the regression line relating the initial to subsequent forward migration indices is presented as the population's chemotactic bias. We were initially interested in understanding the ability of neutrophils to migrate away from one chemoattractant source in response to another, more distant agonist . To this end, we tracked neutrophils migrating from a well containing IL-8 towards a well containing LTB4 (and vice versa). For the chemoattractant sources, we selected a concentration of LTB4 and IL-8 each eliciting optimal neutrophil migration from a distant well. At these concentrations, cells migrate the same distance toward a distant IL-8 source in the presence and absence of a local LTB4 source (and vice versa) . This behavior is observed in spite of the fact that quantitative measurements of chemoattractant concentrations indicate that the mean concentration and gradient slope of the local agonist should itself be able to mediate chemotaxis of neutrophils in the opposite direction . We reasoned that this migration in the context of opposing or even conflicting gradients could represent either true chemotaxis or, possibly, enhanced chemokinesis in the region of chemoattractant overlap. To distinguish between these possibilities, we assessed cell migration behavior in the presence of opposing gradients as follows: cells were placed in a well with either IL-8 or LTB4 (the local well), and the other chemoattractant was added to the distant well 2 mm away. From 75 to 90 min, cells 400–700 μm from the local agonist (1,300–1,600 μm from the distant well) were filmed and their migration bias assessed, exactly as described above for the previous experiment. We found that the mean velocity of migrating cells was the same (24–27 μm/min) whether one or both chemoattractants were present, suggesting that altered motility could not explain cell behavior. Furthermore, neutrophils displayed a true chemotaxis toward a distant IL-8 source in the presence of a local LTB4 source, equivalent to that displayed in the absence of LTB4; similarly, cells chemotaxed towards a distant LTB4 source equivalently, in the presence or absence of a local IL-8 source . Additional control experiments were performed to determine whether IL-8 and LTB4 sources, at the concentrations used here, create functional gradients at a nearby site (400–700 μm). In these control experiments, we allowed naive neutrophils to migrate toward an IL-8 or LTB4 source from a starting well positioned closer to the chemoattractant, so that these cells would be 400–700 μm away from the source during the relevant time interval (75–90 min). Under these conditions, neutrophils exhibited significant chemotaxis to both a local IL-8 source and a local LTB4 source , confirming that in the previous experiment , a functional orienting gradient of the local agonist exists. We conclude that, under these experimental conditions, neutrophils display true chemotaxis to a distant agonist source even when migrating down an effective gradient of a local agonist. The above results suggest that neutrophils can integrate competing directional signals in such a way that they continue to migrate directionally. One possibility is that this integration, in effect, involves a vector sum of the competing orienting signals. If so, we reasoned that it should be possible to demonstrate such vectorial integration by asking neutrophils to respond to two equidistant agonist sources presented at an angle. We adjusted the under agarose assay by cutting three wells in a triangular configuration, and introducing neutrophils into one well and chemoattractants into the other two wells. We performed a series of assays in which neutrophils and chemoattractants were added to the wells and cells were allowed to migrate for 2.5 h. Photographs of cell migration patterns are shown in Fig. 4 . When only one well contained chemoattractant, neutrophils migrated towards that well only. When both wells contained the same chemoattractant, either LTB4 or IL-8, neutrophils migrated furthest in the direction of each chemoattractant well. This is the migration pattern that would be expected if each cell migrated up the steepest local chemotactic gradient encountered. However, notably when one well contained IL-8 and the other contained LTB4, cells migrated furthest in a direction between the two chemoattractant sources. This is the pattern that would be expected if the direction of the entire migrating population is influenced by both chemoattractants. The possibility that this pattern could result from a combined effect of the chemoattractants on cell velocity is unlikely, since cells migrate with about the same velocity in the presence of either chemoattractant as they do the presence of both together . This result confirms that most neutrophils can respond to both agonists, and suggests that the cells' integrated response to two different agonists can result in predominant cell targeting to a point between the sources, which is consistent with a model of vectorial integration of the two signals. We have shown that neutrophils behave as if they can integrate directional information from competing chemotactic signals . On the other hand, in the initial experiments presented in Fig. 3 , we saw that neutrophils can chemotax away from a local chemoattractant source towards a distant source of a different chemoattractant, without a noticeable effect of the local agonist on the cells' chemotactic bias. Why do neutrophils appear to ignore the local gradient in this context? One possibility is that cells somehow prioritize signals from a distant gradient over those from an otherwise equivalent local agonist source: this might be possible, for example, if the strength of the perceived orienting signals (and thus the behavior of cells) is influenced by the cells' prior history. To evaluate this possibility, we altered the conditions of the assay: we placed the IL-8 and LTB4 wells closer together (1.5 mm), and used slightly different amounts of IL-8 and LTB4 (0.5 and 0.1 pmol, respectively), so that cells coming from either direction would enter the common central region (600–900 μm from each well) at 75 min. If the cells' chemotactic bias simply reflects an integration of the gradient vectors present, we anticipated that the cells in the middle region would behave the same way regardless of the direction from which they had migrated. We observed that, under these conditions, cells that had migrated into the central region between the two wells always displayed true chemotaxis towards whichever agonist was presented in the distant well: neutrophils that had migrated from LTB4 into the central region displayed a significant chemotactic bias toward the initially distant IL-8 source, and vice versa. Furthermore, the chemotactic bias cells displayed towards an initially distant chemoattractant source was only slightly lower in the presence of an opposing gradient of the other chemoattractant . One possibility is that cells originating in one chemoattractant source well alter that chemoattractant source in such a way that it no longer generates a functional gradient (i.e., by degrading the chemoattractant). To address this issue, the same experiment was performed with neutrophils starting simultaneously in both chemoattractant source wells. To distinguish cells that originated in the LTB4-containing well from cells that originated in the IL-8–containing well, cells were labeled with either a green or an orange nuclear dye and cell movements were tracked using confocal microscopy. The chemotactic biases of these distinct cells populations were evaluated as cells migrated within a central region between the two chemoattractant sources (cells 550–950 μm from each well at 75 min). Again, we found that cells that had migrated into the central region between the two wells always displayed true chemotaxis towards whichever agonist was presented in the distant well . By the end of the observation period, cells had intermingled significantly . Because both cell populations were present in the central region simultaneously, we conclude that cells from different starting locations display different chemotactic behavior even when experiencing an identical chemotactic field. These results suggest that cellular chemotaxis in the presence of conflicting gradients is influenced by cellular memory, which alters the perceived strength of the orienting signals in a given chemotactic field based on the cells' previous chemoattractant environment, in such a way that cells respond preferentially to gradients of novel chemoattractants. Previously, we have shown that leukocytes can navigate in a step-by-step fashion through complex arrays of chemoattractants . Here, we have sought to understand the mechanisms whereby leukocytes can respond sequentially to regulatory cell–derived chemoattractants. Our experiments reveal two fundamental properties of leukocyte chemotactic responses: an ability of neutrophils to integrate competing directional signals, as if responding to the vector sum of orienting gradients present, and the dependence of the resulting directional responses on the cells' prior history (i.e., on cellular memory of the recent chemoattractant environment). We propose that these interrelated properties allow leukocytes to navigate successfully through complex chemoattractant arrays. We shall discuss the results of our experiments in the context of these concepts. The present studies were initiated to understand the cellular responses that allow neutrophils to migrate efficiently from one agonist source to another . Our analysis revealed that neutrophils display true chemotaxis to a distant chemoattractant source, even when the local agonist source also produces an effective (orienting) reverse gradient at the same position and time. Migrating neutrophils are polarized, displaying a discrete leading edge and trailing uropod, even in the absence of an orienting gradient . Gradients of leukocyte chemoattractants are thought to elicit chemotaxis by determining the direction of a cell's leading edge and, hence, the direction of cell locomotion, independent of cell velocity, adhesivity, or other factors. If cells can integrate orienting signals, then in the presence of overlapping gradients, the direction of cell chemotaxis would be expected to reflect the vector sum (the average magnitude and direction of) the orienting signals present. If the chemoattractant gradients are not aligned, as when cells are migrating away from one source towards another, the orienting signals would compete. In this situation, whichever gradient produces a stronger orienting signal would be expected to determine the cells' direction of orientation and, thus, determine the direction of cells' chemotactic response. The apparent ability of cells to ignore a local chemoattractant source, thus, may reflect an integrated response to the opposing orienting signals, but one in which the cells' chemotactic bias is determined preferentially by the (presumably more compelling) distant gradient. In fact, we were able to demonstrate convincingly that neutrophils can integrate conflicting directional signals by showing that, in the presence of two balanced agonist sources 60° apart, neutrophils migrate furthest in a direction between the agonist sources. This observation not only confirms that most neutrophils can respond efficiently to both of the two agonists used in this study (IL-8 and LTB4), but that at the population level, the direction of cells' displacement is determined by the vector sum of the orienting signals provided by each gradient. In the extreme case, this implies that two opposing gradients (of exactly matched orienting strength) should be able to cancel each other out. In fact, under carefully selected conditions, we have been able to create such a balanced competition in which neutrophils between IL-8 and LTB4 sources behaved as if in the presence of a uniform chemoattractant field . One prediction from these observations is that in the presence of a stable array of regulatory chemoattractants, cells will eventually find a central region in which opposing orienting signals are perceived as equivalent. Within this region, cells would migrate back and forth until they encountered additional signals. Such signals could include a newly introduced chemoattractant source, or other classes of guidance cues such as counterreceptors for leukocyte adhesion molecules . Even if other influences were irrelevant, the phenomenon of directional persistence (and of cellular memory see below) would ensure that in this setting cells would be broadly distributed throughout a region of regulatory cell activation, as in a tissue site of inflammation. In studying neutrophils' chemotactic behavior in the presence of opposing chemoattractant gradients, we discovered that cells presented with identical chemotactic fields exhibit different chemotactic behavior depending upon their history. Neutrophils arriving in a central region between IL-8 and LTB4 sources displayed opposite chemotactic responses, depending on the direction from which they came, even when neutrophils were added simultaneously to both wells and assessed as they migrated towards each other in the same central location. If cells' directional bias is determined by the integration of opposing orienting signals, how can neutrophils display different chemotactic biases when migrating in exactly the same gradient conditions? We propose that these experiments define a phenomenon of cellular memory of the recent chemoattractant environment, in which cells' responsiveness to agonists in the recent chemoattractant environment is selectively diminished, ensuring that they can preferentially respond to newly arising chemoattractant sources within tissues. What is the basis of this change in cellular responsiveness? Neutrophils and other leukocytes are known to adjust their sensitivity to a chemoattractant upon exposure to that chemoattractant. At the chemoattractant concentrations that efficiently elicit chemotaxis (0.01–10× the receptor K d ), the conditions most relevant to our experiments, cells undergo a process known as adaptation. Adaptation is a feature of many sensory systems using G protein–coupled receptors, including animal visual systems , and is thought to reflect adjustment in agonist-specific signaling mediated by homologous receptor desensitization (or resensitization and receptor recycling). An adapting system adjusts its sensitivity according to the background level of stimulation it receives. Early studies showed that neutrophils experiencing slight increases in chemoattractant level exhibit a transient response, and then adjust to the new chemoattractant concentration . This adaptation takes many seconds to several minutes, after which neutrophils can again respond to an additional step increase in chemoattractant concentration. However, the dose of chemoattractant required to elicit a neutrophil response increases as cells adapt to higher ambient chemoattractant concentrations . Cells that have adjusted to a certain chemoattractant concentration can regain their prior sensitivity to lower agonist levels when the chemoattractant is removed, but this process takes time. Neutrophils that experience a sudden decrease in chemoattractant concentration show morphological changes, which subside within 2–6 min, depending on the magnitude of the decrease . The full recovery of chemotactic responsiveness can take even longer. For example, in studies by Goldman and Goetzl 1984 , neutrophils preincubated with low, chemotactic levels of LTB4 (0.3, 1, or 3 nM), washed, and allowed to recover, showed a significant reduction in chemotactic responsiveness to LTB4 even when assayed starting 10 min later. Cells preincubated with slightly higher concentrations (10–30 nM) exhibited no chemotaxis to LTB4 even when assayed after a 10-min recovery period . Cells that have experienced extreme, saturating chemoattractant levels (∼100× the receptor dissociation constant, or K d ) undergo not only receptor desensitization, but also extensive receptor internalization, and can require a long recovery time (20–60 min) to regain former receptor levels and signal transduction efficiency . We hypothesize that the time delay required to readjust cellular sensitivity after a reduction in ligand exposure is the basis for the phenomena we observe, in essence altering cells' perception of the relative strength of local chemotactic signals as a function of the cells' recent chemoattractant environment. A model is shown in Fig. 5 c. How can this model explain the apparent ability of cells to migrate between two chemoattractant sources presented at an angle? Leukocytes and other cell types, such as Dictyostelium amebas, are thought to sense chemotactic gradients using a spatial mechanism, in which they instantaneously calculate the differential occupancy of chemoattractant receptors across the cell body . Although distinct chemoattractant receptors could theoretically each initiate a unique signaling cascade leading to chemotaxis, current studies suggest that signals through different chemoattractant receptors converge on a common chemotaxis-initiating pathway . If different chemoattractant receptors share common intracellular signaling pathways, at any given instant a cell is not likely to be able to tell whether the signals it is receiving are from one agonist or another. Thus, at any instant, neutrophils presented with competing gradients should respond as if in the presence of a single agonist. However, the cells' ability to adjust their sensitivity to different attractants independently can allow vector integration of distinct orienting signals over time. In our model, when two sources of the same chemoattractant are present, a cell simply migrates up the steepest local gradient it encounters. When two different chemoattractants are present, a cell also migrates up the steepest local gradient. However, as it migrates closer to one chemoattractant source and becomes less sensitive to that agonist and, thus, relatively more responsive to the other, it will turn and move towards the second agonist, potentially even across the midline. Thus, the perception and response to complex chemoattractant arrays involves both instantaneous spatial gradient perception at each moment, and changing perceptions of individual gradient vectors over time. In this model, it is the temporal component that allows cells to integrate orienting vectors from different chemoattractant sources. The phenomenon of cellular memory has fundamental implications for directing leukocyte homing in vivo, namely, whenever a cell experiences competing attractant gradients, memory will promote cell migration towards a novel chemoattractant. This model is outlined in Fig. 6 . In this study, we show that neutrophil chemotaxis in overlapping chemoattractant gradients is guided by vector integration of orienting signals, and by cells' memory of their prior chemoattractant environment. (In vivo, of course, these mechanisms would operate in conjunction with regulated adhesion and cell activation to control cell direction.) Our observations support a model in which a leukocyte's chemotactic bias in competing chemotactic gradients is dynamically regulated by its previous experiences. This type of regulation can promote leukocytes' sequential migration to (and navigation through) the gradient sources present within a tissue, allowing combinations of chemoattractants to effectively guide cells to their unique destinations. | Study | biomedical | en | 0.999996 |
10545502 | The cDNA corresponding to the mouse gene of synaptotagmin III C2A-C2B, residues 295–569 (33,000 kD), was subcloned into pET28A using the NdeI-HinDIII cloning site. The expression plasmid was transformed into BL21(DE3). The bacteria were grown in a BIOFLO3000 using ECPYM1 media with 50 mg/ml kanamycin sulfate. Heterologous protein expression was initiated at OD 600 50.0 with 300 mM IPTG for 3 h at 37°C. Cells were harvested and frozen in liquid nitrogen until needed. Native gel electrophoresis was carried out using 10–15% native Phast (Pharmacia) gels with native buffer strips soaked for 2 h in 0.88 M l -alanine, 0.25 M Tris, pH 8.8, and 1 mM EDTA before running the gel. Samples were mixed and allowed to incubate for at least 1 h in 20 mM Hepes, pH 7.8, 150 mM NaCl, 5 mM DTT, and 1 mM EDTA before loading the gel. The concentration of both proteins was determined by amino acid analysis (Keck facility, Yale University). SNARE core complex was expressed in a polycistronic T7 expression vector containing rat synaptobrevin-II, residues 1–96, rat syntaxin-1A, residues 180–262, rat SNAP-25A residues 1–83 and residues 130–206, and purified according to previously published methods . 30 g of frozen bacterial cells were resuspended in 125 ml of extraction buffer (100 mM sodium phosphate, pH 8.0, 200 mM NaCl, and 10% ethylene glycol) with 200 mg of lysozyme and 0.5% Triton X-100. Passing the lysate twice through a pneumatic cell cracker (Microfluidics) at 10,000 lb/in 2 facilitated cell lysis. The histidine-tagged protein was incubated in batch with NTA-Ni (Qiagen) resin overnight at 4°C while slowly mixing. The resin was washed in a column with extraction buffer including 50 mM imidazole until the OD 280 was below 0.05. The tagged protein was eluted with 300 mM imidazole. At this point, 10% ethylene glycol, and 5 mM CaCl 2 were added to the protein solution. Under these conditions, synaptotagmin III precipitated and was removed by centrifugation in a GSA rotor at 10,000 rpm for 20 min. The protein pellet was washed in 25 mM ethanolamine, pH 8.5, 300 mM NaCl, and 10% ethylene glycol. The protein was resuspended in 30 ml of 50 mM sodium phosphate, pH 7.8, and 5 mM EDTA. The histidine tag was removed by cleaving the protein with TEV protease (BRL) overnight at 4°C. The resuspended protein was filtered through a 0.22-μm filter before ion exchange chromatography with a Mono Q 5/5 (Pharmacia) column in 25 mM ethanolamine, pH 8.0. The purified protein eluted from a 0–1-M NaCl gradient at 200 mM NaCl. The protein was concentrated to 40 mg/ml. Synaptotagmin III C2A-C2B crystallized in 1.5 M MgCl 2 and 100 mM MES, pH 6.5, at 20°C using the hanging drop method. Large hexagonal crystals grew after ∼1 wk. Analysis of the systematic absences in the diffraction data narrowed possible space groups choices to either P6 1 22 or P6 2 22: a = 126 Å, b = 126 Å, c = 118 Å. Inspection of the molecular replacement solution of the C2A domain uniquely determined P6 2 22. The Matthews coefficient calculation predicted two molecules in the asymmetric unit given average solvent content; however, only one protein molecule was found resulting in a solvent content of 70%. Native and trimethyl lead acetate (TMLA) data were collected at station BL1-5 at SSRL at a temperature of 20°C. Diffraction data from several crystals were merged to obtain highly redundant and complete native and anomalous derivative data sets. Diffraction data of the derivative were collected at a wavelength of 1.00 Å to take advantage of the lead (Pb) anomalous signal. Diffraction data were integrated and scaled using the DENZO package . Placement of each C2 domain within the asymmetric unit was attempted by molecular replacement as implemented in CNS using the synaptotagmin I C2A domain (residues 140–265; pdb entry, 1rsy) as the search model. Only the C2A domain of synaptotagmin III was located by this technique. The C2B domain was manually positioned in an electron density map calculated from native amplitudes and phases obtained from the molecular replacement solution combined with the initial SIRAS (single isomorphous replacement, anomalous scattering) phase probability distribution. The C2A and C2B domains were separately refined by rigid body refinement to R free and R values of 48 and 46%, respectively. Three heavy atom sites were localized by an automated Patterson search method and confirmed by difference Fourier maps. Two of the heavy atom peaks corresponded to TMLA ions in the Ca +2 -binding loops of each domain. The third site mapped to a minor site on the C2A domain. Because of the structural similarity of the two domains, it was difficult to correctly assign each domain to the electron density at this early stage. The two domains were distinguished by identifying a unique cysteine residue (Cys523) in the C2B domain. This cysteine residue is involved in a disulfide linkage between crystallographically related C2B domains. The model was fit to density modified electron density SIRAS maps ( Table ), as well as the model-phase combined SIRAS electron density maps. The program O was used to build the initial model . Initially, the residues of the C2A domain of synaptotagmin I were changed to the homologous residues for the corresponding synaptotagmin III residues. The electron density of the C2B domain was smeared along the plane of the β-sandwich, especially at the distal end of the domain. This effect is a consequence of the pivoting motion of the C2B domain about the inter-domain linker. This motion would also explain the observed diffuse scatter in the diffraction pattern (not shown). We relied on the primary sequence similarity of the C2B domain with the C2A and PKC-β domains to aid initial model building in the most disordered regions. The seven-residue α-helix between the between strands β7 and β8 of the C2B domain was constructed from the available electron density. The model phases were used as prior phase probability distributions to feed back into the heavy atom model refinement resulting in significantly improved SIRAS phases. The MLHL target function was used with the SIRAS experimental phases. Refinement included several rounds of conjugate gradient minimization, torsion angle simulated annealing at 3,000 K , grouped B-factor refinement for both domains, and individual B-factor refinement for the C2A domain only ( Table , A and B). Refinement progress was monitored by a concomitant drop in R free and R. All phasing and refinement calculations were carried out with CNS . The bulk solvent model used a density level (κ sol ) of 0.27 e − /Å 3 and a B-factor of 25 Å 2 . The final model had 98.7% of all residues in allowed regions. Three residues in the C2B domain are reported as disallowed (Ala 553, Lys 557, and Ser 475). Due to the disorder in a portion of the C2B domain, several side chain rotamers could not be absolutely determined and were set to those found in common with the PKC-β (1a25) and the synaptotagmin I (1rsy) C2 domains. It should be noted, however, that the side chain rotamers in the Ca +2 -binding region of the C2B domain could be unambiguously assigned . The Mg +2 and the sulfate ions were included in the model after the protein tracing was completed. It should be noted that at this resolution one cannot exclude the possibility that some of the Mg +2 electron density peaks correspond to ordered water molecules. However, the observed Mg +2 sites correspond to known sites in the divalent cation-binding sites of other C2 domains. The last few COOH-terminal residues of synaptotagmin III are predicted to be α-helical by secondary structure prediction . Although they are present in the expression construct used for crystallization, they were disordered in the crystal structure. The cytosolic portion of synaptotagmin III contains two tandem C2 domains joined by a short seven-residue linker . Each of the two homologous C2 domains consists of an eight-stranded Greek key β-sandwich with type I (S-type) C2 topology . The Ca +2 -binding pocket (CalB) of each domain is located at the apex of the fold and the divalent cations are cradled in between three conserved loops. The fold of each C2 domain contains a series of α-β bulges , which are unique to C2 domains. This motif determines the overall shape of the domain and may position key residues to surface accessible locations. The Ca +2 -binding regions of both C2 domains are directed toward each other in the crystal structure. Only ∼300 Å 2 of surface area is shared between the two domains, which is likely too small to represent an important interaction in solution. It is therefore unlikely that this contact has physiological function. Thus, we predict that the two C2 domains are largely independent of each other in solution. The Ca +2 -binding loop 3 of C2B is disulfide linked to another C2B domain that is related by crystallographic symmetry to another molecule. This covalent linkage does not affect the overall loop conformation since the Ca +2 -binding loops of the superimposed C2A and C2B domains are very similar. The relative orientation of the two C2 domains represents the conformation of synaptotagmin III C2A-C2B favored by crystallization. The observed partial disorder of the C2B domain in this crystal form suggests that the relative orientation and position of the two C2 domains is variable in solution. This anisotropic disorder is confined to the plane of the β-sandwich, and the electron density of the β-sheets of the C2B domain is somewhat smeared out. However, the electron density for the C2B domain at the apex of the fold, including the Ca +2 -binding pocket, is well defined, allowing sidechain interpretation of the Ca +2 -binding region of the C2B domain . The C2A and C2B domains are structurally similar with the exception of the α-helix between the strands β7 and β8 of each C2 domain . The seven-residue α-helix of the C2B domain is not present in either the C2A domain of synaptotagmin I, the isolated C2 domain of PKC-β, or the type-II (P-type) C2 domain of PLC-δ1. This conserved α-helical insertion has also been reported in the isolated C2B domain of rabphilin-3A and may have functional importance . The root-mean-square difference (Rmsd) between the rabphilin C2B and the synaptotagmin III C2B domains computed over all Cα atoms is 1.5Å. As in the structure of the C2A domain of synaptotagmin I and the isolated C2 domain from protein kinase C, the C2A domain of synaptotagmin III contains a cis-proline (Pro 411) that precedes the β-strand containing the polybasic region. The homologous region in C2B, however, does not contain this proline. Instead, it uses a non-proline β-turn to accommodate the residues leading into the polybasic strand. In the C2B domain, this insertion shifts the polybasic region to a more central location on strand β4. Synaptotagmin III crystallized in the presence of 1.5 M MgSO 4 . Although Mg +2 could potentially mimic Ca +2 binding, the aspartate residues in the C2A domain that have been shown by solution NMR and x-ray crystallography to pivot upon divalent cation binding, are not in the Ca +2 -bound conformation . Under these crystallization conditions, the synaptotagmin III C2A domain is in the unliganded conformation. However, three peaks in electron density difference maps were found in the Ca +2 -binding pocket of the C2A domain. These peaks coincide with the positions of the three calcium ions in the PKC-β C2 structure and were interpreted as Mg +2 . The Mg +2 could be responding to the negative electrostatic field from the aspartate residues within the divalent cation-binding pocket of the C2A domain without proper coordination of the aspartate residues. In the C2B domain, significant electron density, consisting of a single 5σ peak, was also found in the vicinity of the Ca +2 -binding region . This peak corresponds to the high affinity Ca +2 site observed in the crystal structures of the synaptotagmin I C2A, the PKC-β C2, and the PLC-δ1 C2 domains. Although the divalent cation coordinating aspartate residues are present in the C2B domain, only one Mg +2 binds in the divalent cation-binding pocket, despite the very high Mg +2 concentration used for crystallization. The interpretation of this electron density peak in terms of a divalent cation-binding site is supported by the observed substitution of the site by a trimethyl lead ion in the TMLA derivative. The linker between C2 domains for most of the synaptotagmin isoforms, including synaptotagmin III, is seven to nine residues in length . The amino acid composition of synaptotagmin III suggests a rather flexible linkage with two contiguous glycine residues. The primary sequence of the linker is not conserved among the synaptotagmin homology group . The inter-domain linker may be more rigid for the synaptotagmin isoforms that have fewer glycine residues and more proline residues, such as synaptotagmin VII or synaptotagmin VIII . The rigidity of the inter-domain linker in some synaptotagmin isoforms may position the C2 domains for docking to vesicle-fusion related protein complexes, or otherwise restrict the range of motion possible between the two C2 domains. Other tandem C2 domain containing proteins such as rabphilin or rim have much longer linkers, thus providing more flexibility between C2 domains. Synaptotagmin III is characterized by promiscuous binding to various accessory proteins and membrane components . The two C2 domains of synaptotagmin have different binding partners and binding affinities. The x-ray crystal structure of the cytosolic domain of synaptotagmin III provides a structural explanation of these disparities between the two C2 domains. Despite the lack of Ca +2 in the crystallization condition, this structure could still mimic some of the Ca +2 -binding properties of C2B domains in the synaptotagmin homology group. In the crystal structure, the C2B domain associates with only one Mg +2 . The reduced divalent cation-binding capacity of the C2B domain would leave this area of the molecule with a residual negative charge relative to the C2A domain. This difference between the C2A and C2B domains may explain some of the biochemical differences observed in in vitro experiments. Overall, the C2A domain of synaptotagmin III has a more uniform electrostatic surface potential than the C2B domain . The surface of the C2B domain possesses distinctly basic and acidic areas . These charged areas may be important for the Ca +2 -independent interactions observed in the isolated C2B domain. The Ca +2 -binding pocket of the C2B domain is chemically similar to the Ca +2 -binding pocket of the C2A domain; however, the shape of the pocket is very different . In principle, this can be explained by either a difference in backbone conformation between the two domains or by a difference in sidechain packing. Superposition of the two domains does not reveal a significant deviation in the backbone position; however, one cannot rule out more subtle backbone differences within the coordinate error of the crystal structure. Although synaptotagmin III has been implicated in Mg +2 -dependent phospholipid binding , the crystal structure indicates that 1.5 M Mg +2 is unable to induce rotamers of the aspartic acid residues similar to those found in C2A domains complexed with Ca +2 . In the presence of Ca +2 , crystallographic and NMR studies indicated that Asp 466 (and homologous residues in other synaptotagmin isoforms) pivot to coordinate the highest affinity Ca +2 in the C2A domain . Therefore, the Mg +2 in the synaptotagmin C2A-C2B structure are probably responding to the negatively charged surface of the C2A domain rather than coordinating with specific residues. The ionic radius of Mg +2 may also be too small to induce a Ca +2 -like coordination. A sulfate ion from the crystallization medium is located near Lys 356 of the C2A domain of synaptotagmin III. This ion may mimic phospholipid binding to synaptotagmin, since this face of the C2A domain interacts with the phospholipid bilayer . The crystal structure of the C2A domain of synaptotagmin I also contained a sulfate ion derived from the crystallization . The sulfate ion in this structure is in a different position; however, it is associated with the same face of the protein. This indicates that the electrostatic potential is positively charged at this location on the protein. Only one molecule is present in the asymmetric unit of this crystal form. The crystallographic symmetry together with primary sequence analysis of the available synaptotagmin isoforms may provide clues to the mechanism of C2B domain homo- and heterodimerization. Two crystallographically related molecules in this hexagonal crystal form direct their divalent cation-binding pockets toward the sixfold crystallographic axis. The resulting crystal packing contacts include the sequence Asp-Phe-Asp (386–388), which includes two of the divalent cation-binding residues in C2 domains. These three residues are conserved in all C2A domains with the exception of synaptotagmin VII. In the C2B domain, the homologous motif is Asp-Tyr-Asp (520–522), with few exceptions among the other synaptotagmin isoforms. In the crystal structure of the synaptotagmin III C2A-C2B domains, an alternate rotamer of Tyr 521 can be modeled to coordinate Asp 466 of the crystallographically related molecule to form a hydrogen bond. Aspartate 466 anchors the divalent cation-binding chain in the Ca +2 -binding pocket of C2 domains. This putative interaction may provide an initial nucleation point for self-association. The sequence variability present in the other isoforms of synaptotagmin, for example, synaptotagmin VII, VIII, and XI, may modulate their individual binding properties to other synaptotagmin isoforms. The core of the SNARE complex, composed of synaptobrevin-II (1–96), syntaxin-1A (180–262), SNAP-25A (1–83), and SNAP-25A (130–206), interacts with the C2A and C2B domains of synaptotagmin III independent of divalent cations . We have modeled the association between these two moieties using the following arguments: first, the position of the presynaptic membrane restricts the possible contacts between the two C2 domains of synaptotagmin and the SNARE complex. The syntaxin component of the SNARE complex embeds its transmembrane α-helix into the presynaptic membrane; likewise, the synaptobrevin transmembrane α-helix is anchored in the vesicle phospholipid membrane. Our model, therefore, puts the synaptobrevin α-helix on top of the complex and the syntaxin α-helix on the bottom relative to the presynaptic membrane bilayer on the bottom of Fig. 6 . The palmitoylated SNAP-25 linker would also contact the presynaptic membrane, so it localizes to the membrane associated with syntaxin. Second, the divalent cation-binding loops (CalB) of the C2 domains of PLA2 contact phospholipid bilayer with some residues embedded in the bilayer itself . Ca +2 in the CalB region either directly binds phospholipid headgroups through Ca +2 bridging or indirectly binds by quenching the electrostatic charge of the CalB domain, in situ . This restriction would allow the C2A and C2B domains of synaptotagmin to straddle the fusion complex and to interact simultaneously with the presynaptic membrane. Third, the unique electrostatic properties of the SNARE complex and synaptotagmin allow the positively charged, polybasic region of the C2 domains to interact with the negatively charged surface at the center of the SNARE complex . Fourth, the cupped shape of the polybasic region of the C2 domains provides a favorable interaction surface with the cylindrical SNARE complex. Fifth, the length and flexibility of the inter-domain linker restrict the possible C2 domain orientation with respect to the SNARE complex. Most isoforms of synaptotagmin have inter-domain linkers of eight to nine residues in length . The C2 domains presumably act as independent rigid bodies, so the inter-domain linker governs the available flexibility between them. The amino acid composition of this linker is not conserved among synaptotagmin isoforms. The proportion of glycine residues and therefore, the degree of flexibility possible between C2 domains varies by isoform. Some of the linker regions of synaptotagmin contain proline residues; therefore, the C2 domains of these isoforms are probably more restricted in their possible orientations. Specific regions of the SNARE complex are likely to be involved in synaptotagmin C2 domain interactions. The NH 2 -terminal domain of syntaxin interacts with the C2A domain of synaptotagmin I . The COOH-terminal 220–240 residues of syntaxin bind in a Ca +2 -dependent manner to the cytoplasmic domain of synaptotagmin . The C2B domain of synaptotagmin also binds to SNAP-25. This interaction is not affected by the deletion of the COOH-terminal 26 residues by BoNT/E suggesting that the NH 2 -terminal region of SNAP-25 is not involved in binding the C2B domain. Therefore, the C2B domain of synaptotagmin probably interacts with adjacent regions of SNAP-25 and syntaxin on the surface of the synaptic fusion complex in a Ca +2 -independent fashion . According to the zipper model of SNARE-mediated fusion , the SNARE proteins associate from the NH 2 to COOH terminus as the neurotransmitter-filled synaptic vesicle and presynaptic membranes approach each other. In the early stages of fusion, synaptotagmin may act as a negative regulator of exocytosis by preventing the completion of the “zippering” action of the SNARE complex by binding to the COOH-terminal, membrane attachment side of the SNARE complex through surface electrostatic interactions on the fusion complex, independent of divalent cations. Indeed, synaptotagmin may bind with higher affinity to the partially assembled SNARE complex than to the fully assembled complex. When an action potential or other fusion signal triggers a Ca +2 flux, as in the case of neuronal exocytosis, synaptotagmin may then bind to the presynaptic phospholipid membrane in a Ca +2 -dependent manner in preference to the SNARE complex. Therefore, synaptotagmin binding to the SNARE complex or the presynaptic membrane would be mutually exclusive properties of synaptotagmin. Once synaptotagmin is associated with the membrane, the SNARE complex would then be available to mediate fusion between the vesicle membrane and the presynaptic phospholipid membrane. | Study | biomedical | en | 0.999997 |
10545503 | The spleen-derived murine DC line D1 has been described previously . It was cultured in Iscove's modified Dulbecco's medium (Sigma Chemical Co.) supplemented with 10% endotoxin-free FCS (Life Technologies, Inc.) and 30% R1 medium (granulocyte/macrophage colony-stimulating factor [GM-CSF]–transfected NIH-3T3 fibroblast conditioned medium). Cells were passaged twice a week in 145-mm nontissue culture–treated petri dishes (4–5 × 10 6 cells per dish). To control their nonactivated state, the level of cell surface expression of MHC class II and costimulatory molecules was checked regularly by FACS ® as described . Fresh DCs were generated from bone marrow (BM) as described previously . In brief, cells obtained from the femur BM of C57/Bl6 mice were cultured for 2–3 wk in granulocyte/macrophage colony-stimulating factor (GM-CSF)–containing medium, as described for D1 cells. Tumor cell line TS/A (spontaneously arising undifferentiated mammary carcinoma, H-2 d ; kind gift from G. Forni, University of Turin, Turin, Italy) was cultured in RPMI 1640 supplemented with 10% endotoxin-free FCS, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, nonessential amino acids, and sodium pyruvate. Antibodies used here were: mouse anti–I-A b mAb (Y3P) for FACS ® analysis and immunoprecipitation; rat mAb anti–mouse MHC II for EM; rabbit antiserum to the cytosolic sequence of mouse MHC II α chain for Western blot. Rat anti–mouse invariant chain (Ii) (IN-1 mAb), rabbit anti–mouse calnexin (kindly provided by A. Helenius, Swiss Federal Institute of Technology, Zürich, Switzerland), mouse mAb anti–mouse annexin II (kindly provided by Dr. V. Gerke, Institut für Medizinische Biochemie, Münster, Germany), rabbit anti-hsp84 (Affinity BioReagents), and rat anti–glycoprotein (gp) 96 (SPA850) for Western blot; rat mAb anti–mouse CD9 (PharMingen) and anti-hsc73 (SPA815) (StressGen Biotechnologies Corp.) for Western blot, EM, and immunoprecipitation; rat mAb anti–mouse Mac-1 (M1/70) and rabbit anti–mouse annexin II antiserum (kindly provided by V. Gerke) for immunoprecipitation and EM; and FITC-conjugated rat anti–mouse CD40 (PharMingen) for FACS ® analysis. Secondary antibodies were: FITC-conjugated anti–mouse Ig (Jackson ImmunoResearch Laboratories, Inc.) for FACS ® analysis; HRP-conjugated secondary antibodies from Pierce for Western blotting; and polyclonal anti–rat antibody (DAKO), and/or protein A gold (purchased from Dr. J.W. Slot, Department of Cell Biology, Utrecht University, The Netherlands) for EM. Tumor growth assay was performed as described previously . In brief, BALB/c (H-2 d ) or syngeneic nude mice were injected intradermally with 10 5 TS/A cells per mouse. At day 4 after tumor inoculation, mice were injected intradermally with 3–5 μg of exosomes per mouse in the lower ipsilateral flank. Exosomes were prepared from 24-h supernatant of BALB/c bone marrow–derived DCs (BM-DCs) exposed to peptides eluted from surface MHC class I molecules of tumor cells by acid treatment (acid-eluted peptide [AEP]). As a control, AEP from normal splenic cells were fed to BM-DCs before exosome purification. Tumor size was monitored weekly for 1 mo. D1 cells, either immature or mature after treatment for 24 h with LPS, were fixed with 2% paraformaldehyde in phosphate buffer 0.2 M, pH 7.4 (PB), for 2 h at room temperature. Ultrathin sections of cells fixed and processed for ultracryomicrotomy , as well as whole mounts of exosomes , were immunogold-labeled as described previously. Observations were made with a CM120 Twin Phillips electron microscope. Exosomes were prepared either from the supernatant of a 3-d-old DC culture, or from fresh culture medium incubated for 24 h with 3-d-old DCs. We could not evidence any difference in the overall composition of exosomes purified from 3-d or from 24-h supernatants. Exosomes were purified as described previously , by three successive centrifugations at 300 g (5 min), 1,200 g (20 min), and 10,000 g (30 min) to eliminate cells and debris, followed by centrifugation for 1 h at 110,000 g . The exosome pellet was washed once in a large volume of PBS, centrifuged at 110,000 g for 1 h, and resuspended in 50–200 μl of PBS with 0.01% sodium azide. The amount of exosomal proteins recovered was measured by Bradford assay (Bio-Rad). As different batches of FCS used for tissue culture contain variable amounts of endogenous bovine exosomes (W. Stoorvogel, personal communication), the batch used for DC culture was carefully characterized in terms of amount of bovine exosomes and markers expressed by these exosomes. Approximately 10% of the exosomal proteins recovered from a D1 or BM-DC supernatant come from FCS. Two antibodies used here or in a previous study recognize both murine and bovine proteins present in exosomes: anti-hsc73 and anti-TfR (H68.4 hybridoma). Therefore, the actual presence of the murine, DC-derived protein in exosomes was demonstrated by immunoprecipitation from metabolically labeled, DC-derived exosomes , and by Western blots performed on exosomes produced by DCs grown in medium depleted of bovine exosomes by overnight centrifugation at 110,000 g (data not shown). Under these conditions, TfR was detected but not enriched in exosomal preparations (data not shown). Flotation of exosomes on a continuous sucrose gradient was performed as described , but in an SW41 rotor. Fractions of the gradient (1 ml each) were diluted in 2 ml of PBS, centrifuged for 1 h at 100,000 g , and the pellet was loaded on a 10% SDS gel for Western blot analysis. Total proteins were obtained from D1 cells lysed in 50 mM Tris, pH 7.5, 0.3 M NaCl, 0.5% Triton X-100, 0.1% sodium azide, with a cocktail of antiproteases (chymostatin, leupeptin, aprotinin, pepstatin [CLAP], 100 μM each; Sigma Chemical Co.), and cleared from nuclei by centrifugation at 10,000 g . To separate cytosol and total membranes, D1 cells were homogenized in 10 mM triethanolamine, 1 mM EDTA, 10 mM acetic acid, 250 mM sucrose, pH 7.4 (TEA-sucrose), supplemented with CLAP, by 60 passages through a 25-G needle. The supernatant, cleared from nuclei and cell debris by centrifugation at 1,200 g , was centrifuged for 1 h at 100,000 g ; total membranes were recovered in the pellet and cytosol in the supernatant. Proteins (1–10 μg) were separated on 10 or 12% SDS-PAGE, blotted on Immobilon (Millipore), and detected by Western blot using an enhanced chemiluminescence detection kit (Boehringer Mannheim). Metabolic labeling of D1 cells or BM-DCs was performed: cells were starved for 1 h at 37°C in methionine/cysteine-free RPMI supplemented with 30% dialyzed R1 medium (labeling medium), and incubated overnight at 37°C in fresh labeling medium supplemented with 5% FCS and 10–20 μCi/ml [ 35 S]methionine/cysteine (Promix 35 S; ICN). Labeling medium was removed, then cells were washed and further incubated for 24 h in complete medium at 37°C. Exosomes were purified from the supernatant; total lysates, cytosol, and total membranes were prepared from the same cells. Proteins (20,000 cpm of each sample) were separated on a 12% SDS-PAGE, dried, and autoradiographed. The peak profile of each lane was obtained using NIH Image software. Alternatively, 200,000 cpm of exosomes were loaded on a continuous sucrose gradient. The fractions were then run on a 10% SDS-PAGE, dried, and autoradiographed. For immunoprecipitation, 3 × 10 6 cpms of cell lysates or exosomes were diluted in 1 ml lysis buffer, precleared for 2 h in the presence of 50 μl protein G–Sepharose, and precipitated with specific antibodies coated on protein G–Sepharose. 30 μg of proteins from exosomes or total lysates was run on an 8–15% SDS-PAGE and stained with Coomassie blue (Bio-Rad). The major bands in the exosomes preparation were excised from the gel, and in-gel digested with trypsin as described previously . An aliquot of the digest solution was analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS), using a Bruker Biflex mass spectrometer (Bruker-Franzen Analytik) equipped with a scout multiprobe inlet and a gridless delayed extraction ion source. Ion acceleration voltage was 19.5 kV and the reflectron voltage was 20.0 kV. For delayed ion extraction, a 6.2-kV potential difference between the probe and the extraction lens was applied. Mass spectra of peptide mixtures were acquired as the sum of ion signals generated by irradiation of the target with 100 laser pulses. They were calibrated using ion signals from trypsin autodigestion peptides . Monoisotopic peptide masses were assigned and used in database searches. Typical search parameters, using the Microsoft FIT program, were as follows: maximum allowed peptide mass error of 100 ppm; consideration of one incomplete cleavage per peptide; no restriction was placed on the isoelectric point of the protein, and a protein mass range of 0–200 kD was allowed. Subcellular fractionation of D1 cells was performed as described previously for murine B lymphoma cells . In brief, cells homogenized in TEA-sucrose were first fractionated on a discontinuous sucrose gradient, where the low density membrane (LDM) fraction was recovered at the junction between 1 and 0.25 M sucrose, and the high density membrane (HDM) fraction at the junction between 1.04 and 1 M sucrose. After mild trypsin digestion (5 ng trypsin [Sigma Chemical Co.] per μg of proteins, 10 min at 37°C, stopped by soybean trypsin inhibitor, 10 ng/ng trypsin), LDMs were loaded on a free-flow electrophoresis (FFE) chamber (Dr. Weber, GmbH, Ismaning, Germany). Fractions were collected, pooled pairwise, and analyzed for protein content (Bradford assay; Bio-Rad) and β-hexosaminidase activity . 15 pools of fractions, within 10 fractions of the protein and β-hexosaminidase activity peaks, were kept for further analysis. They were centrifuged at 10,000 g for 1 h, and the pellets were resuspended in reducing SDS sample buffer and run on SDS-PAGE for Western blot analysis. We have recently shown that murine BM-DCs secrete exosomes . Exosomes were purified by ultracentrifugation from supernatants of BM-DCs exposed to peptides eluted from MHC class I molecules at the surface of a tumor cell line. Injection of tumor peptide-pulsed exosomes into mice bearing the tumor induced a strong delay in tumor growth, whereas injection of exosomes pulsed with normal spleen–eluted peptides had no effect . This antitumor response was only observed in immunocompetent mice; in nude mice that lack T lymphocytes, tumor growth was not affected by injection of exosomes pulsed with tumor peptides . Therefore, DC-derived exosomes elicit T cell–dependent immune responses resulting in reduced tumor growth and tumor eradication . To unravel the molecular mechanisms of exosome action, we used the well-characterized growth factor–dependent murine spleen DC line, D1 . D1 cells show all the attributes of fresh immature DCs, including high endocytic capacity and abundant MHC class II–containing lysosomal compartments. As shown by EM, immature D1 cells displayed numerous characteristic MVBs containing internal vesicles labeled by anti–MHC class II antibodies . Such MVBs were also observed apposed or in close proximity to the cell surface, suggesting their possible fusion with the plasma membrane and the secretion of exosomes. Exosomes were indeed isolated from D1 culture supernatants by differential centrifugation as described previously . After the last ultracentrifugation step, the pellet was washed once in PBS. Thus, we obtained routinely 15–20 μg of exosomal proteins (as measured by Bradford assay) from the supernatant of a 24-h culture of 2 × 10 7 immature D1 cells. Whole-mount EM preparations of exosomes showed a population of vesicles , similar in size and morphology to BM-DC–derived exosomes . As noted above for DC-derived exosomes, these vesicles are more heterogeneous in size and MHC class II labeling than B-EBV–derived exosomes (40–90 vs. 50–80 nm in diameter, respectively). Western blot analysis showed that exosomes secreted by D1 cells are, like exosomes produced by B-EBV or by BM-DCs , enriched in MHC class II, and devoid of Ii, which associates with newly synthesized MHC class II in the ER. D1-derived exosomes are also devoid of calnexin (an ER resident protein). Like exosomes from B-EBV cells , D1-derived exosomes migrate on a continuous sucrose gradient at a density of 1.14–1.20 g/ml , confirming their vesicular nature. Therefore, exosomes produced by D1 cells resemble exosomes produced by BM-DCs or B-EBVs. We decided to use D1 cells to analyze the regulation of exosome production during DC maturation and to characterize the molecular composition of exosomes. In immature DCs, most MHC class II and Ii molecules are found in late endocytic compartments . As shown in Fig. 2 A, many of these compartments display intraluminal membrane vesicles. Upon maturation, these compartments disappear, and MHC class II molecules are redistributed to the plasma membrane . Accordingly, we observed that after LPS treatment of D1 cells for 24 h, the number of MHC class II–positive compartments, as well as the amount of MHC class II in the remaining compartments, decreased (on average, the number of MVBs per cell profile decreased from five to eight in immature, to one to two in mature D1 cells) . Concomitantly, MHC class II labeling at the cell surface is highly increased, and the plasma membrane displayed numerous processes . Although the precise pathway of MHC class II transport to the plasma membrane during this maturation process is still unknown, it is possible that direct fusion of endocytic compartments with the plasma membrane participates in this process. In this case, increased exosome production should occur upon DC maturation. We tested this possibility by quantifying the amount of exosomal proteins produced by immature D1 cell in the presence or absence of LPS. Unexpectedly, we observed a lower production of exosomes in the presence of LPS (a 35% reduction) . Furthermore, if maturation was induced for 16 h, before the 24-h time period of exosome collection, the amount of produced exosomes was reduced by 67%, as compared with immature D1 cells . At the end of the total 40-h LPS treatment, D1 cells showed all the signs of maturation: increased surface expression of MHC class II and costimulatory molecules, and decreased internalization activity . Similar results were obtained with murine BM-DCs and with human monocyte–derived DCs (data not shown). Therefore, production of exosomes is developmentally regulated: it is effective in immature DCs and reduced upon maturation, when endocytic activity of DCs decreases. These results also suggest that exosome formation is an active process, requiring the selection of a limited set of proteins, whose identity defines exosomes as an independent cellular compartment and whose functions determine their biological roles. Therefore, we undertook an extensive characterization of the protein composition of exosomes. D1 cells were metabolically labeled overnight with [ 35 S]methionine/cysteine, washed once, and fresh nonradioactive medium was added to the cells for 24 h. Metabolically labeled exosomes were purified from this supernatant. In four independent experiments, the amount of radioactivity recovered in exosomes was 0.17 ± 0.1% of the radioactivity incorporated into cells. The protein composition of exosomes from metabolically labeled cells was analyzed by SDS-PAGE and autoradiography, and compared with that of whole D1 cells, cytosol, or total cellular membranes. As shown in Fig. 5 A, exosomes displayed a unique protein composition pattern. At least seven proteins (of ∼180, 90, 70, 58, 43, 32, and 27 kD) were strongly enriched in exosome preparations compared with whole cells, cytosol, or total membranes. At least eight other major proteins from whole cells, cytosol, or total membranes were either absent or less abundant in exosomes . Interestingly, two of the bands enriched in exosomes (70 and 58 kD) are not detected in the cytosol or in the cell lysates, suggesting a particularly strong mechanism of enrichment of this protein in exosomes. Similar analyses were performed on metabolically labeled primary BM-DCs, and the pattern of bands obtained was very similar to that of exosomes from D1 cells (data not shown). All the proteins present in exosomes comigrated on a continuous sucrose gradient at the expected density (1.15 g/ml), confirming that all the proteins in the exosome pellets are associated to vesicular structures of similar density. Therefore, DC-derived exosomes accumulate a unique subset of cellular proteins. To identify these proteins, 30 μg of exosomes was loaded on an 8–15% gradient SDS gel; the pattern of bands stained by Coomassie brilliant blue was very similar to that of metabolically labeled exosomes . All of these major bands were excised, trypsin-digested, and the peptides generated were analyzed by MALDI-TOF-MS. The peptide profiles generated from the different bands were compared with the theoretical tryptic peptide profiles of known proteins from the databases . The results are summarized in Table . Among the identified proteins, three are transmembrane (Mac-1 α chain, MHC II β chain, and CD9), one is secreted and peripherally associated to membranes (MFG-E8), and four are cytosolic, often found in association with membranes (Gi2α, annexin II, gag from MRV provirus, and hsc73). In two cases (MFG-E8 and gag), the theoretical molecular weight of the identified protein was not consistent with its migration on the SDS gel. Two forms of MFG-E8, due to various degrees of glycosylation, have been described in mice and cows , probably accounting for the migration of MFG-E8 as three separate bands in our exosome preparation . Gag is first translated as a 60-kD precursor and subsequently cleaved into three mature products: the gag peptides generated from band 10 all matched the mature 30-kD core shell protein. The presence of hsc73, annexin II, MHC II, Mac-1, and CD9 in exosomes from D1 cells and primary BM-DCs was quantified by Western blot and immunoprecipitation from metabolically labeled cells . Similar amounts of exosomes and cell lysates (2 and 6 μg) or the same number of counts (3 × 10 6 cpm) were analyzed by Western blot and immunoprecipitation, respectively. MHC II and CD9 were highly enriched (>10-fold) in exosomes compared with total cells. Mac-1 was enriched by 5–10-fold, annexin II and hsc 73 by 2–3-fold. These results confirm the MALDI-TOF-MS analysis and show that exosomes accumulate a defined set of cellular proteins. Immunoelectron microscopy analysis of whole-mounted exosomes showed staining for MHC II, CD9, and Mac-1, but not for annexin II and hsc73, although the antibodies used stained D1 cells ultrathin cryosections (data not shown). These results suggest that in contrast to MHC class II, CD9, and Mac-1, hsc73 and annexin II are not exposed at the surface of exosomes but contained within their lumen. The MALDI-TOF-MS analysis of band 4 revealed a mixture of the MFG-E8 protein and another protein of potential interest in our study of exosome-mediated antitumor effects: the 70–kD hsp family member, hsc73. Several groups have reported that three hsp family members, hsc73, gp96, and hsp84, induce immune responses and rejection of established tumors . Since the presence of hsc73 in exosomes could be relevant for exosome's antitumor effects, we analyzed in more detail the presence of different hsp family members in DC-derived exosomes. As shown in Fig. 7 A and 8 A, when the same amount of proteins from total cells or from exosomes were analyzed by Western blot, approximately three times more hsc73 could be detected in the exosomal preparations than in D1 total cell lysates. The two other main hsps generating antitumor immune responses were either absent (gp96), or present, but not enriched (hsp84) in exosomes, compared with cell lysates . In murine fibroblasts, hsc73, or a closely related protein, accumulates in lysosomes , raising the possibility that the presence of hsc73 in exosomes results from its selective accumulation in endocytic compartments. Therefore, we analyzed the subcellular distribution of the three main hsps involved in antitumor immunity, gp96, hsp84, and hsc73. Subcellular fractionation of D1 cells was performed first by discontinuous sucrose flotation to separate cytosol and HDMs (ER, some plasma membrane, and Golgi apparatus) from LDMs (endosomes, lysosomes, plasma membrane, and Golgi apparatus). The LDMs were then further fractionated by FFE, to separate endosomes and lysosomes from the other cell membranes . As expected, gp96 was found in the HDMs and LDMs, but was absent from the cytosol . In contrast, hsp84 was detected in the cytosol and the HDMs (which, because of the setting of the sucrose gradient, is often contaminated with cytosol), but totally absent from the LDMs. hsc73 was present in all three fractions, consistent with its proposed presence in the cytosol and endocytic pathway. Before the FFE, LDMs were mildly trypsin digested. This treatment generated a 60-kD tryptic fragment of hsc73, and a 80-kD fragment of gp96 . The LDMs were then further fractionated by FFE. Most cellular membranes are not negatively charged, and consequently, the bulk of cellular proteins are not shifted to the anode . Negatively charged endosomal and lysosomal membranes, as detected by β-hexosaminidase enzymatic activity (a lysosomal resident enzyme), were found in the fractions deviated towards the anode . Western blot analysis of the FFE fractions, using anti–MHC class II antibodies, showed that in D1 cells, as in human and mouse DCs , abundant MHC class II molecules are present in the endocytic pathway . To localize hsc73 and gp96 in the fractions, the same filters were hybridized with the corresponding specific antibodies. As expected, gp96 was only present in unshifted FFE fractions, containing plasma, ER, and Golgi membranes. In contrast, most hsc73 was found in shifted FFE fractions, together with endosomes and lysosomes . Therefore, hsc73 accumulates in endocytic compartments in D1 cells, probably accounting for the selective presence of hsc73 in exosomes. DC-derived exosomes elicit potent T cell–dependent immune responses in mice . This striking effect on the immune system in vivo prompted us to analyze the molecular structure of exosomes. Thus, here we identify eight major proteins from DC-derived exosomes. Fig. 9 shows a scheme of our current idea of the nature and topology of the major proteins present in exosomes. Exosomes contain a defined subset of cytosolic proteins, most likely involved in exosome function and/or biogenesis . Exosomes also accumulate membrane proteins potentially involved in their association to target cells or in T cell activation . The most abundant exosomal protein, MFG-E8, binds to integrins (αvβ3 and αvβ5) expressed in DCs and macrophages, and could target DC-derived exosomes to other APCs. Another exosomal protein, the hsp 70 family member hsc73, is a potent inducer of antitumor immune responses in vivo, suggesting a mechanism for exosome's antitumor effects . The presence of hsc73 in exosomes is interesting in relation to two aspects of exosome biology: their biogenesis and their biological effects in vivo. We show that in D1 cells, a significant fraction of hsc73 is associated to endocytic compartments , as suggested in other cell types such as fibroblasts . Furthermore, the two other hsps analyzed here, gp96 (also known as grp94) and hsp84 (or hsp90), are not associated to endosomes and lysosomes , and do not accumulate in exosomes . These results are consistent with the idea that exosomes form in endocytic compartments. The topography of exosome-associated hsc73 is unclear. As a cytosolic protein, hsc73 should be inside the vesicles. Consistent with this possibility, exosome-associated hsc73 was partially protected from trypsin digestion (data not shown), and was not detected by immunoelectron microscopy on whole mounts of exosomes , suggesting that it resides inside exosomes. However, hsc73 may also be taken up by a putative specific receptor and accumulate on MVB internal vesicles, and thereby on the surface of exosomes. Dice and co-workers have shown that hsc73 is involved in binding misfolded proteins and addressing them to lysosomes, suggesting that hsc73 may translocate across lysosomal membranes . Moreover, in exosomes secreted by reticulocytes, the two major proteins (on Coomassie blue–stained gels) are TfR and hsc73 : hsc73 was postulated to associate with the TfR in order to help its segregation into exosomes. However, hsc73 in exosomes from DCs may play a totally different role. The presence of hsc73 in exosomes of APCs is most interesting in the light of results showing that hsps induce antitumor immune responses . hsc73 is associated with endogenous peptides, and injection of hsc73 purified from the tumor in tumor-bearing mice stimulates potent cytotoxic T lymphocyte immune responses and tumor rejection . Furthermore, hsc73-associated peptides are efficiently transferred to APCs for MHC class I and II antigen presentation . This may be due to the presence of a receptor for hsc73 at the surface of macrophages and DCs . hsc73 can also induce macrophages to activate T cells independently of antigen . The role of exosomes in peptide transfer between cells and their capacity to activate macrophages and DCs are under examination. Indeed, it is still unclear whether exosomes interact directly with T lymphocytes or require the presence of an additional APC. Although direct stimulation of T cells by exosomes was observed in several antigenic systems in vitro , it was always inefficient, compared with stimulation induced by intact APCs. In contrast, our recent results show that T cell stimulation by exosomes is much more efficient in the presence of DCs (Regnault, A., A. Lozier, C. Théry, G. Raposo, S. Amigorena, and L. Zitvogel, manuscript in preparation), suggesting an indirect mode of action for exosomes in vivo. In any case, to induce immune responses, exosomes must interact with target cells: T lymphocytes or APCs. From this point of view, the strong enrichment of MFG-E8 in exosomes is very interesting. MFG-E8 was originally described at the surface of milk fat globules . It has two EGF-like domains, with an integrin-binding, Arg-Gly-Asp motif and a phosphatidylserine-binding, factor VIII–like, domain. Our preliminary results show that exosomal membranes expose phosphatidylserine (Théry, C., B. Hugel, and J.-M. Freyssinet, unpublished data), suggesting that MFG-E8 may bind exosomes through phospholipids. The human and bovine homologues of MFG-E8 also interact with αvβ3 and αvβ5 integrins . Interestingly, αvβ3 and αvβ5 are expressed at the surface of macrophages and immature DCs, respectively, and mediate the phagocytosis of apoptotic bodies for clearance or for antigen presentation by MHC class I molecules . Therefore, MFG-E8 may target DC-derived exosomes to other APCs rather than to T lymphocytes. Furthermore, several other major exosome components could be involved in exosome–T cell or exosome–APC interaction. The presence and abundance of several members of the tetraspan protein family (CD63, CD81, CD82) in exosomes of B-EBVs or DCs have been reported recently . CD63 and CD82 interact with several membrane proteins, including integrins and MHC class I and II molecules, and probably potentiate cell–cell interactions . We show here that another tetraspan family member, CD9, is most likely the major tetraspan in DC-derived exosomes (see Table ). CD9 also associates to other transmembrane proteins, and is a cofactor potentiating the interaction of an EGF-like growth factor and its receptor . Therefore, CD9 could participate in the interaction of exosomes with either APCs or T cells in vivo. Another component of exosomes potentially involved in their interaction with cells is the α chain of Mac-1 (a β2 integrin also known as the type 3 complement receptor, CR3) . Since we have coprecipitated this chain with its β chain counterpart from exosomes , it is likely that functional Mac-1 is present at the surface of exosomes. Observations made by EM using an anti–Mac-1 antibody on preparations of intact exosomes as well as the known orientation of MHC class II molecules at the surface of B-EBV– or DC-derived exosomes , suggest that Mac-1 is present at the surface of exosomes in a conformation suitable for binding to its ligands. Therefore, Mac-1–expressing exosomes could be addressed to ICAM-1– or ICAM-2–expressing cells (the known ligands for Mac-1) such as DCs, lymphocytes, or endothelial cells. The presence in exosomes of plasma membrane–associated proteins, such as Mac-1 or CD9, could be explained by the extremely high constitutive internalization rate (including endocytosis and macropinocytosis) in immature DCs . As a result, most plasma membrane proteins are actively internalized and continuously recycled. This property may be important for the generation of exosomes, since mature DCs, with reduced internalization abilities, produce less exosomes than immature DCs . Concerning the cytosolic proteins found in exosomes, all of them are involved in vesicle budding or fusion of intracellular compartments. Annexin II plays a role in early endosome fusion, and in fusion of secretion granule with the plasma membrane . The Gi2α subunit of heterotrimeric G proteins has been found in phagosomes (Garin, J., unpublished observations), but its role is still unclear. The eventual presence in exosomes of the β and γ subunits, which normally associate with Giα, will be tested. Finally, detection of a murine retrovirus gag protein in exosomes may reflect the presence of endogenous retroviruses in D1 cells, as in most laboratory mice strains . Gag is a cytosolic protein associated to membranes by its myristoylated NH 2 terminus. The possible association of MRV gag with endocytic membranes has not been investigated so far, but budding of other retroviruses, such as HIV, into MVBs has been observed (Raposo, G., unpublished observation). However, morphological EM analysis failed to reveal any viral particle in our exosome preparations (G. Raposo, not shown). The work described here represents the first extensive analysis of the protein composition of a subendocytic compartment. The identification of most major components gives new critical insights into the function of DC-derived exosomes. It confirms their endocytic origin and suggests novel possibilities for their biogenesis, their cellular targeting, and their modes of action in vivo. The selective production of exosomes by immature DCs suggests that, in vivo, exosomes are produced in peripheral tissues, and not in the secondary lymphoid organs, where DCs migrate after maturation to stimulate T cells. Therefore, exosomes most likely do not directly activate T lymphocytes, but rather sensitize other DCs, which have not encountered antigens themselves for T cell stimulation. Through transfer of MHC–peptide complexes, antigens, or hsp-associated peptides, exosomes could represent a novel means of communication between cells of the immune system. | Study | biomedical | en | 0.999996 |
10545504 | DME, calf serum, and Lipofectamine were purchased from GIBCO BRL. FBS was purchased from Gemini Bio-Products Inc. Myelin basic protein was prepared from spinal cord (Pelfreeze) as previously described . Methyl cellulose, agarose, and all other reagents were purchased from Sigma Chemical Co. unless otherwise specified. The plasmids CMV5 HA-ERK2 , IL2Rβ1, IL2Rβ3 , CD2FAK, CD2FAK K454R, CD2FAK Y397F , FRNK and pcDNA3 FAK wt , pDCR ras G12V, and RSV Hyg are as described. The src construct containing an activating point mutation at tyrosine 527 was obtained from Dr. Jean Wang (University of California, San Diego). NIH 3T3 cells were cultured in DME supplemented with 10% bovine calf serum. FAK +/+ and FAK −/ − mouse embryo fibroblasts (MEFs) were cultured in DME supplemented with 10% FBS. For transfections, cells were plated at a density of 4 × 10 5 cells per 6-cm dish 24 h before transfection. Cells were transfected with Lipofectamine (GIBCO BRL) as previously described . 24 h after transfection, cells were transferred to medium containing 0.5% serum for an additional 24 h for adherent cells. Cells that were suspended for 24 h were trypsinized 24 h after transfection and placed in suspension in DME media containing 0.5% methyl cellulose, 0.4% serum over 1% agar-coated dishes as previously described . Cells were stimulated by the addition of serum to 10% for 10 min, extracted in lysis buffer , and assayed for Erk2 and MEK 1 activity. For transformation assays, cells were transfected using Lipofectamine with 0.4 μg of RSVHyg and 0.8 μg of two different cDNAs to give 2.0 μg total DNA. Vectors were the empty control vector, pDCR ras G12V, IL2β1, and CD2FAK. After 24 h, the cells were fed fresh medium and allowed to grow for 2 d. Cells were trypsinized and 1/20 of the total was replated in either normal growth medium, minimal medium, or soft agar to measure foci formation as previously described . Cells were also replated in medium containing 200 μg/ml Hygromycin B (Boehringer Mannheim) to measure transformation efficiency. Soft agar colony volume was determined by visually measuring colony diameter against a scale, and then calculating the volume according to the formula, V = 4/3πr 3 . Minimal media foci size was determined by trypsinizing the foci and replating them in soft agar. The number of soft agar colonies were counted after 2 wk to determine the number of transformed cells per original minimal medium focus. For assays of transfected hemagglutinin-tagged (HA) Erk2 activity, anti–HA (12CA5) antibody purified over an HA peptide affinity column was used for immunoprecipitations. Erk activities were immunoprecipitated from 150 μg of cell lysates using 0.5 μg of anti–HA antibody. For all assays, Erk activation was normalized to the amount of Erk2 protein immunoprecipitated. One third of each immunoprecipitation was run on a 10% SDS–polyacrylamide gel that was transferred to Hybond C (Amersham) and immunoblotted using the anti–Erk2 antibody (C-14; Santa Cruz Biotechnology) to measure the amount of Erk2 protein. The remaining two thirds were used to measure Erk2 activity to measure the in-gel kinase assay as described . Activities of endogenous Erks 1 and 2 from FAK +/+ and FAK −/ − MEFs were measured by running 5 μg of total cell lysate on gels for assay by the in-gel kinase assay method. In brief, samples were run on 12.5% SDS–polyacrylamide gels containing 0.25 mg/ml myelin basic protein and renatured. Kinase reactions were performed soaking gels in kinase buffer containing 25 μCi/ml γ-[ 32 P]ATP and 10 μM cold ATP. Gels were washed exhaustively and analyzed by autoradiography. Autoradiographs were quantitatively analyzed using a model I.S. 1000 digital imaging system from Alpha-Innotech Corp. Endogenous MEK-1 was immunoprecipitated from 100 μg of cell lysate using anti–MEK-1 (Santa Cruz Biotechnology). Immunoprecipitates were washed three times in lysis buffer and once in kinase buffer , 10 mM Tris, pH 7.5, 10 mM MgCl 2 , and 1 mM DTT. One fifth of the samples were used to measure the amount of MEK-1 protein by Western blotting, four fifths were used for kinase assays. MEK kinase activity was measured in kinase buffer containing 25 μM ATP, 5 μCi γ-ATP, and 2 μg of kinase-dead GST-ERK2 , for 30 min at room temperature. Samples were electrophoresed on 10% SDS–polyacrylamide gels, which were dried and analyzed by autoradiography. Our strategy for identifying components of the pathway that mediates the integrin requirement for growth factor activation of MAP kinase was to screen for activated mutants that could specifically restore MAP kinase in suspended cells. As an initial test of the strategy, HA-tagged Erk2 was coexpressed with a chimera that contains the cytoplasmic tail of the integrin β1 subunit fused to the extracellular and transmembrane domain of the Tac subunit of the IL-2 receptor (IL2Rβ1) . This chimera induces FAK phosphorylation in suspended cells, indicating that it signals constitutively . Previous work showed that cellular responses to serum or the purified mitogen platelet–derived growth factor or lysophosphatidic acid were similarly modulated by cell adhesion . Thus, cells were stimulated with 10% serum for convenience, and because it mimics normal culture conditions. When cotransfected with a control plasmid, the activity of the transiently transfected HA-Erk2 was strongly stimulated by serum in adherent cells, but was minimal in suspended cells . This behavior mimics the endogenous MAP kinase , indicating that the transfected Erk is regulated normally. By contrast, cells cotransfected with HA-Erk2 and IL2Rβ1 showed strong activation in both the adherent and suspended cells. Expression of IL2Rβ3 also rescued serum induction of MAP kinase activity in suspension, but expression of an IL2α5 construct had no effect (not shown). Neither IL2Rβ1 nor IL2Rβ3 directly induced Erk activity in the absence of serum or altered its activation in adherent cells. These results show that a constitutively activated integrin β1 cytoplasmic tail can substitute for cell adhesion and specifically restore serum activation of MAP kinase in suspended cells. The IL2Rβ1 construct induces phosphorylation of FAK in suspended cells ; thus, FAK was an obvious candidate to mediate this effect. To test if FAK was involved, a FAK chimera (CD2FAK) that has constitutively high kinase activity was coexpressed with the HA-tagged Erk2 construct. Expression of CD2FAK also completely restored serum activation of Erk2 in suspended cells , but like the IL2Rβ1, did not directly activate MAP kinase in the absence of serum or alter activation of Erk2 in adherent cells. Kinase-defective CD2FAK (K454R) had no effect on MAP kinase, indicating that tyrosine kinase activity is required. It is well established that activity of endogenous FAK is regulated by integrin-mediated adhesion , though soluble mitogens also stimulate FAK in some systems . We also observed that kinase activity of FAK immunoprecipitated from adherent 3T3 cells was much higher than from suspended cells, and that addition of serum caused only a slight increase in either case (data not shown). Western blotting of whole cell lysates indicated no change in the levels of FAK or appearance of proteolytic fragments after 24 h in suspension (data not shown). These results are in agreement with published data and support the idea that FAK is regulated by cell adhesion. Next, we tested whether FAK was required for the effect of integrins on this pathway. HA-Erk2 was coexpressed with the FAK tyrosine autophosphorylation mutant (CD2FAK Y397F) or the COOH terminus of FAK, termed FRNK, both of which have been shown to function as dominant negatives . These mutants were tested for their ability to block effects of the IL2β1 chimera and the endogenous integrins that bind to ECM in adherent cells in culture. Results from these experiments showed that the Y397F FAK mutant blocked activation of MAP kinase in adherent cells by 80% . A similar result (79% inhibition) was obtained using the dominant negative FAK construct, FRNK. Dominant negative FAK also blocked the rescue of Erk2 activity by IL2β1 in suspended cells . These results demonstrate that the integrin-dependent signal that promotes serum activation of MAP kinase requires FAK. To test the role of FAK in this pathway without relying on overexpression of mutant proteins, we examined adherent and suspended polyclonal fibroblasts from FAK −/ − mouse embryos. FAK-positive MEFs behave similarly to 3T3 cells in that the activation of MEK 1, Erk1, and Erk2 was strongly dependent on cell adhesion . By contrast, the FAK −/ − cells showed no adhesion dependence for the activation of the MAP kinase pathway. The absolute level of kinase activation in the FAK −/ − cells was lower than in FAK +/ + cells, equivalent to 46% for MEK1 and 61% for Erk activity relative to FAK +/+ cells. However, the baseline for MAP kinase activity in the FAK −/ − cells in the absence of serum was 72% higher than in FAK +/+ cells; serum induction of MAP kinase activity was, therefore, 3.4-fold in FAK −/ − cells compared with 9.5-fold in FAK +/+ cells. Importantly, no reduction in the activation of MEK or Erks occurred when the FAK −/ − cells were placed in suspension. Thus, although these cells may have partially adapted for the loss of FAK, they have completely lost the adhesion dependence for the regulation of MAP kinase by serum. We also noted that Raf in both FAK +/+ and FAK −/ − cells showed a strong gel shift upon addition of serum, independent of whether cells were adherent or suspended (not shown). These results agree with our previous data showing that adhesion acts at the step between Raf and MEK . To confirm that FAK was responsible for these differences, FAK −/ − cells were transiently cotransfected with wild-type (wt) FAK and HA-Erk2. Expression of wt FAK in the FAK −/ − cells completely restored the adhesion dependence for the activation of the HA-Erk2, whereas cotransfection with empty vector had no effect . These data demonstrate that FAK is absolutely required for the adhesion dependence of MAP kinase activation by serum. The ability of the IL2Rβ1 and CD2FAK chimeras to maintain serum activation of MAP kinase in suspended cells suggests that these constructs should promote anchorage-independent growth. Our previous work showed that oncogenic V12 ras strongly activates MAP kinase, but that this activation is still dramatically decreased in suspended cells . Thus, increasing MAP kinase in suspended ras -transformed cells should enhance colony formation in semisolid medium. To test this hypothesis, ras G12V was cotransfected with either active FAK (CD2FAK) or with the IL2Rβ1 construct. Cell growth was assayed under adherent and nonadherent conditions ( Table and Table ). Expression of IL2Rβ1 or CD2FAK did not increase the number of ras transformants (foci), consistent with the idea that activated ras transforms cells with high efficiency ( Table .) Nor were these constructs sufficient to induce anchorage independence or foci in low serum in the absence of ras , which is consistent with data showing that activation of MAP kinase alone transforms cells very poorly . However, cotransfection of CD2FAK or IL2Rβ1 enhanced the growth rate of ras -transformed cells in suspension (Table II.). Importantly, this effect occurred without altering growth of adherent ras -transformed cells, where endogenous integrins already promote MAP kinase activation by serum. These data are consistent with the hypothesis that these constructs specifically enhance anchorage-independent growth by allowing transforming ras to activate the MAP kinase pathway to its full potential in nonadherent cells. FAK was originally identified as a protein whose tyrosine phosphorylation increased in v-src transformed cells . Furthermore, oncogenic src was shown to stimulate FAK activity in both adherent and suspended cells . Transforming variants of src also induce activation of endogenous ras . These results suggest the interesting prediction that by activating both FAK and Ras, v-src might efficiently promote activation of MAP kinase in suspended cells. Thus, to further test the role of FAK in transformation, the activity of MAP kinase in cells transfected with activated src was assayed. These experiments showed that MAP kinase activity in suspended src -expressing cells was 96 ± 4% of that in adherent cells, compared with 16 ± 2% for suspended ras -transfected cells ( n = 4). Thus, anchorage-independent activation of FAK correlates with anchorage-independent activation of MAP kinase. To determine if FAK activity was required for the adhesion-independent activation of MAP kinase by activated src , we cotransfected src and HA-Erk2 along with either a dominant negative FAK (FRNK) or an empty control vector. Coexpression of dominant negative FAK inhibited Erk2 activation in the src -transfected cells by 60–70% in either suspended or adherent cells . Consistent with our results in which FRNK inhibited the serum activation of MAP kinase in adherent cells, coexpression of FRNK also strongly inhibited ras activation of MAP kinase in adherent cells, but had only a weak effect in suspended ras cells. We also tested whether colony formation was inhibited when src was cotransfection with the FRNK construct. However, no decrease in soft agar growth was observed (data not shown). This result may demonstrate that FAK is not essential to transformation by activated src , or may only indicate that inhibitory levels of FRNK are difficult to achieve in stable cotransfectants. First, our data demonstrate that the integrin signal that mediates the serum activation of the MAP kinase pathway emanates from the β cytoplasmic domain of the integrin receptor. This conclusion is based on the result that an activated IL2Rβ1 chimera containing only the cytoplasmic domain from the integrin was sufficient to restore MAP kinase in suspended cells. However, this construct did not induce serum-independent MAP kinase activity. Thus, the contributions of integrins and growth factor receptors to the MAP kinase pathway can be separated, and the IL2Rβ1 activates only the integrin component of the pathway. Second, we found that this integrin signal is mediated by FAK. Expression of the IL2Rβ1 chimera, which was previously shown to induce FAK phosphorylation , restored activation of MAP kinase in suspended cells. Consistent with this result, we found that expression of a constitutively active FAK was also sufficient to restore MAP kinase induction in suspension. Conversely, dominant negative FAK constructs inhibited the activation of MAP kinase induced by serum in adherent cells or the rescue by IL2Rβ1 in suspended cells. These conclusions were confirmed in FAK −/ − MEF cells, which showed a complete loss of the effect of cell adhesion. Expression of FAK restored the integrin regulation of MAP kinase induction by serum in FAK −/ − cells, indicating that the differences in FAK −/ − cells were not due to secondary genetic alterations. These results are all the more surprising in light of recent findings that FAK is not essential for the transient activation of MAP kinase that occurs when suspended cells are replated on FN or other ECM proteins . Therefore, these results suggest that FAK may be more crucial for the synergism with growth factor induction of MAP kinase than it is for the direct integrin induction of MAP kinase activity. However, it should be noted that the experimental conditions employed for these published studies of direct integrin activation of Erk differ from ours in some respects. FAK is activated by a variety of ECM proteins that bind different integrins, suggesting that the effect we describe here should relatively widespread, so that many different matrices can promote growth factor activation of MAP kinase. However, we have not compared different ECM proteins for their potency in this regard. A number of signaling molecules known to associate with FAK in focal adhesions such as paxillin and Cas represent potential downstream targets for FAK. However, further work will be required to elucidate this pathway. A critical role for FAK in growth factor induction of MAP kinase activity is likely to explain the loss of cell cycle progression when FAK is inhibited , and may be important in the embryonic lethality of FAK −/ − mouse embryos. MAP kinase has been demonstrated to regulate cell migration through the enhancement of myosin light chain phosphorylation . Thus, the requirement for FAK activity in the efficient activation of MAP kinase could also be related to effects of FAK on cell motility, as demonstrated in a number of studies . Previous studies have shown that FAK and src family kinase function in a highly cooperative manner . Thus, the Y397F mutant of FAK that does not bind c-src functions as a strong dominant negative for FAK, whereas the K454R kinase defective mutant that still binds c-src does not . Our result that the Y397F but not the K454R mutant blocks FAK signaling in adherent cells is, therefore, consistent with previous studies, and most likely reflects the ability of the K454R mutant to be phosphorylated by other kinases (or endogenous FAK), enabling it to bind c-src. Therefore, it is likely that src family kinases make a critical contribution to the effects described here, either by activating FAK or by directly phosphorylating key downstream substrates. Third, our data implicate FAK in oncogenic transformation by v-src . Constitutive FAK activation is not sufficient to transform cells but substantially potentiates anchorage-independent growth induced by v- ras without altering growth of adherent ras cells. It is important to note in this regard that while ras -transformed cells form colonies in soft agar, their growth is markedly slower in suspension than when adherent. v-src , which induces constitutive activation of FAK , as well as activation of ras stimulates MAP kinase in an adhesion-independent manner. MAP kinase activation by v-src was inhibited by dominant negative FAK in both adherent and suspended cells, indicating that FAK contributes to v-src induction of MAP kinase under both conditions. Tumorigenesis is thought to be a multistep process. Deregulation of FAK could greatly enhance the tumorigenicity of transformed cells in vivo via effects on both growth and motility. Anchorage-independent growth is the in vitro characteristic that correlates most closely with in vivo tumorigenicity . Motility plays a critical role in invasion and metastasis. FAK expression correlates with motility in human melanoma cell lines , and its activity and expression increase in metastatic tumors . Elevated FAK expression also correlated with the invasive potential of tumors . These data support the notion that elevation of FAK levels or activity can contribute to progression of human tumors. In mammalian cells, cellular functions such as growth, gene expression, and migration are controlled by multiple external stimuli, and understanding how these inputs are integrated is an important goal in cell biology. Our results identify a point of intersection between integrin and growth factor pathways, and identify FAK as a key component of the integrin but not the growth factor arm of this pathway. These data also represent the first example where oncogene function can be understood in terms of specific activation of an integrin-mediated event. Therefore, this work contributes to our understanding of both normal cell regulation and the subversion of these pathways in cancer. | Other | biomedical | en | 0.999996 |
10545505 | Human newborn arterial SMC were isolated from the thoracic aorta as described previously . SMC between passages 5 and 9 were cultured on the surface or in the presence of the following collagen type I preparation: polymerized collagen gels (1.0 mg/ml final concentration) prepared by neutralization of the collagen solution (Vitrogen 100, Collagen Corp.) with 1/6 volume of 7× DME concentrate, dilution to a final 1× DME solution, and incubation at 37°C for 24 h. Monomer collagen-coated dishes were prepared by incubating with 0.1 mg/ml of collagen solution in 0.1 M acetic acid for 24 h. Monomer collagen-coated dishes were washed twice with DME before cell seeding. Degraded type I collagen was prepared by incubating polymerized collagen gels (prepared as above) with 2.5 mg/ml collagenase type 1 (Worthington Biochemical Corp.) for 30 min at 37°C. After digestion, collagenase activity was inhibited by the addition of an equal volume of 1× DME containing 2% plasma-derived serum . Fibronectin was purified from human plasma as previously described . Fibronectin-coated dishes (0.625 μg/cm 2 ) were prepared by overnight incubation at room temperature. Calpain and caspase inhibition studies were performed with two inhibitors: calpain inhibitor 1 (ALLN; Calbiochem-Novabiochem Corp.) and the caspase inhibitor benzyloxycarbonyl Val-Ala-Asp fluoromethyl ketone (ZVAD-fmk; Alexis Biochemicals). SMC were preincubated with ALLN or ZVAD (100 μm) for 3 h or overnight before treatment with degraded collagen in the presence of ALLN or ZVAD. To block integrin function, SMC were preincubated at 37°C for 30 min with nonblocking (P1H6) and blocking (P1H5) Fab antibody fragments against α2, blocking anti-α3 Fab antibody (P1B5), blocking αvβ3 (LM609; Chemicon) at (5 μg/200,000 cells) and αv blocking (cyclic penRGD) and control (RGE) peptides (100 μM) (GIBCO BRL). Monoclonal antibody supernatants for α2 and α3 were provided by W.G. Carter (Fred Hutchinson Cancer Research Center, Seattle, WA). Matrix metalloproteinase inhibition studies were performed with recombinant human tissue inhibitors of metalloproteinase (TIMP1 and TIMP2; 3 μg/ml; Oncogene Research Products). Antibodies for Western blot detection, immunoprecipitation, and immunostaining included: 2–18 N pp125 FAK and 903–1058 C pp125 FAK (Santa Cruz Biotechnology), 354–534 N pp125 FAK (Transduction Laboratories), α-actinin (Sigma), p130cas, paxillin (Transduction Laboratories), talin-8d4 (Sigma), and vinculin (Calbiochem-Novabiochem Corp.). Anti–mouse and –rabbit peroxidase-conjugated secondary antibodies were purchased from Vector Laboratories Inc. SMC were plated on monomeric collagen- or fibronectin-coated plastic chamber slides for 24 h and either left untreated or treated with degraded collagen fragments for 1–5 min. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 in PBS and washed serially in PBS, 0.15 M glycine/PBS + 0.02% NaN 3 , and PBS. Cells were blocked in 5% normal goat serum, 0.05% Tween in PBS for 30 min and incubated with primary antibodies to paxillin and vinculin for 1 h at room temperature followed by several washes in PBS and subsequent incubation with FITC-labeled secondary antibodies (Vector Laboratories Inc.). Cells were also incubated with FITC-labeled phalloidin (Sigma). Immunostaining of cells was analyzed by confocal microscopy. Cells were washed twice with PBS and lysed in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 1 mM NaF, 0.1 mM Na 3 VO 4 , 10 mM β-glycerophosphate, 0.5 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) for 30 min on ice. Cell lysates were clarified by centrifugation at 27,000 g for 20 min, and protein concentration was determined using the BCA protein assay (Pierce). Lysates were separated on 10% or 7.5% SDS-page; proteins were transferred to Immobilon membrane (Millipore) and immunoblotted with specific antibodies. All immunoblots were visualized by enhanced chemiluminescence (ECL, Amersham Corp.). For pp125 FAK coimmunoprecipitation studies, cell lysates were precleared with protein A-agarose (Santa Cruz Biotech.), incubated with 2 μg of the 2-18 N pp125 FAK antibody, and immunoblotted with specific antibodies against p130cas, paxillin, and vinculin. Triton X-100–soluble (cytoplasmic) and –insoluble (cytoskeletal) fractions were prepared as previously described , with the exception of modification of Triton X-100 and radioimmunoprecipitation assay (RIPA) lysis buffers. In brief, SMC were lysed in Triton X-100 lysis buffer (20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 5 mM EGTA, 0.4 mM leupeptin, 0.2 mM Na 3 VO 4 , and 0.1 mM PMSF) for 1 h at 4°C. Triton X-100 insoluble and soluble extracts were separated by centrifugation at 15,000 g for 5 min. The cytoskeletal pellet was washed twice with Triton-free lysis buffer, and proteins were extracted using RIPA buffer (10 mM Tris-HCl [pH 7.2], 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, and 1 mM Na 3 VO 4 ). In vitro transcription and translation of pp125 FAK were performed with the TNT ® -coupled reticulocyte lysate system (Promega) and [ 35 S]methionine (1,000 Ci/mmol, Amersham Corp.), according to the manufacturer's instructions. The expression plasmid for chicken pp125 FAK was given by J.T. Parsons (University of Virginia, Charlottesville, VA). Of the reaction, 1/25 was used as a substrate and incubated with a range of 0.25–2 activity units of purified calpain I and calpain II (Calbiochem-Novabiochem Corp.) for 30 min at 30°C in reaction buffer (50 mM Tris-HCl, pH 7.4, 10 mM CaCl 2 , 5 mM β-mercaptoethanol, and 30 mM NaCl) in the presence and absence of the human recombinant endogenous calpain inhibitor, calpstatin (30 μM; Calbiochem-Novabiochem Corp.). Reactions were terminated by the addition of 4× SDS sample buffer. Common molecular mass standards (Bio-Rad Laboratories) were used to determine Rf values and proteolytic fragment size for both in vitro and cellular pp125 FAK cleavage analysis. Vitrogen (Collagen Corp.) concentration was adjusted to 1 mg/ml and neutralized after dialysis against 10 mM borate, 0.2 M CaCl 2 , pH 8. The vitrogen solution was then radiolabeled by acetylation with [ 3 H]acetic anhydride (NEN Life Science) as described previously . 3 H-labeled collagen degradation was assayed by modification of a previously described procedure . In brief, an aliquot of radiolabeled vitrogen was used to generate polymerized fibrillar collagen gels, as described above. SMC were cultured on the labeled polymerized collagen, and at subsequent time points after cell seeding culture supernatants were analyzed for degraded 3 H-labeled collagen fragments by liquid scintillation spectroscopy. Cell lysates were prepared in lysis buffer as described above and serum-free conditioned media was collected from SMC cultures. Samples were prepared in nondenaturing loading buffer and separated on 10% SDS–polyacrylamide gel impregnated with 1 mg/ml gelatin. After electrophoresis, gels were washed twice in 2.5% Triton X-100 for 30 min, briefly rinsed with water, and incubated for 24 h at 37°C in collagenase buffer (50 mM Tris-HCl buffer, pH 7.5, 200 mM NaCl, and 10 mM CaCl 2 ). Gels were subsequently fixed and stained in Coomassie blue fixative solution (50% methanol and 10% acetic acid containing 0.25% Coomassie blue R250). Human arterial SMC cultured on polymerized type I collagen fibrils are arrested in the G1 phase of the cell cycle and do not respond to growth factor stimulation, whereas SMC on monomeric type I collagen proliferate in response to growth factors . We hypothesized that matrix alteration or degradation may be necessary to release cells from a nonpermissive state, such as culture on polymerized collagen. To test whether degraded type I collagen has unique properties, we added degraded collagen to SMC that had been plated on monomeric collagen or fibronectin for 24 h. Within 5 min after addition of degraded collagen, the SMC round up but remain loosely attached . This cell rounding is reversible; after incubation with degraded collagen for 24 h, SMC attach and spread if the degraded collagen is removed and the cells are plated on fresh monomeric collagen or fibronectin-coated dishes (results not shown). To evaluate effects on focal adhesions, we immunostained SMC cultures with antibodies against paxillin and vinculin . On monomeric collagen, SMC exhibit abundant vinculin- and paxillin-containing focal adhesion structures , while these structures are almost totally absent when SMC are treated for only 5 min with degraded collagen . The loss of focal adhesions is also reversible, and focal adhesions recover within 3 h after removal of degraded collagen and replating of SMC on fresh monomeric collagen (data not shown). Because degraded type I collagen reversibly alters cell spreading and focal adhesion formation, we asked whether it alters a major component of focal adhesions, focal adhesion kinase (pp125 FAK ). Incubation with degraded collagen (1 mg/ml concentration) rapidly induces pp125 FAK processing, resulting in a significant reduction in the levels of native pp125 FAK , as monitored by Western analysis with a polyclonal antibody to the NH 2 terminus of pp125 FAK . This reduction in native pp125 FAK is accompanied by an increase in levels of 90-, 50-, and 42-kD bands. By immunoblotting the same lysates with an antibody to pp125 FAK directed against residues 354–534 , we detect 90- and 40-kD fragments . Using a polyclonal antibody against the COOH terminus of pp125 FAK , we identify a cleaved 35-kD fragment . This cleavage of pp125 FAK is reversible, and native pp125 FAK is restored to maximal levels within 3 h when the degraded collagen is removed and the SMC are replated on fresh monomeric collagen (data not shown). The cleavage of pp125 FAK is also specific for treatment with degraded collagen, and is not observed upon addition of BSA or laminin treated with collagenase (results not shown). pp125 FAK cleavage mediated by caspases has been demonstrated in Jurkat T cells and endothelial cells . Cleavage of pp125 FAK has also been observed in platelets, where it is mediated by the calcium-activated protease calpain I, which results in proteolytic fragments of 90, 45, and 40 kD , similar in size to those observed when SMC are treated with degraded collagen. To determine whether calpain and/or caspases are responsible for pp125 FAK processing, we treated SMC with degraded collagen in the presence of the calpain inhibitor ALLN (100 μM) or the broad caspase inhibitor ZVAD (100 μM). As shown in Fig. 3 , inhibition of calpain partially reduces processing of native pp125 FAK , whereas the caspase inhibitor (ZVAD) has no influence on pp125 FAK processing. To identify which calpain(s) is capable of cleaving pp125 FAK , we incubated in vitro translated pp125 FAK with purified calpain I or calpain II. As shown in Fig. 4 A, both calpain I and II induce dose-dependent processing of pp125 FAK to 90-, 50-, 40-, and 35-kD fragments. The proteolytic activity of either calpain I or II can be blocked by incubation with the specific endogenous calpain inhibitor calpstatin (30 μM). The size of the proteolytic pp125 FAK fragments obtained in vitro correlates with the proteolytic cleavage of pp125 FAK , observed when SMC are treated with degraded collagen fragments . The putative proteolytic fragments are illustrated in Fig. 4 B with the epitopes of pp125 FAK antibodies used to characterize the cleavage. We asked whether the ability of degraded collagen to promote pp125 FAK cleavage was mediated through ligation with cell surface integrin receptors. SMC cultured on fibronectin were preincubated with Fab fragments of blocking (P1H5) or nonblocking (P1H6) antibodies against the α2 integrin subunit, blocking α3 integrin subunit antibody (P1D6), blocking αvβ3 antibody (LM609), or αv-blocking (cyclic RGD) or control (RGE) peptides, followed by 30 min incubation in the presence of degraded collagen fragments (0.25 mg/ml). Preincubation with nonblocking anti-α2 Fab fragments, a blocking anti-α3 Fab, blocking anti-αvβ3, or RGE peptide did not prevent pp125 FAK cleavage. However, when degraded collagen was added to SMC preincubated with either blocking anti-α2 Fab, cyclic RGD peptide, or α2 Fab and cyclic RGD peptide in combination, the induction of pp125 FAK cleavage was suppressed . Changes in pp125 FAK cleavage with integrin blockade were quantified by densitometric scanning of both native 125 kD pp125 FAK and the 90-kD cleavage fragment. Densitometry values from three separate experiments were combined and are shown in Fig. 5 B. Asterisks indicate significant difference from control + degraded collagen ( P < 0.05 by Student's t test). Although suppression of pp125 FAK cleavage by preincubation with αv-blocking cyclic RGD peptide is a consistent observation, statistical analysis of three quantified experiments reveal that this effect is not quite significant. The blocking anti-αvβ3 is ineffective because the SMC do not express detectable αvβ3 . These results suggest that α2- and to some extent, αv-containing integrins may contribute to the induction of pp125 FAK cleavage stimulated by degraded collagen fragments. Three other focal adhesion proteins, paxillin, talin, and α-actinin, have been shown to be substrates for calpain . We asked whether they are also cleaved in SMC exposed to degraded collagen. Native paxillin levels (68 kD) are slightly reduced in SMC cultured with degraded collagen, and a 55-kD band appears . Inhibition of calpain (ALLN, 100 μM) prevents processing to the 55-kD paxillin fragment, whereas caspase inhibition with ZVAD (100 μM) has no effect. Similarly native talin (230 kD) also appears to be proteolytically cleaved giving rise to a 190-kD fragment in SMC treated with degraded collagen . The generation of a 190-kD cleavage fragment of talin is identical in size to a previously identified calpain-mediated cleavage product of talin . Inhibition of calpain but not caspase activity suppresses talin cleavage . In contrast to paxillin, talin and pp125 FAK , α-actinin is not processed in response to degraded collagen , nor is actin, another known substrate of calpain (data not shown). The COOH-terminal cleavage fragment of pp125 FAK (35 kD) contains the focal adhesion targeting (FAT) sequence and second proline-rich domain (p2) of pp125 FAK , sites involved in pp125 FAK interaction with other focal adhesion proteins, such as p130cas, paxillin, and vinculin . We asked whether the ability of pp125 FAK to associate with other focal adhesion signaling molecules is altered in SMC after treatment with degraded collagen. Coimmunoprecipitation studies using the 2–18 NH 2 -terminal pp125 FAK antibody demonstrate a significant reduction in the association of paxillin, vinculin, and p130cas with pp125 FAK in SMC cultured with degraded collagen compared with cells on monomeric collagen . Control coimmunoprecipitation assays using a normal rabbit IgG were performed in parallel with pp125 FAK immunoprecipitations, and rabbit IgG did not associate with paxillin, vinculin, or p130cas in either untreated or degraded collagen-treated SMC samples (results not shown). The subcellular localization of pp125 FAK between cytoplasmic and cytoskeletal fractions is also altered in SMC treated with degraded collagen . Significantly less native pp125 FAK is in the cytoskeletal fraction, and the NH 2 -terminal proteolytic fragments of pp125 FAK are predominantly found in the cytoplasmic fraction. Levels of native pp125 FAK in the cytoplasmic fraction are only slightly reduced in response to treatment with degraded collagen, suggesting that cytoskeleton-associated pp125 FAK may be more sensitive to calpain-mediated processing. To directly test the role of calpain activity in focal adhesion disassembly and cell rounding induced by degraded collagen, SMC cultured on fibronectin were preincubated with or without calpain inhibitor 1 (ALLN, 100 μM) for 18 h before addition of degraded collagen (0.25 mg/ml). Immunostaining with antibodies against paxillin and FITC-labeled phalloidin demonstrate that, in the absence of calpain inhibition, SMC round up and retain limited numbers of focal adhesions one minute after the addition of degraded collagen . In contrast, SMC preincubated with the calpain inhibitor round up to a lesser extent after incubation with degraded collagen, and many of these cells contain more focal adhesions than cells not treated with calpain inhibitor . SMC incubated with degraded collagen in the absence of calpain inhibition do not maintain organized stress fibers , whereas cells preincubated with the calpain inhibitor retain organized actin stress fibers and a spread morphology after treatment with degraded collagen . Culture of cells, including SMC, for extended periods on polymerized collagen or three-dimensional matrices, is associated with gradual dissolution of the matrix mediated by induction of metalloproteinase activity . Therefore, we asked whether extended culture of SMC on polymerized collagen is associated with collagen breakdown and alterations in pp125 FAK . Using 3 H-labeled polymerized collagen, we monitored the release of collagen fragments at sequential time points after addition of SMC. Collagen degradation is observed as early as 6 h after plating on polymerized collagen, and increases at subsequent time points . Evaluation of pp125 FAK demonstrates cleavage fragments as early as 18 h after plating on polymerized collagen , which increases with time. The cleavage of pp125 FAK on polymerized collagen can be significantly suppressed by incubation with the calpain inhibitor ALLN (100 μM) . Calpain-mediated cleavage of paxillin and talin can also be observed in response to long-term culture of SMC on polymerized collagen (results not shown). We hypothesized that the pp125 FAK cleavage observed when SMC are cultured on polymerized collagen may be initiated as a result of generation of degraded collagen fragments from an induction of endogenous SMC MMP activity. Gelatin zymography demonstrates that pro and active forms of MMP2 are increased in SMC cultured on polymerized collagen as early as 6 h after plating . Western blotting of cell lysates also demonstrates significant increases in intracellular levels of both MMP1 and MMP2 when SMC are cultured on polymerized collagen (data not shown). Addition of recombinant TIMP1 and TIMP2 (3 μg/ml) to SMC cultured on polymerized collagen for 24 h significantly reduces both the induction of MMP2 activity as determined by gelatin zymography and subsequent degradation of 3 H-labeled polymerized collagen (data not shown). Western blotting of total cell lysates demonstrates that inhibition of MMP activity and collagen degradation by TIMP1 and TIMP2 is accompanied by a significant reduction in the extent of pp125 FAK cleavage . The possibility that structural changes in the ECM may be necessary for cell migration during tissue remodeling and tumor invasion is supported by two examples of matrix degradation products promoting distinct migratory responses. MMP2 digestion of laminin 5 confers unique signaling properties that promote cell migration, which are not possessed by native laminin 5 . MMP2 cleavage of the laminin 5 γ2 chain exposes a cryptic promigratory site on laminin 5. This altered form of laminin 5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues. It has also been shown that thrombin cleavage of osteopontin enhances its haptotactic activity , and the NH 2 -terminal cleavage fragment contains a cryptic adhesive sequence recognized by α9β1 integrin . The studies presented in this report demonstrate unique signaling properties of degraded collagen primarily mediated through the integrin α2β1 that rapidly promotes focal adhesion disassembly and cell rounding. Previous work has proposed that denaturation or metalloproteinase degradation of collagen reveals cryptic RGD integrin-binding motifs . MMP degradation of collagen differentially affects α2β1 binding to the collagen fragments and reveals a cryptic β3 integrin binding motif, which induces specific integrin-signaling events in SMC, resulting in increased tenascin-C expression . In our studies, the molecular changes mediated by the interaction with degraded collagen fragments are not mimicked by RGD peptides, nor mediated by αvβ3. The effects of degraded collagen are independent of the substrate (and integrins) involved in cell adhesion. Studies have postulated that limited proteolysis of components of the focal adhesion complex by proteolytic enzymes is a potential mediator for focal adhesion disassembly . Particularly interesting candidate enzymes are the calpains, a family of intracellular, calcium-activated cysteine proteases, consisting of two ubiquitously expressed isozymes, calpain I and II . Calpains are localized to sites of focal adhesions in several cell types , and have also been demonstrated to cleave a number of focal adhesion proteins in platelets or in vitro, including pp125 FAK , pp60 src, paxillin, talin, actin, and α-actinin . However, a direct link between calpain-mediated proteolytic activity and disassembly of focal adhesion complexes in adherent cell populations has not been established. In this study, we demonstrate that proteolytic cleavage of pp125 FAK , paxillin, and talin is rapidly induced in adherent vascular SMC treated with degraded collagen fragments, and can be observed in longer term SMC cultures on polymerized collagen after MMP induction and the endogenous generation of degraded collagen fragments. Calpain inhibitor studies and in vitro cleavage assays indicate that pp125 FAK , paxillin, and talin cleavage is mediated by calpain. Cleavage of other focal adhesion proteins, including known calpain substrates, such as α-actinin and actin is not observed, indicating that calpain proteolysis induced by degraded collagen is fairly selective for the focal adhesion components pp125 FAK , paxillin, and talin. Our kinetic studies demonstrate that calpain proteolysis of pp125 FAK , paxillin, and talin is observed within 5 min after addition of degraded collagen and occurs in parallel with focal adhesion disassembly and loss of cell attachment. The mechanisms that regulate calpain proteolytic activity in vivo are still poorly understood. Studies have proposed that translocation of calpain to the plasma membrane and focal adhesion sites promotes activity in response to elevated calcium . Integrin ligation with ECM ligands increases intracellular calcium levels , and rapid initiation of calpain-mediated cleavage of substrates is observed in response to integrin αIIbβ3 ligation on platelets . The rapid kinetics of calpain-mediated cleavage of pp125 FAK , paxillin, and talin that we observe in SMC in response to exposure to degraded collagen fragments is consistent with the time course of integrin-mediated calpain activation observed previously . Our integrin-blocking studies further suggest that initiation of pp125 FAK cleavage is at least partially dependent on the association of the collagen fragments with α2- and possibly also αv-containing integrins. This raises the possibility that degraded collagen fragments possess specific integrin-signaling properties, including initiation of calpain activation. However, the precise downstream signaling mechanisms responsible for initiating calpain cleavage of pp125 FAK , paxillin, and talin remain to be elucidated. Numerous studies have established pp125 FAK phosphorylation as a key step in regulating focal adhesion turnover . Autophosphorylation of pp125 FAK on tyrosine 397 generates a high affinity binding site for the src family kinases . Formation of a pp125 FAK /src signaling complex enhances the catalytic activity of both pp125 FAK and src and recruitment of other focal adhesion proteins, further promoting assembly of the focal adhesion signaling complex . Our studies suggested that calpain-mediated cleavage of pp125 FAK should dissociate the COOH-terminal focal adhesion targeting sequence (FAT) and second proline-rich domain from the NH 2 -terminal catalytic kinase and integrin binding domains. Further, we demonstrate that calpain cleavage of pp125 FAK induced by degraded collagen impairs the ability of pp125 FAK to function as an adapter protein and to associate with paxillin, p130cas, and vinculin. pp125 FAK cleavage also results in decreased levels of native pp125 FAK associated with the cytoskeletal fraction and accumulation of NH 2 -terminal cleavage fragments in the cytoplasmic fraction. This translocation of NH 2 -terminal fragments to the cytoplasm is most likely a consequence of cleavage of the COOH-terminal FAT sequence and second proline-rich domain, which are required for localization of pp125 FAK to focal adhesion sites . Thus, intracellular translocation of pp125 FAK , combined with impaired ability to associate with and recruit other focal adhesion proteins, strongly suggests that calpain cleavage represents an important mechanism for regulating pp125 FAK activity and focal adhesion disassembly. Previous studies suggest that pp125 FAK activity may also be regulated by expression of an alternatively spliced form called pp125 FAK -related non-kinase (FRNK) . FRNK is a truncated 41-kD protein identical to the COOH-terminal domain of pp125 FAK . It is not catalytically active because it lacks the kinase domain, but localizes to focal adhesions through protein associations mediated by the COOH-terminal proline-rich domains and FAT sequence . Previous studies suggest FRNK may regulate pp125 FAK activity by acting as a competitive inhibitor . Our results demonstrate that calpain cleavage of pp125 FAK generates a 35-kD COOH-terminal fragment consisting of the COOH-terminal FAT sequence and second proline-rich domain and thus is structurally similar to FRNK. Due to very low levels of the 35-kD COOH-terminal fragment, we have been unable to detect whether it localizes to the cytoskeletal fraction or is associated with other focal adhesion proteins, such as paxillin. The domain structure of the 35-kD COOH-terminal fragment indicates that it has the potential to act as a competitive inhibitor of pp125 FAK , and further contribute to disassembly of focal adhesions that accompanies calpain cleavage of pp125 FAK . However, its effectiveness in such a role would depend on the maintenance of this fragment at high levels within focal adhesion sites. Tyrosine phosphorylation of pp125 FAK is not required for focal adhesion assembly and cell spreading in response to SMC adhesion to a fibronectin substrate . This is consistent with our studies, which demonstrate that, with adhesion of SMC to distinct collagen or fibronectin substrates, there is no significant regulation of pp125 FAK tyrosine phosphorylation. We, therefore, propose that, under certain conditions, the primary mechanism for regulating pp125 FAK activity and focal adhesion assembly in SMC is mediated through calpain-dependent proteolytic processing, rather than tyrosine phosphorylation. The calpain-mediated proteolysis impairs recruitment of other signaling proteins, decreases association with the cytoskeletal fraction, and generates fragments structurally similar to FRNK that may further contribute to focal adhesion disassembly by acting as competitive inhibitors of pp125 FAK . Our studies demonstrate that pretreatment of SMC with a calpain inhibitor before incubation with degraded collagen fragments suppresses the cell rounding and loss of substrate attachment. The influence of calpain inhibition on focal adhesion disassembly is less clear, as many of the weakly attached SMC, after treatment with degraded collagen, are lost during immunostaining, and only the most adherent cells remain attached and are evaluated by confocal microscopy. However, results presented in this study indicate that pretreatment with calpain inhibitors before addition of degraded collagen appears to enhance the number of focal adhesion structures, compared with cells not pretreated with the calpain inhibitor. More significantly, calpain inhibition maintains substrate anchorage, a spread morphology, and organized actin stress fibers in all cells incubated with degraded collagen fragments. These results further implicate calpain cleavage of pp125 FAK , paxillin, and talin in promoting focal adhesion disassembly and cell rounding induced by degraded collagen. Our data are also consistent with previous studies demonstrating that proteolytic cleavage of pp125 FAK occurs in parallel with cell rounding and loss of substrate attachment . Our studies have focused primarily on the influence calpain cleavage has on pp125 FAK function and focal adhesion integrity. However, calpain cleavage of paxillin and talin is also likely to have significant consequences on focal adhesion complexes and may contribute to disassembly of focal adhesions observed when SMC are treated with degraded collagen fragments. Focal adhesions are dynamic structures that are assembled and disassembled at different stages in the cell cycle, indicating their possible involvement in regulation of cell proliferation . It has also been proposed that focal adhesion assembly and disassembly control cell migration by regulating attachment to the substratum at the leading edge with detachment from the posterior end of the cell . Cells derived from pp125 FAK−/− embryos exhibit a reduced capacity for cell migration and have more focal adhesions than pp125 FAK+ cells . Thus, pp125 FAK may play a critical role in the regulation of focal adhesion turnover and cell migration. During the development of atherosclerotic and restenotic lesions, the ECM surrounding SMC is modified by increased MMP activity derived from both vascular SMC and inflammatory monocytes . Modulation of the vascular ECM may contribute to transition of SMC to a “synthetic” phenotype, which results in enhanced SMC proliferation, migration, and lesion progression . Our demonstration that degraded collagen fragments promote calpain-mediated disassembly of SMC focal adhesions suggests the possibility that, in localized sites of MMP expression, SMC anchorage to the surrounding ECM may be disrupted, thereby promoting their migration and invasion into the arterial intima. Loss of focal adhesion contacts with the surrounding ECM may contribute to enhanced SMC proliferation by releasing SMC from a nonpermissive matrix environment, such as polymerized collagen fibrils . In conclusion, our results characterize an alternative to phosphorylation of pp125 FAK for regulating pp125 FAK activity and focal adhesion assembly in a viable adherent cell population, one dependent on calpain-mediated cleavage of pp125 FAK , paxillin, and talin. We further demonstrate that degraded collagen fragments initiate this cleavage through integrin signals distinct from those of native collagen. | Study | biomedical | en | 0.999997 |
10545506 | Breast carcinoma cell lines were obtained from American Type Culture Collection (ATCC) and maintained in DME with 10% FBS (SKBr3, MDA-MB-435, MDA-MB-436, BT-549, and Hs578t) or MEM with 10% FBS (MDA-MB-453 and BT-20). The cell lines MCF-7 and MDA-MB-231 were obtained from Dr. Mary J.C. Hendrix (University of Iowa, Iowa City, IA) and maintained in DME with 10% FBS. The cell lines SUM 159PT and SUM 149 were kindly provided by Dr. Steve Ethier and generated by the University of Michigan Human Breast Cell/Tissue Bank and Data Base. They were maintained in Ham's F-12 with 5% FBS supplemented with insulin (5 mg/ml) and hydrocortisone (1 mg/ml). The cell line SUM 1315 was obtained from the same source and maintained in Ham's F-12 with 5% FBS supplemented with insulin (5 mg/ml) and EGF (10 ng/ml). HT1080 cells were obtained from ATCC and maintained in DME with 10% FBS. To transfect MDA-MB-435 with E-cadherin, the calcium phosphate transfection kit (Stratagene) was used, according to manufacturer's protocol. For electroporations (BT-20 cells), 10 6 cells were washed with PBS and resuspended in electroporation buffer (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 , 10 mM KH 2 PO 4 , 25 mM Hepes, 2 mM EGTA, 5 mM MgCl 2 ) supplemented with 2 mM ATP and 5 mM glutathione. After a 5 min incubation on ice, the cells were electroporated at 500 μF and 380 V in a BioRad gene pulser. Cells were immediately plated in a 100-mm dish in complete medium. Floating cells were removed and fresh medium was added 24 h after electroporation; puromycin was added to the culture for selection of clones 48 h after electroporation. For transfection of N-cadherin, a restriction fragment containing nucleotides 442–3362 was ligated into the expression vector pLK-pac . The E-cadherin construct has been described previously . The human cadherin-11 cDNA was provided by Drs. S. Takashita and A. Kudo . Unless otherwise stated, all reagents were from Sigma Chemical Co. Rabbit polyclonal antibodies (Jelly) against human E-cadherin extracellular domain , and mouse mAbs against E-cadherin (HECD1; a kind gift of Dr. Masatoshi Takeichi, Kyoto University, Kyoto, Japan) and N-cadherin , have been described previously. The mouse mAb against β-catenin (6E3) was made as described by Johnson et al. 1993 . The mouse mAbs against cadherin-11 were kindly provided by Dr. Marion Bussemakers (University Hospital Nijmegen, The Netherlands). The diacylglycerol lipase inhibitor, RHC80267, was purchased from BIOMOL. Monolayers of cells were washed with PBS at room temperature and extracted on ice with 2.5 ml/75 cm 2 flask 10 mM Tris acetate, pH 8.0, containing 0.5% NP-40 (BDH Chemicals Ltd.), 1 mM EDTA, and 2 mM PMSF. The cells were scraped, followed by vigorous pipetting for 5 min on ice. Insoluble material was removed by centrifugation at 15,000 g for 10 min at 4°C. Cell extracts were resolved on 7% SDS-PAGE as described , transferred electrophoretically to nitrocellulose, and immunoblotted as described using primary antibodies followed by ECL, according to the manufacturer's protocol (Pierce Chemical Co.). For the purpose of loading equal amounts of protein onto SDS-PAGE, quantification was done using the BioRad protein assay reagent according to the manufacturer's protocol. Cells were grown on glass coverslips, fixed with Histochoice (Amresco), washed three times with PBS, and blocked for 30 min with PBS supplemented with 10% goat serum. Coverslips were exposed to primary antibodies for 1 h, washed three times with PBS, and exposed to species-specific antibodies conjugated to FITC or rhodamine for 1 h. Cells were viewed using a Zeiss Axiophot microscope equipped with the appropriate filters, and photographed using Kodak T-MAX 3200 film. Living cells were viewed using a Zeiss Axiovert microscope and photographed using Kodak T-MAX 400 film. For motility assays, 5 × 10 5 cells were plated in the top chamber of noncoated polyethylene teraphthalate (PET) membranes (6-well insert, pore size 8 mm; Becton Dickinson). For in vitro invasion assays, 3 × 10 4 cells were plated in the top chamber of Matrigel-coated PET membranes (24-well insert, pore size 8 mm; Becton Dickinson). In motility and invasion assays, 3T3 conditioned medium was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h and those that did not migrate through the pores in the membrane were removed by scraping the membrane with a cotton swab. Cells transversing the membrane were stained with Diff-Quick (Dade). Cells in ten random fields of view at 100× were counted and expressed as the average number of cells/field of view. Three independent experiments were done in each case. The data were represented as the average of the three independent experiments with the SD of the average indicated. When cells were induced with dexamethasone to express a transgene, the control cells were treated with the same level of dexamethasone. To inhibit FGF receptor signaling, cells were treated with RHC80267 (which inhibits the activity of diacylglycerol lipase) at a concentration of 10–40 μg/ml 3T3 conditioned culture medium during the 24 h of the assay. E-cadherin has been termed a tumor suppressor, mainly because cells derived from E-cadherin–negative epithelial tumors tend to be invasive, whereas cells derived from E-cadherin–positive tumors tend not to be. In the case of cells derived from breast carcinomas, the majority of E-cadherin–negative cells are invasive . However, an increasing number of exceptions to this rule are becoming evident. Our laboratory has recently shown that expression of an inappropriate cadherin by an oral squamous carcinoma cell line influences expression of E-cadherin and the cellular phenotype . This observation led us to hypothesize that the invasiveness of some breast cancer cells may be due to an increase in the expression of an inappropriate cadherin, possibly N-cadherin, rather than to a decrease in the expression of E-cadherin. To test this hypothesis, we surveyed a large number of cell lines, many of which had been characterized previously, for expression of E- and N-cadherin. The data, which are summarized in Table , supported our notion that invasiveness is correlated with N-cadherin expression, rather than lack of E-cadherin expression. Fig. 1 is an immunoblot of extracts of the cell lines presented in Table . Equal amounts of protein were loaded in each lane. The samples were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for E-, N-, or P-cadherin, cadherin-11, and β-catenin. Fig. 2 presents phase micrographs of the living cells to compare the morphologies of breast cancer cells expressing the various members of the cadherin family. MCF-7 cells expressed E-cadherin, had low invasion rates, and presented an epithelial-like morphology. BT-20 cells expressed both E- and P-cadherin, had low invasion rates, and presented an epithelial-like morphology. In contrast, E-cadherin–negative cell lines did not present an epithelial morphology, but rather appeared as fibroblast-like cells with less obvious cell–cell interactions. Even the SUM149 cell line that expressed a small amount of E-cadherin, along with substantial amounts of P-cadherin, did not have the epithelial appearance typified by the MCF-7 and BT-20 cell lines. SUM1315 cells, which expressed P-cadherin, along with a small amount of cadherin-11, also had a fibroblastic appearance with minimal cell–cell interactions. However, these fibroblastic, N-cadherin–negative cell lines had low motility and invasion rates . The N-cadherin–expressing cell lines all displayed a fibroblastic phenotype, as typified by MDA-MB-435, MDA-MB-436, and SUM159 . Cell lines that did not express any cadherin, as typified by SKBr3, displayed a fibroblastic phenotype much like the N-cadherin–positive cells, however, they were less adhesive to the substratum than were cadherin-expressing cells. In addition, they tended to float in the medium upon reaching confluency and when undergoing mitosis. In this study, we hypothesized that the invasive behavior of some breast cancer cell lines may be due to expression of N-cadherin, rather than to lack of expression of E-cadherin. To test this hypothesis, we performed invasion assays on Matrigel-coated membranes and motility assays on uncoated membranes. Fig. 3 presents data from representative cell lines. The N-cadherin–expressing cell lines, SUM159 and MDA-MB-435, were substantially more invasive and more motile than the E-cadherin–expressing line (MCF-7), the E/P-cadherin–expressing cell lines (BT-20 and SUM149), and the P-cadherin–expressing line . The cell line that did not express any cadherins, SKBr3, was no more motile nor invasive than were the E-cadherin–expressing cell lines BT-20, MCF-7, and SUM 149. Together, these data suggest that, in these cells, N-cadherin acts to promote motility and invasion, rather than E-cadherin acting to suppress these activities. Since the cell lines in this study were derived from separate tumors and, thus, are likely to be descendents of different cell types, we sought to manipulate expression of specific cadherins in representative cell lines to determine if the invasive phenotype was due to N-cadherin or to other cellular aspects. We chose two cell lines for these studies: BT-20, which expresses E- and P-cadherin and has a low rate of invasion, and MDA-MB-435, which expresses N-cadherin and is highly invasive. When BT-20 cells were transfected with N-cadherin (BT-20N), they expressed levels of N-cadherin that were comparable to MDA-MB-435; however, they did not undergo a morphological change , nor did they downregulate the expression of E-cadherin to any significant level. Fig. 4B and Fig. C , show that E- and N-cadherin colocalized at cell–cell borders, suggesting that both cadherins are active at the cell surface. When equal amounts of protein from extracts of BT-20 and BT-20N cells were resolved by SDS-PAGE and immunoblotted for cadherin expression, it could be seen that the BT-20N cells slightly downregulated E-cadherin, that the two cell lines expressed equal levels of P-cadherin, and that the BT-20N cells expressed levels of N-cadherin that were comparable to the invasive N-cadherin–expressing cells depicted in Fig. 1 . In addition, β-catenin coimmunoprecipitated equally well with either E- or N-cadherin in these cells . BT-20 cells were unusual in that they expressed high levels of both E- and N-cadherin and, thus, were an ideal cell line in which to test the hypothesis that it is the expression of N-cadherin, not the lack of E-cadherin, that promotes cell motility and invasion in some breast cancer cells. As predicted, motility and invasion rates for BT-20N were five- to eightfold higher than the rates for nontransfected BT-20 cells . Although BT-20N cells were not as motile as the N-cadherin–expressing MDA-MB-435 cells , they were almost as invasive . Since the BT-20N cells expressed high levels of E-cadherin, and were highly motile and invasive, we had good evidence that E-cadherin did not inhibit invasion in these cells and, thus, does not act as an invasion suppressor in all breast cancer cells. However, to further test this idea, we transfected N-cadherin–expressing MDA-MB-435 cells with E-cadherin (MDA-MB-435E) to see if E-cadherin would decrease the invasive nature of these cells. In this experiment, we sought to obtain clones that expressed high levels of E-cadherin, but still retained a significant level of N-cadherin. Fig. 6 D shows the levels of expression of E- and N-cadherin in several clones. Clone 2 was chosen for subsequent studies because it expressed the highest level of E-cadherin and, in addition, showed a two- to threefold reduction in N-cadherin expression, compared with the parental cells. Although these cells expressed very high levels of E-cadherin, they did not display a typical epithelial morphology, and closely resembled the parent cell line . Both E- and N-cadherin were localized to regions of cell–cell contact . When the MDA-MB-435E cells were tested for motility and invasion, they were not significantly different from the parental MDA-MB-435 cells , even though β-catenin was associated with the transfected E-cadherin, as well as the endogenous N-cadherin . Hazan et al. 1997 suggested that N-cadherin–expressing breast cancer cells invade the stroma because they associate with the N-cadherin–expressing stromal cells. In our studies, we employed an in vitro invasion assay in which the cells invade an extracellular matrix that does not include any stromal cells. Thus, we can make the important statement that, in our studies, N-cadherin actively promotes invasion and motility. In Hazan et al. 1997 , the investigators showed that N-cadherin–expressing breast cancer cells coaggregated with N-cadherin–expressing fibroblast-like cells. Since it has been suggested that it is the entire complement of cadherins expressed by a cell that determines its ability to associate with other cells, and that even cells expressing different levels of the same cadherin can sort from one another , we sought to determine if the BT-20N cells that express N-, E-, and P-cadherin would segregate from an N-cadherin–expressing fibroblast cell line, HT1080. Equal numbers of BT-20 cells and HT1080 cells, or BT-20N cells and HT1080 cells, were mixed together and allowed to settle on glass coverslips. They were then prepared for immunofluorescence analysis using antibodies against E- or N-cadherin. In the immunofluorescence analysis of the BT-20/HT1080 cocultures, E-cadherin stained only the BT-20 cells and N-cadherin stained only the HT1080 cells. Fig. 7A and Fig. B , show that these two cell lines effectively segregated from one another as expected. In the immunofluorescence analysis of the BT-20N/HT1080 cocultures, antibodies against E-cadherin stained only the BT-20N cells, whereas antibodies against N-cadherin stained both the BT-20N cells and the HT1080 cells. Fig. 7C and Fig. D , show that the BT-20N cells and the HT1080 cells effectively segregated from one another, even though both cell lines express N-cadherin. Thus, epithelial cells that express N-cadherin along with other cadherins have not necessarily gained the ability to intermix with stromal cells. In the course of our studies on breast tumor cell lines, we characterized one atypical line (MDA-MB-231) that did not express E-, P-, or N-cadherin, but nonetheless was invasive ( Table ). Since MDA-MB-231 cells expressed significant levels of β-catenin, a protein that is not stable in cadherin-negative cells, we suspected that this cell line expressed another member of the cadherin family of proteins, possibly one that is closely related to N-cadherin. We therefore analyzed RNA from this line with degenerate PCR primers designed to amplify all cadherins and found that it expressed cadherin-11 mRNA. Expression of cadherin-11 protein was confirmed by immunoblotting data with a cadherin-11–specific mAb, in agreement with recent data . Like N-cadherin, cadherin-11 is expressed by some mesenchymal cells . Interestingly, cadherin-11 is expressed in some epithelial cells of the human placenta, and it has been suggested that cadherin-11 plays a role in mediating trophoblast–endometrium interactions as the cytotrophoblasts invade the uterine wall . Thus, one idea is that cadherin-11 could act in a manner similar to N-cadherin in promoting cell motility and invasion in breast cancer cells. To test this idea, we transfected cadherin-11 into BT-20 cells (BT-20Cad-11 cells). Like the BT-20N cells, BT-20Cad-11 cells retained the morphology of their parent line, even though they expressed high levels of cadherin-11 at cell–cell borders . As predicted, cadherin-11–expressing BT-20 cells were more invasive and motile than the parental BT-20 cells . Interestingly, the cadherin-11–expressing cells were not as invasive or motile as the N-cadherin–expressing cells. For example, the MDA-MB-231 cells were not as motile as the MDA-MB-435 cells . More significantly, the BT-20 cells transfected with cadherin-11 did not become as motile as they did when transfected with N-cadherin. This may be due to differences between the two cadherins, or differences in expression levels of the transfected cadherins. It is reasonable to speculate that the level of expression of the inappropriate cadherin is relevant since the cell line SUM1315 expresses a small amount of cadherin-11, yet is not invasive. The laboratories of Frank Walsh and Patrick Doherty have shown that N-cadherin promotes neurite outgrowth from cerebellar neurons . In addition, they showed that N-cadherin–mediated neurite extension was dependent on FGF receptor signaling, but was independent of ligand . Walsh and Doherty thus proposed a model whereby the FGF receptor was induced to dimerize in the absence of FGF via interaction with N-cadherin . Dimerization of the FGF receptor results in receptor cross phosphorylation that initiates a number of signal transduction pathways. The pathway relevant to N-cadherin–dependent neurite outgrowth involves the generation of arachidonic acid from diacylglycerol, by the action of diacylglycerol lipase. The Walsh and Doherty laboratories showed that the diacylglycerol lipase inhibitor, RHC 80267, prevented neurite extension on N-cadherin–transfected 3T3 cells, thus implicating this type of FGF receptor signaling in N-cadherin–dependent neurite extension . We hypothesized that the N-cadherin–mediated cell motility we observed in epithelial cells may also be acting through FGF receptor signaling. To test this hypothesis, we treated MDA-MB-435 cells, BT-20 cells, and BT-20N cells with varying levels of RHC80267 to determine if it would influence the motility of these cells in the transwell assay. RHC80267 inhibited cell motility in both N-cadherin–expressing cell lines in a dose-dependent manner . Importantly, this inhibitor had no effect on the motility of the N-cadherin–negative BT-20 cells. Although these data are consistent with the hypothesis that N-cadherin dependent cell motility is mediated through FGF receptor signaling in a manner similar to N-cadherin–dependent neurite outgrowth, additional experiments must be done to further support this notion. Thus, we are continuing to investigate the mechanism whereby N-cadherin mediates motility in epithelial cells. To determine if cadherin-11 and N-cadherin promote cell motility through a similar pathway, we treated MDA-MB-231 and BT-20cad11 cells with RHC80267, and compared motility rates between treated and nontreated cells . The diacylglycerol lipase inhibitor decreased the motility of cadherin-11–expressing cells in a dose-dependent manner. Cadherin-11–expressing cells are less motile than MDA-MB-435, and the inhibitor is less effective in decreasing the motility of the cadherin-11 expressing cells, suggesting there may be some differences in the respective signal transduction pathways, possibly in growth factor receptor levels or isoforms. Previously, our laboratory showed that expression of different cadherin family members by squamous epithelial cells markedly effected morphology , i.e., when oral squamous epithelial cells expressed N-cadherin, they converted to a fibroblastic phenotype concurrent with decreased cell–cell adhesion. Thus, when we turned our attention to breast cancer cells for the present study, we were interested not only in the expression of various cadherins by these cells, but also in whether these cadherins influenced the morphology of the cells. We were not surprised to find that breast cancer cells endogenously expressing N-cadherin displayed a fibroblastic phenotype with tenuous cell–cell contacts, whereas breast cancer cells endogenously expressing E-cadherin displayed a typical epithelial morphology. We were, however, surprised to find that transfection of N-cadherin into the E-cadherin–expressing BT-20 breast cancer cell line had no effect on morphology, even though it had a dramatic effect on cell behavior. Equally surprising was the fact that forced expression of E-cadherin had no effect on the morphology of the fibroblastic N-cadherin–expressing MDA-MB-435 cells. Thus, the breast cancer cell lines examined in this study behaved very differently from the oral squamous epithelial lines that we characterized previously. Interestingly, the oral squamous epithelial cells downregulated E-cadherin when they were forced to express N-cadherin, suggesting an inverse relationship between these cadherins. In contrast, the breast cancer cells continued to express their endogenous cadherin(s) when transfected with a different cadherin. The continued expression of endogenous cadherin may account for the lack of morphological change in the transfectants. Thus, the breast cancer cells differ from the oral squamous epithelial cells in two very important ways: first, the oral squamous epithelial cells appear to coregulate cadherins in an inverse manner, whereas these cadherins are independently regulated in breast cancer cells; and second, expression of E-cadherin by the oral squamous epithelial cells is sufficient for epithelial morphology, whereas epithelial morphology in the breast cancer cells appears to depend on other factors, in addition to E-cadherin. In the present study, we have demonstrated that N-cadherin (or cadherin-11) expression in human breast carcinoma cells promotes an invasive phenotype. By transfecting the BT-20 cells with these nonepithelial cadherins, we have provided evidence for a direct role of these cadherins in cell motility and invasion. Previous studies have correlated the expression of N-cadherin or cadherin-11 with invasion in breast cancer cells. However, in this study, we took the important next step and used transfection studies to show that a cell line that has a low invasion rate could be converted to a highly invasive cell by expression of N-cadherin or cadherin-11. The BT-20 breast cancer cell line provided an important tool for these studies since they did not downregulate E-cadherin when forced to express N-cadherin. Thus, we can conclude that, even in cells expressing high levels of E-cadherin, N-cadherin (or cadherin-11) can promote motility, suggesting that, in this regard, both N-cadherin and cadherin-11 are dominant over E-cadherin. A study by Sommers et al. 1994 supports this idea. These authors showed that transfection of E-cadherin into the E-cadherin–negative breast cancer cell lines, BT549 and HS578, did not decrease the invasive capacity of these cells. These authors suggested that the transfected E-cadherin was not functional; however, these authors were unaware of the fact that the BT549 and HS578 cell lines express N-cadherin. A previous study using MDA-MB-435 cells showed that transfection of E-cadherin into these cells reduced their capacity to form tumors when injected into the foot pads of nude mice . In contrast to our study, these authors showed that E-cadherin–transfected clones of MDA-MB-435 cells underwent a morphological change upon E-cadherin expression. In addition, they showed that E-cadherin–transfected clones were less tumorigenic in their assay than the parental cells. One difference in the study of Meiners et al. 1998 and ours is that they did not assay for N-cadherin expression in their E-cadherin–positive clones of MDA-MB-435 transfectants. Our study clearly demonstrates that N-cadherin influences the behavior of the cells, and that cells retaining N-cadherin do not undergo a morphological or behavioral change upon expression of E-cadherin. Thus, one possible explanation for the difference between these two studies is that the cells in the Meiners' study did not express N-cadherin. The point of our study was to determine if N-cadherin was capable of influencing the behavior of epithelial cells, even if they expressed E-cadherin, thus, we were particularly careful to select cell lines that retained N-cadherin expression after transfection with E-cadherin . One puzzling aspect of cell lines derived from metastatic tumors is that they often express E-cadherin and appear to be relatively normal epithelial cells. A possibility suggested by the present study is that such cells may have upregulated the expression of N-cadherin during the process of metastasis. Our results suggest that expression of N-cadherin would confer on these cells the capacity to invade, even though they continued to express E-cadherin. In this regard, expression of an inappropriate cadherin like N-cadherin (or other related cadherins) may be a better gauge of the clinical state of a tumor than is decreased expression of E-cadherin. Some of the E-cadherin–negative breast cancer cells expressed endogenous P-cadherin. These cells had a fibroblastic morphology similar to that of the N-cadherin–expressing cells; however, they were not highly invasive, suggesting that P-cadherin confers upon breast cancer cells characteristics different from those conferred by either E- or N-cadherin. P-cadherin is expressed in the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. Radice et al. 1997 recently showed that P-cadherin deficient mice develop age-dependent hyperplasia and dysplasia of the mammary epithelium, and suggested that P-cadherin may play a role in maintaining the normal phenotype of breast epithelial cells. One possibility is that the P-cadherin–expressing tumor cells were derived from the myoepithelium, rather than from the true epithelium. E-cadherin has been termed an invasion suppressor because transfection of this protein into some E-cadherin–negative invasive carcinoma cells resulted in decreased invasive capacity. Our prediction is that at least some of these cell lines cells expressed a cadherin, like N-cadherin or cadherin-11, and overexpression of E-cadherin resulted in downregulation of the endogenous cadherin, as we saw with the oral squamous epithelial cells. Thus, we hypothesize that the invasion suppressor role of E-cadherin arises in part from its ability to decrease the level of N-cadherin in certain, but not all, tumors. In the present study, cell lines that did not express any classical cadherins, as evidenced by lack of β-catenin protein, as well as lack of detectable cadherin, had low invasion rates. Our hypothesis, that loss of E-cadherin alone does not necessarily increase invasive capacity in breast carcinoma cells, is supported by the observation that function-blocking antibodies against E-cadherin did not confer a highly motile, invasive phenotype on MCF-7 cells, a breast cancer cell line that is E-cadherin–positive and N-cadherin–negative . The current study suggests that, in some carcinoma cells, expression of N-cadherin, or a similar cadherin such as cadherin-11, may actually be necessary for increased motility and invasion. A recent clinical study suggested that inactivation of E-cadherin is an early event in the progression of lobular breast carcinomas . We might suggest that a subsequent event would be activation of the expression of an inappropriate cadherin, such as N-cadherin or cadherin-11. Understanding the mechanism by which N-cadherin promotes motility in epithelial cells is important if we are to develop treatments that will decrease the invasiveness of tumor cells. A number of studies have shown that epithelial cells can be induced to scatter in response to growth factors, such as hepatocyte growth factor and members of the FGF, EGF, and TGF families . Walsh, Doherty, and coworkers have established, through extensive studies on FGF receptor and cell adhesion molecules, that N-cadherin and the FGF receptor cooperate to induce neurite outgrowth in cerebellar neurons . These authors have proposed a scheme for activation of the kinase activity of the FGF receptor through cis interactions with N-cadherin, via an HAV domain in the FGF receptor and an HAV interaction domain in the fourth extracellular domain of N-cadherin . In addition, it has been proposed that the cadherins form lateral dimers in the plane of the membrane , which could result in dimerization of the FGF receptor, and subsequent activation of the signal transduction pathway. We based the studies presented herein on the model presented by Walsh and Doherty, and proposed that interaction of N-cadherin with the FGF receptor in N-cadherin–expressing epithelial cells may result in increased motility, similar to that seen by treating epithelial cells with growth factors. To test this hypothesis, we interfered with the N-cadherin–dependent FGF receptor signal transduction pathway proposed by Walsh and Doherty by inhibiting a downstream enzyme, diacylglycerol lipase. We showed that inhibiting diacylglycerol lipase decreased motility of N-cadherin–expressing cells in a dose-dependent manner while having no effect on the motility of N-cadherin–negative cells. Thus, our data strongly support the notion that N-cadherin promotes motility in breast cancer cells by activating growth factor receptor signal transduction pathways. Continued efforts in our laboratory are aimed at further defining the signal transduction pathway(s) that mediate cadherin-dependent motility in epithelial cells. At first glance, it might seem unlikely that expression of an additional cell adhesion molecule would confer a motile and invasive phenotype upon an epithelial cell. However, motile cells, such as fibroblasts and myoblasts, express N-cadherin and a switch from E- to N-cadherin occurs in the chick embryo when epiblast cells ingress through the primitive streak to form the mesoderm . Another interesting cadherin switch occurs during establishment of the human placenta, where fetal cytotrophoblast cells invade the vasculature of the uterus. During this invasive process, the cytotrophoblast cells downregulate the expression of E-cadherin and upregulate vascular/endothelial (VE) cadherin . Thus, it is feasible to suggest that increased expression of a nonepithelial cell cadherin, such as N-cadherin, could increase the invasive potential of tumor cells. Ongoing studies in our laboratory are designed to determine how N-cadherin differs from E-cadherin in its ability to induce cell motility. We hypothesize that E-cadherin does not have the ability to interact with the relevant growth factor receptors, and we are preparing chimeric molecules between E- and N-cadherin to test this hypothesis. An important message from the present studies is that cadherins may not function identically in different cell types. The fact that cadherins may act differently in different cell types is particularly evident when comparing the current study with earlier studies showing that mouse L cells or S180 fibroblasts attained an epithelial morphology when transfected with either E- or N-cadherin . It will be important in future studies to consider the cellular makeup, as well as the complement of cadherin family members, when interpreting data on cellular morphology and behavior. | Study | biomedical | en | 0.999997 |
10545507 | The following peptides were synthesized, coupled to keyhole limpet hemocyanin, and injected into New Zealand white rabbits: β521, NH 2 -CTGSPHTSPTHGGGRPM; pan-db, NH 2 -CRVEHEQASQPTPEKAQQNP. Both antibodies were affinity-purified from the sodium sulphate precipitated IgG fraction of rabbit sera on immobilized peptide columns following the manufacturer's recommendations (Sulfolink, Pierce and Warriner). The antidystrophin antibody 2166 was raised against the last 17 amino acids of murine dystrophin and detects all dystrophin isoforms. The antidystrophin antibody 2401 was raised against the alternatively spliced, hydrophobic COOH terminus of dystrophin, and detects Dp71. The antiutrophin polyclonal antibody, URD40, was raised against the distal rod domain of mouse utrophin and is preabsorbed against dystrophin. The SYN1351 antisyntrophin mAb was kindly supplied by Prof. Stan Froehner . The MANDRA1 antidystrophin mAb was kindly supplied by Prof. Glenn Morris . The PSD-95 and Munc-18 mAbs were purchased from Transduction Laboratories. The other dystrobrevin antisera, α1CT-FP and βCT-FP, have been described previously . Adult rats and mice were given an overdose of sodium pentobarbital (60 mg/kg i.p.) and the tissue was fixed by transcardiac perfusion with either 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), Bouin's fixative, or 70% ethanol in distilled water. The brains were removed, postfixed overnight at 4°C, dehydrated, and paraffin-embedded. Sections were cut at 10–20 μm in the transverse or sagittal planes and mounted on gelatin-coated slides. Sections were stained with rabbit antibodies, β521 (1:200), α1CT-FP (1:500), or 2166 (1:500), and mouse monoclonal antizebrin II (1:200) at the dilutions shown . Indirect peroxidase immunocytochemistry was performed on the slide as described previously , with either rabbit anti–mouse IgG or goat anti–rabbit IgG conjugated to HRP as appropriate as the secondary antibody, and diaminobenzidine as the chromogen. Coverslips were applied with Permount. Images of sections and whole-mounts were digitally captured using a Sensys Camera (Optikon Corp. Ltd.) running under V for Windows. Montages were constructed using Adobe Photoshop 4.0. The images were cropped and corrected for brightness and contrast, but not otherwise manipulated. For fluorescence microscopy, 10-μm fixed sections were cut onto SuperFrost Plus slides (BDH). The sections were air dried and blocked in 10% normal donkey serum in TBS (150 mM NaCl, 50 mM Tris, pH 7.5) for 30 min. The sections were incubated with the primary antibody for 1 h, washed twice for 5 min in TBS, and then incubated with a 1:400 dilution of CY3-conjugated donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) for another hour. Slides were washed twice for 5 min in TBS and mounted in Vectashield (Vector Laboratories) fluorescence medium containing 4′,6-diamidino-2-phenylindole (DAPI). Slides were viewed with a Leica DMRE fluorescence microscope. For peptide blocking experiments, β521 was preincubated with the immunizing peptide (25 μM in TBS) or an unrelated peptide for 1 h at room temperature before application to the section. Subcellular fractions, including synaptic membranes (SMs) and PSDs, were prepared from rat forebrain homogenates as described previously . 20 μg of protein from each fraction was separated on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and incubated with an appropriate antibody. Crude synaptosomes were prepared from mouse brain following the method described by Blackstone et al. 1992 . The crude synaptosomes were resuspended in PBS and stored at −20°C. Proteins were solubilized from synaptosome pellets by resuspending them in 1 M Tris HCl, pH 8.0, 100 mM NaHCO 3 , pH 11.5, or 2% detergent (SDS, sodium deoxycholate, Tween-20, Triton X-100, N -lauroylsarcosine, digitonin) in PBS. After incubating the synaptosomes at 4°C for 30 min, the suspension was placed in a bench-top centrifuge and spun at 50,000 g av for 30 min at 4°C. Soluble proteins extracted in the supernatant were removed and the resulting pellet was resuspended in the original volume of PBS. After the addition of an equal volume of 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% [vol/vol] glycerol, 5% [vol/vol] 2-mercaptoethanol) to each sample, the protein extracts were denatured and resolved by SDS-PAGE. The distribution of specific proteins contained in each fraction was determined by Western blotting with an appropriate antibody. Tissue extracts from mouse and rat brain, and rat liver were prepared by homogenizing ∼2 g of freshly dissected tissue in 10 ml of 50 mM Tris, pH 7.4, 2.5 mM EDTA, plus protease inhibitors (Sigma Chemical Co.). The homogenate was centrifuged at 141,000 g av for 45 min at 4°C and the resulting pellet was rehomogenized in 10 ml of RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% [vol/vol] Triton X-100, 1% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] SDS, 2.5 mM EDTA, plus protease inhibitors). After incubation on ice for 30 min, the homogenate was centrifuged at 141,000 g av for 45 min at 4°C. Approximately 1 mg of RIPA-soluble protein in 500 μl was incubated with 4 μg of affinity-purified polyclonal antibody for 6 h at 4°C on a blood mill. Immune complexes were captured by the addition of 50 μl of goat anti–rabbit conjugated magnetic beads (MagnaBind, Pierce and Warriner). After an overnight incubation at 4°C, the beads were extensively washed in RIPA wash buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 0.2% [vol/vol] Triton X-100, 0.2% [wt/vol] sodium deoxycholate, 0.02% [wt/vol] SDS, plus protease inhibitors). Immunoprecipitated proteins were eluted by boiling in 100 μl of SDS/urea buffer (4 M urea, 3.8% SDS, 20% [vol/vol] glycerol, 75 mM Tris, pH 6.8, 5% [vol/vol] 2-mercaptoethanol). The immunoprecipitated proteins were separated on 8 or 10% SDS polyacrylamide gels, transferred to nitrocellulose, and detected with antibodies using standard methods. To establish the locations of α- and β-dystrobrevin in the CNS, we produced antibodies that were isoform-specific. Since the sequences of the dystrobrevins are very similar, a peptide was chosen from the β-dystrobrevin sequence that had minimal homology to the α-dystrobrevin sequence . The β-dystrobrevin antiserum, β521, was characterized extensively for cross-reactivity with α-dystrobrevin by immunoblotting and on sections . On immunoblots of fusion proteins containing the COOH termini of α- or β-dystrobrevin, β521 only detected β-dystrobrevin. By contrast, the pan-db antibody was raised against a sequence that is common to both proteins and detects both proteins on blots . On sections of mouse hippocampus region CA1, β521 stained the dendrites, soma, and nuclei of pyramidal neurons . All labeling was blocked by preincubation with the immunizing peptide , but not by preincubation of β521 with an unrelated peptide (data not shown). To determine the cellular and subcellular localization of β-dystrobrevin in the brain, coronal sections of rat and mouse brain were stained with antibody β521. The distribution of immunoreactivity was essentially identical in rats and mice, and did not depend on the method of fixation. Furthermore, preincubation of β521 with the immunizing peptide completely abolished immunolabeling (data not shown). β-Dystrobrevin immunoreactivity was detected in neurons. In the isocortex , the reaction product is deposited most prominently in the somata (including the nuclei) and the dendrites of the pyramidal cells . Other cortical neurons are also stained. Similarly, in the hippocampus , immunoreactivity is most prominent in CA1, CA2, and CA3 neurons, where the reaction product is also found in the initial segments of dendrites. Neuronal somata are stained in other hippocampal regions as well. Somatal staining also characterizes the cerebellum , where the Purkinje cells, the molecular layer interneurons, and the granule cells are immunoreactive. In all cases, there is also weak axonal staining. Examples are shown for the brainstem, where strong immunoreactivity is detected in axon fascicles associated with the spinal trigeminal tract and in the internal capsule . The α1CT-FP antibody was used to locate α-dystrobrevin-1 in brain sections. As for β-dystrobrevin, there were no significant species differences, and the same distribution was seen with all methods of fixation used. α-Dystrobrevin-1 is expressed by perivascular astrocytes in a pattern that is reminiscent of utrophin immunoreactivity . Seen at low magnification, reaction product is deposited throughout the brain . In the isocortex, the only prominent staining is associated with the vasculature . In the hippocampus, in addition to staining of blood vessels, there is prominent astroglial immunoreactivity . There are also conspicuous regional differences in staining intensity. Most reaction product is deposited in the dentate gyrus and CA2/CA3, with much less immunoreactivity in CA1. A similar combination of perivascular and astroglial immunoreactivity is seen in the cerebellum . At low magnification, α-dystrobrevin-1 immunoreactivity is clearly laminar, with moderate staining of the molecular layer, speckled staining in the granular layer, and little, if any, immunoreactivity in the white matter tracts . No regional differences are apparent in the cerebellum. The granular layer immunoreactivity is confined to the vasculature, and there is no staining of granule cells . At higher magnification, the reaction product in the molecular layer is concentrated in the Bergmann astroglial cells, where both the somata and the radial glial processes are stained. The Purkinje cells were not immunoreactive. The two major dystrophin isoforms present in brain are dystrophin and Dp71. In addition to these products, Dp140 is also found as a minor component in brain extracts. To investigate the location of dystrophin-immunoreactive proteins in the brain, sections of rat and mouse brain were stained with the antibody 2166 that detects all dystrophin isoforms. This antibody gave a complex staining pattern attributable to the presence of three different proteins . In the isocortex, 2166 labeled neuronal somata and blood vessels . In the cerebellar cortex, Purkinje cell somata are labeled, along with dendrites in the molecular layer . There is weak labeling of granule cells and little, if any, labeling in the white matter . A novel feature of the dystrophin isoform expression pattern is that the levels of staining intensity vary in a consistent fashion between Purkinje cells. At low magnification, the labeling of Purkinje cell dendrites is nonuniform and appears as a symmetrical and reproducible parasagittal banded appearance when viewed in the transverse plane . This distribution is reminiscent of the pattern revealed by immunostaining for the well-known Purkinje cell band marker zebrin II. When the pattern of dystrophin isoform expression is compared with that of zebrin II . It is evident that the Purkinje cells which preferentially express dystrophin are zebrin II negative. This pattern of dystrophin immunoreactivity in cerebellar striations is similar to the high microtubule-associated protein (MAP) 1A expressing subset . The differential location of β- and α-dystrobrevin-1 in neurons and glia, respectively, suggests that the dystrobrevins could be independent, cell type-restricted ligands for dystrophin and its isoforms in the brain. To test this hypothesis, we examined the subcellular distribution of the dystrobrevins and the molecular organization of dystrobrevin-containing protein complexes. The subcellular distribution of the dystrobrevins was determined by immunoblotting a range of subcellular fractions, including SMs and PSDs prepared from rat forebrain . Immunoblots were incubated with a panel of antibodies specific for the dystrobrevins, dystrophin and its isoforms, the syntrophins, and the PSD-enriched protein, PSD-95 . β-Dystrobrevin was found to be highly enriched in the PSD fraction, when compared with the light membrane (LM) and SM fractions . The LM fraction comprises membrane fragments primarily of nonsynaptic origin. This result was confirmed using the β-dystrobrevin–specific antibody, β521 (data not shown). By contrast, α-dystrobrevin-1 was found in most fractions and was only moderately enriched in the PSD fraction, when compared with the LM and SM fractions . In control experiments, the enrichment of the PSD protein, PSD-95, was determined in the same subcellular fractions. As expected, PSD-95 was highly enriched in the PSD fraction, compared with the LM and SM fractions . To determine whether the potential dystrobrevin-binding proteins were PSD-enriched, duplicate blots were incubated with antidystrophin and antisyntrophin antibodies. Dystrophin and Dp71 were enriched in the PSD fraction . Furthermore, Dp140, which is thought to be located in perivascular astrocytes , was not PSD-enriched. The enrichment of Dp71 in the PSD was confirmed with the MANDRA1 mAb that is raised against the COOH terminus of dystrophin . On longer exposures, dystrophin was detected in the PSD preparations with MANDRA1 (data not shown). Also, the syntrophins were found to be PSD-enriched . The SYN1351 mAb detects a doublet of proteins of ∼59-kD that could correspond to the two syntrophin isoforms (α and β2) found in brain . Thus, several proteins that are potentially associated with β-dystrobrevin are PSD-enriched. To examine the biochemical association of the dystrobrevins with SMs in brain, synaptosomes were prepared and solubilized with a panel of detergents and buffers. The experiments were not carried out on PSDs due to the highly insoluble nature of this material. The distribution of the dystrobrevins in subcellular fractions was determined by immunoblotting. Interestingly, the dystrobrevins were found in most fractions, including the soluble fraction and the nuclear pellet , whereas the majority of dystrophin and Dp71 was found in the crude membrane and synaptosomal fractions . Most proteins that are PSD-enriched are only solubilized using high concentrations of ionic detergents . To determine the extractability of the dystrobrevins from crude synaptosomes, washed crude synaptosomes were resuspended in a variety of buffers or in PBS containing different detergents ( Table ). α- and β-dystrobrevin, dystrophin, and Dp71 were completely solubilized in 2% SDS and 2% N -lauroylsarcosine. However, α-dystrobrevin-1 was partially solubilized in 2% deoxycholic acid also, whereas β-dystrobrevin, dystrophin, and Dp71 were insoluble in this detergent. These data suggest that β-dystrobrevin and dystrophin are tightly associated with cytoskeletal structures, predominantly the PSD. In control experiments, the extractability of PSD-95 was also determined. PSD-95 was only extracted in 2% SDS ( Table ). To examine the interactions between β-dystrobrevin and the dystrophin family of proteins in brain, reciprocal immunoprecipitations were performed on rat brain and liver using a panel of polyclonal antibodies. For these experiments, rat tissue was used because rat β-dystrobrevin has a slightly higher relative mobility on SDS-PAGE, compared with mouse β-dystrobrevin. This enables a better separation of β-dystrobrevin from the rabbit IgG heavy chain. Rat liver was used as a control tissue because it is an abundant source of β-dystrobrevin and can therefore be used to check the efficacy of the antibodies used for immunoprecipitation. In brain, β-dystrobrevin was only coimmunoprecipitated with the antidystrophin antibody 2166 . In reciprocal experiments, β521 coimmunoprecipitated dystrophin . On long exposures, Dp71 was also detected, albeit weakly, in β-dystrobrevin immunoprecipitates . Although Dp140 was immunoprecipitated by 2166, no Dp140 was coimmunoprecipitated with β-dystrobrevin. Furthermore, 2401 and URD40 failed to coprecipitate β-dystrobrevin . In the liver, β-dystrobrevin coimmunoprecipitated with Dp71, Dp71ΔC, and utrophin . These data show that β-dystrobrevin is specifically associated with dystrophin in the brain, but can also bind to Dp71 or utrophin in the liver. To determine the composition of α-dystrobrevin-1–containing protein complexes in the brain, α-dystrobrevin-1 was immunoprecipitated from rat brain with a panel of polyclonal antibodies . α-Dystrobrevin-1 was coimmunoprecipitated by 2166 and URD40 . However, the relative amounts of α-dystrobrevin-1 immunoprecipitated by each antibody were very different, suggesting that the major complex formed is between α-dystrobrevin-1 and Dp71. Reciprocal immunoprecipitations confirmed the association of α-dystrobrevin-1 with Dp71 and utrophin . No α-dystrobrevin-1 was immunoprecipitated by β521, showing that β-dystrobrevin and α-dystrobrevin-1 are in different protein complexes. Furthermore, Dp140 and dystrophin failed to immunoprecipitate with α-dystrobrevin-1 or utrophin (data not shown). The syntrophin family of proteins have been shown to bind directly to α-dystrobrevin in muscle . To investigate this interaction in brain, we examined the association of the dystrobrevins with syntrophin in the presence and absence of dystrophin and Dp71. The pansyntrophin antibody, SYN1351, was used to immunoprecipitate syntrophin-containing protein complexes from RIPA-extracted crude membranes prepared from the brains of normal C57 mice, dystrophin–deficient mdx mice, and mdx 3Cv mice, which lack dystrophin and all the COOH-terminal dystrophin isoforms . The two predominant dystrobrevin isoforms present in brain, α-dystrobrevin-1, and β-dystrobrevin are precipitated with SYN1351 . Furthermore, a protein corresponding to α-dystrobrevin-2 was also precipitated by SYN1351. The amount of protein precipitated from each mouse strain appeared to be similar, indicating that the interaction between the dystrobrevins and syntrophin was unperturbed by the absence of dystrophin and Dp71 . As expected, no dystrophin cross-reactive proteins were immunoprecipitated with SYN1351 from the brains of mdx 3Cv mice . To exclude the possibility that nonspecific protein precipitation had occurred, duplicate Western blots were incubated with an mAb raised against the synaptic vesicle protein, Munc18. Munc18 was not detected in immunoprecipitates from any of the mouse strains, indicating that the SYN1351 antibody specifically immunoprecipitated dystrobrevin and dystrophin-containing complexes . Furthermore, no apparent difference in dystrobrevin immunoreactivity in the brains of normal mice, compared with dystrophin-deficient mice, was found (data not shown). In control experiments, we investigated the association of syntrophin with α-dystrobrevin in skeletal muscle extracts prepared from normal and mdx mice. By contrast to the situation in brain, the absence of dystrophin results in a dramatic reduction in the amount of α-dystrobrevin-1 and -2 associated with syntrophin . These results are consistent with the reduction of α-dystrobrevin immunoreactivity at the sarcolemma of dystrophin-deficient muscle . Thus, the lack of dystrophin in muscle affects the association of α-dystrobrevin and syntrophin, whereas in the brain, this interaction is apparently unaltered by the absence of dystrophin or Dp71. In this paper, we have presented several lines of evidence showing that β-dystrobrevin is a component of a DPC in neurons. To the best of our knowledge, this is the first example of a protein that is directly and specifically associated with dystrophin in the brain. We have shown that the closest relative of β-dystrobrevin, α-dystrobrevin-1, in common with several other components of the DPC, is associated with perivascular astrocytes and other glial cells. Thus, despite extensive sequence homology, both proteins are differentially distributed in the brain, where they form distinct protein complexes. In the brain, β-dystrobrevin is found in neurons in the cortex, hippocampus, and cerebellum, where it is associated with neuronal somata, dendrites, and nuclei . This location is similar to that described for dystrophin . The location of β-dystrobrevin and dystrophin in the same types of neurons supports our proposal that these proteins form part of a neuronal DPC-like complex. However, there are some distinct differences between the neuronal locations of β-dystrobrevin and dystrophin. For example, in the cerebellum, β-dystrobrevin is predominantly expressed in granule cells and in Purkinje cell somata . By contrast, dystrophin immunoreactivity was not found in granule cell neurons, but predominated in the Purkinje cell somata and dendrites and in perivascular astrocytes . Interestingly, β-dystrobrevin is found at sites that are not reported to express dystroglycan, such as granule cells and axons . Since dystroglycan is a bind partner for dystrophin and its isoforms, these data could suggest that a proportion of β-dystrobrevin is not associated with DPC-like complexes in the brain. The association of β-dystrobrevin with some neuronal nuclei reflects this theme . This result is supported by the presence of significant amounts of β-dystrobrevin in the nuclear pellet, compared with the content of Dp71 and dystrophin in the same fraction . β-dystrobrevin lacks an obvious nuclear localization signal, but does have some sequence features typical of nuclear proteins, such as the zinc finger-like ZZ domain . It is interesting to speculate that β-dystrobrevin may translocate from the neuronal membrane to the nucleus. This mechanism has been described for a number of proteins that are involved in the acquisition of long-term memory . A similar mechanism has been demonstrated for β-catenin, a protein that plays a dual role in cadherin-mediated cell adhesion at specialized submembranous sites and in Wnt-signaling in the nucleus. Like β-dystrobrevin, β-catenin is found at the membrane, in the cytoplasm, and nucleus, but has no obvious nuclear import signal . While it is difficult to envisage a role for members of the DPC in the nucleus, it should be emphasized that the recently characterized syntrophin-binding protein, SAST (syntrophin-associated serine/threonine kinase), is also found in the nuclei of hippocampal pyramidal neurons . Our data and the data of Lumeng and colleagues raise the possibility that DPC-like complexes may be present in the nucleus. Clearly, establishing a role for these proteins in the nucleus warrants further investigation. We have shown that β-dystrobrevin colocalizes and coimmunoprecipitates with dystrophin in neurons. Furthermore, β-dystrobrevin, dystrophin, and syntrophin are PSD-enriched. These data strongly suggest that β-dystrobrevin is a neuronally expressed dystrophin-associated protein. Additionally, β-dystrobrevin coimmunoprecipitates with syntrophin and interacts with the syntrophin family of proteins in the yeast two-hybrid system (Benson, M.A., and D.J. Blake, unpublished results). Our data suggest that the DPC-like complex in neurons is composed of dystrophin, β-dystrobrevin, and syntrophin . Therefore, by analogy to the situation in muscle, this complex may be involved in the etiology of cognitive impairment in DMD patients. The molecular identity of other components of this complex is currently unknown. However, a wealth of circumstantial evidence suggests that nNOS may also be associated with the dystrophin–β-dystrobrevin complex in neurons. In the brain, α-syntrophin interacts with nNOS through reciprocal PDZ domains . Furthermore, nNOS also binds to the PSD proteins, PSD-93 and PSD-95, which are involved in N -methyl d -aspartate (NMDA) receptor clustering . This interaction places nNOS in proximity to the NMDA receptor at the postsynaptic membrane of excitatory synapses . Our finding that β-dystrobrevin and the syntrophins are PSD-enriched and present in a complex offers a separate mechanism to locate nNOS to the postsynaptic membrane. Therefore, it is a distinct possibility that the DPC-like complex at the neuronal membrane contains nNOS bound to syntrophin, β-dystrobrevin, and dystrophin . This interaction, coupled with the role of nitric oxide in the calcium-dependent processes of neurotoxicity, could render dystrophin-deficient neurons more susceptible to metabolic or physiological insults . This hypothesis is supported by the findings that dystrophin-deficient neurons have enhanced susceptibility to hypoxia-induced loss of synaptic transmission and have increased intracellular calcium . Hypoxia and hyperexcitation cause parallel increases in intracellular calcium in neurons that can ultimately lead to cell death. Consistent with these ideas, an increase in neuronal cell loss has been described in some patients with DMD . We have shown that α-dystrobrevin-1 is found in perivascular astrocytes and in Bergmann glia of the cerebellum . The pattern of α-dystrobrevin-1 immunoreactivity in the brain is reminiscent of utrophin that was shown to be associated with the cerebral microvasculature and, in particular, the foot processes of perivascular astrocytes . Other proteins known to be associated with the DPC, such as dystroglycan , laminin-2 , the dystrophin isoform Dp140 , and agrin are expressed in perivascular regions of the brain. The accumulation of α-dystrobrevin-1 and the DPC components at these sites may play a role in the pathogenesis of viral infections of the central nervous system since α-dystroglycan has been shown to be a receptor for several arenaviruses . Thus, DPC-like complexes in perivascular astrocytes may form part of the permeability barrier between the circulatory system and the brain. In astrocytes, α-dystrobrevin-1 is predominantly associated with Dp71 and syntrophin. However, a minor complex is also formed between α-dystrobrevin-1 and utrophin. Therefore, it is possible that different DPC-like complexes are maintained within glial cells. It is noteworthy that Dp140, a protein that is expressed in glial cells associated with brain vasculature , does not copurify with α-dystrobrevin-1 in brain extracts . Since α-dystrobrevin-1 is only found in glial cells, a large proportion of Dp71 must also be expressed by glia, accounting for the perivascular staining of the antidystrophin antibody 2166 in the brain . Thus, the DPC-like complex containing α-dystrobrevin-1 in glia is predominantly composed of α-dystrobrevin-1, Dp71, and syntrophin . There is also some evidence that Dp71 may be present in neurons, especially in the dentate gyrus . It is possible that neuronally derived Dp71 is associated with β-dystrobrevin because a small amount of Dp71 was detected amongst the proteins immunoprecipitated by β521 . Furthermore, we have shown that Dp71 is PSD-enriched . However, some glial-derived proteins, such as α-dystrobrevin-1, are clearly present in PSD preparations. PSDs are known to absorb proteins of a nonneuronal origin, such as glial fibrillary acidic protein, during their isolation , which could account for the presence of α-dystrobrevin-1 and Dp71 in the PSD fractions. Central to the pathogenesis of muscular dystrophy is the disruption of the DPC in muscle. In dystrophin-deficient muscle, there is a dramatic reduction in the levels of DPC at the sarcolemma, possibly due to a failure of this complex to assemble . However, to the best of our knowledge, no one has examined the assembly of DPC-like complexes in other tissues. In this paper, we have shown that the dystrobrevin-syntrophin complex in the brain is apparently unaffected by the absence of dystrophin and Dp71 . Furthermore, dystrobrevin-immunoreactivity appears normal in the brains of dystrophin-deficient mice (Blake, D.J., unpublished results). By contrast, in muscle this complex is destabilized in the absence of dystrophin . This difference between the assembly or stability of DPC-like complexes in muscle and brain may explain why the absence of dystrophin or Dp71 in the brain produces a relatively mild phenotype, compared with the devastating muscle pathology seen in patients with DMD and the mdx mouse. The analysis of several mouse mutants has elegantly demonstrated that the absence of individual components of the DPC complex in muscle often results in muscular dystrophy . Therefore, it can be argued that the each component of the DPC contributes directly to the pathogenesis of muscular dystrophy. However, our data raises the possibility that the assembly of DPC-like complexes in neurons and glia is largely unaffected by the absence of dystrophin and its isoforms. While these findings seem to preclude a contributory role for β-dystrobrevin in the cognitive dysfunction that affects some DMD patients, it should be emphasized that, due to the refractory nature of the PSD, subtle differences in the assembly of the dystrophin–dystrobrevin complex at postsynaptic sites may go undetected. In conclusion, we have shown that there are fundamental differences in the composition of DPC-like complexes in neurons and glia, and between the assembly and stability of the DPC in muscle and brain. The identification of β-dystrobrevin as a binding partner for dystrophin and their enrichment in PSDs suggests that both proteins are components of central synapses and thereby define the neuronal DPC. This discovery is particularly relevant because a large proportion of DMD patients have mild cognitive impairment. The identification of β-dystrobrevin in multiple subcellular compartments in the brain could invoke another route for the propagation of extra- or intracellular signals to different regions of the neuron. It is tempting to speculate that some of the cognitive difficulties experienced by DMD patients may be caused by alterations in the β-dystrobrevin–dystrophin complex at PSDs. | Study | biomedical | en | 0.999997 |
10545508 | A cDNA clone was constructed, corresponding to the bovine rod photoreceptor NCKX1 that encoded a fusion between the highly conserved TM1–TM5 and TM6–TM11 regions. The clone was 32 P-radiolabeled using the Prime-It II Random Primer Labeling Kit (Stratagene). We used this 32 P-radiolabeled DNA probe to screen a Drosophila genomic library (a gift of K. Moses, Emory University, Atlanta, GA, and G.M. Rubin, University of California, Berkeley, Berkeley, CA). The genomic library was screened according to the methods described in Sambrook et al. 1989 . We obtained several genomic clones. A 1.3-kb EcoRI and XbaI genomic fragment was subcloned into pBluescriptII KS (Stratagene), 32 P-radiolabeled, and used to screen a Drosophila eye-enriched lambda ZAPII cDNA library (gift of C.S. Zuker, University of California, San Diego, San Diego, CA) . A set of overlapping cDNAs encompassing the entire Nckx30C transcript was isolated and sequenced. No other cDNAs were identified. The full-length Nckx30C cDNA was constructed and the DNA sequence was determined by fluorescent-based sequencing methods (Applied Biosystems). The BLAST search program (http://www.ncbi.nlm.nih.gov/BLAST) and the CLUSTAL W multiple sequence alignment program were used for sequence similarity analysis (http://pbil.ibcp.fr/NPSA/npsa_clustalw.html). The sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AF190455. The full-length Nckx30C cDNA was cloned into the novel insect cell vector pEIA as an EcoRI fragment, and this was used for the stable transfection of High Five insect cells as described . High Five cells (BTI-TN-5B1-4) derived from Trichoplusia ni egg cell homogenates were purchased from Invitrogen. These cells do not display endogenous exchanger activity. Potassium-dependent sodium/calcium exchange activity was measured in High Five cells transformed with Nckx30C cDNA. The cells were loaded with sodium via the sodium-potassium ionophore, monensin, in a medium containing high sodium, 150 mM NaCl, 80 mM sucrose, 0.05 mM EDTA, and 20 mM Hepes, pH 7.4, according to the methods described for rod outer segments . The ionophore was removed and the sodium-loaded cells were washed with and resuspended in 150 mM LiCl, 80 mM sucrose, 0.05 mM EDTA, and 20 mM Hepes, pH 7.4. The cells were diluted 10-fold in media containing 80 mM sucrose, 20 mM Hepes, pH 7.4, and either 150 mM KCl, 150 mM NaCl, or 150 mM LiCl. 45 Ca uptake was initiated by addition of 35 μM CaCl 2 and 1 μCi 45 Ca. 45 Ca uptake in High Five cells was measured with a rapid filtration method with the use of borosilicate glass fibers over a time-course of 5 min as described previously . The ice-cold washing medium contained 140 mM KCl, 80 mM sucrose, 5 mM MgCl 2 , 1 mM EGTA, and 20 mM Hepes, pH 7.4. Total RNA was prepared from the heads and bodies of 0–7-d-old Drosophila w1118 and eya lines using the Ultraspec RNA isolation system (Biotecx). PolyA + RNA was isolated by affinity chromatography on oligo(dT) cellulose columns using the FastTrack 2.0 System (Invitrogen, Inc.). PolyA + RNA from third instar larvae and 0–24-h embryos (Canton S strain) was purchased from Clontech. PolyA + RNA (10 μg) from each sample was run on a denaturing 0.9% agarose gel for 3 h 130V/cm. The gel was stained with ethidium bromide and photographed on a UV transilluminator. The mRNA was transferred overnight by capillary action in 20× saline sodium citrate (SSC) to a positively charged nylon membrane (Nytran Plus), and fixed by UV cross-linking. The membrane was probed with [ 32 P]UTP antisense riboprobes (Maxiscript In Vitro Transcription kit; Ambion, Inc.). Four subclones of Nckx30C were constructed and used for riboprobe production; 1.3 kb BamHI-BamHI (5′ end), 500 bp BamHI-ClaI (TM1–TM5), ClaI-ClaI (loop region), and ClaI-EcoRV (TM6–TM11) . The antisense and sense riboprobes were incubated with the membranes using the NorthernMax hybridization buffer (Ambion, Inc.), the membranes were washed with 0.2× SSC, 1% SDS at 65°C, and exposed to Kodak X-OMAT X-ray film (Eastman Kodak). All four antisense probes displayed the same labeling pattern. No signal was detected with the sense probes. A 250-bp mouse β-actin antisense riboprobe was used as a control for the amount of RNA loaded (Maxiscript In Vitro Transcription kit; Ambion, Inc.). Polytene chromosome squashes ( w m f strain) were prepared as described . A 1.3-kb EcoRI and XbaI genomic fragment was nick-translated using biotin-16-UTP (Boehringer Mannheim Corp.). Hybridization with the biotinylated probe was carried out in 600 mM sodium chloride, 1× Denhardt's, 50 mM magnesium chloride, and 50 mM sodium phosphate, pH 6.8, at 37°C overnight. Samples were washed with 2× SSC, 0.1% Triton X-100, and PBS, pH 7.2 . Hybrids were detected using Vectastain ABC (Vector Laboratories, Inc.). Labeling was identified at a single cytological position on chromosome 2L, 30C5-7. Heads from the w1118 strain of flies were embedded in Tissue-Tek OCT Compound (Miles, Inc.) and frozen on dry ice. Eyes were fixed in 4% paraformaldehyde, infiltrated with 2.3 M sucrose, and frozen in liquid nitrogen according to methods described previously . In situ hybridizations were carried out on 14-μm head sections and 0.5-μm eye sections in 50% formamide, 5× SSC, 0.5 mg/ml sheared herring sperm DNA, 0.05 mg/ml heparin, 0.25% CHAPS, 0.1% Tween 20, 1 mg/ml yeast torula RNA, 1× Denhardt's, at 55°C overnight. Embryo and imaginal disc in situ hybridizations were carried out essentially as described in Panganiban et al. 1994 . In brief, embryos (Canton S strain) were collected, dechorionated, fixed, and processed for in situ hybridization as described by Panganiban et al. 1994 . Proteinase K was not used. Embryos were hybridized in 50% formamide, 5× SSC, 500 μg/ml sheared salmon sperm DNA, 0.1% Tween 20, 1 mg/ml glycogen. Hybridization was carried out at 55°C for 24–30 h. Probe concentrations were 90 ng/300 μl. The larval imaginal discs were dissected from crawling third-instar larvae, but left attached to the cuticle. The discs were fixed, permeabilized with proteinase K at 25 μg/ml for 3 min at room temperature, and postfixed. Probe concentrations were 120 ng/300 μl. Digoxigenin-labeled sense and antisense riboprobes were generated by in vitro transciption of five DNA templates as recommended by the digoxigenin/UTP supplier (Boehringer Mannheim Corp.). Five Nckx30C cDNA probes were used: 1.3 kb BamHI-BamHI (5′ end), 500 bp BamHI-ClaI (TM1–TM5), ClaI-ClaI (loop region), ClaI-EcoRV (TM6–TM11), and the full-length Nckx30C cDNA clone . Calx cDNA was provided by E. Schwarz (Columbia University, New York, NY) . Chaoptin cDNA was provided by D. Van Vactor (University of California, Berkeley) and S.L. Zipursky (University of California, Los Angeles, Los Angeles, CA) . After transcription, the reaction mix was treated with DNase and the riboprobes were hydrolyzed in carbonate buffer for 2 min at 80°C. All riboprobes were quantified by analysis in denaturing 0.8% agarose gels and by dot blot analysis. All detection steps were as described in Tautz and Pfeifle 1989 . All Nckx30C antisense probes showed the same labeling pattern. No signal was detected with the sense probes. The NCKX-type exchangers are a novel group of calcium extrusion proteins. NCKX1 function was initially described in the outer segments of retinal rod photoreceptor cells . Antibodies directed to NCKX1 were used to screen a bovine retinal expression library, and NCKX1 cDNA was cloned and sequenced . A second isoform, NCKX2, was cloned from rat brain, and was shown on the basis of in situ hybridization to be widely expressed in various regions of the brain . To isolate Drosophila Nckx30C , we constructed a cDNA probe from the bovine rod photoreceptor NCKX1 that encoded a fusion between the highly conserved TM1–TM5 and TM6–TM11 regions. We used this probe to screen the Drosophila genomic library and obtained a 1.3-kbp EcoRI-XbaI fragment that corresponds to an EcoRI site at the 5′ end of the gene. The XbaI site is located in the intron. This 1.3-kbp genomic fragment was used to screen the cDNA library. A set of overlapping cDNAs encompassing the entire Nckx30C transcript was isolated and sequenced. The composite cDNA has a single open reading frame that encodes a protein of 856 amino acids . We compared the derived amino acid sequence of Nckx30C with the human NCKX1 and rat NCKX2 . There is 66 and 71% identity in two clusters of amino acids that corresponds to the predicted TM domains of human rod photoreceptor cell NCKX1 and rat brain NCKX2, respectively. A partial restriction map for Nckx30C is shown in Fig. 4 A. The hydropathy analysis confirms that Drosophila NCKX contains 11 hydrophobic regions that correspond to the 11 predicted TM helices in NCXK1 and NCKX2 . As with NCKX1 and NCKX2, there is a large cytoplasmic loop located between TM5 and TM6 in Nckx30C . This large cytoplasmic loop displays very little amino acid identity with the corresponding cytoplasmic loops of NCKX1 or NCKX2 . The number of amino acids that make up the hydrophilic loops and the two sets of membrane-spanning segments are shown in Fig. 4 C. One feature distinguishes NCKX30C from other members of the NCKX superfamily. Hydropathy analysis revealed that NH 2 -terminal to TM1 is a region that may contain two additional membrane-spanning segments not observed in either NCKX1 or NCKX2. The correct identity of this NH 2 -terminal sequence for Nckx30C was confirmed by 5′ rapid amplification of cDNA ends (RACE) starting with a primer corresponding to sequence of TM1, and by PCR using first-strand cDNA. We cytologically mapped Nckx to 2L at 30C5-7 by in situ hybridization to polytene chromosomes, and based on the chromosomal location have named the gene Nckx30C . We examined the functional properties of NCKX30C in High Five cells. The strategy for these experiments takes advantage of properties of both NCX and NCKX. First, NCX and NCKX are bidirectional in their ability to mediate both calcium efflux (forward exchange) and calcium influx (reverse exchange). The direction of transport is dictated by the direction of the TM sodium gradient. Normally, the inward sodium gradient removes calcium from the cell (forward exchange). However, when external sodium is removed, the outward sodium gradient drives calcium into the cell (reverse exchange). Therefore, we measured the properties of reverse exchange of NCKX30C by measuring 45 Ca uptake in sodium-loaded cells. We tested for NCKX activity by using three different manipulations of the cation gradient that are known to prevent NCKX activity. The cells were loaded with sodium via the sodium-potassium ionophore, monensin, in a medium containing high sodium, according to the methods described for rod outer segments . The ionophore was removed and the sodium-loaded cells were washed with and resuspended in low sodium buffer as described in the Materials and Methods. The cells were diluted 10-fold in media containing 80 mM sucrose, and 20 mM Hepes, pH 7.4, and either 150 mM KCl, 150 mM NaCl, or 150 mM LiCl. 45 Ca uptake was initiated by addition of 35 μM CaCl 2 and 1 μCi 45 Ca. The first condition causes the release of internal sodium by the action of monensin, a sodium(-potassium) selective ionophore. Fig. 5 A shows that 45 Ca uptake by NCKX30C was strongly inhibited by the addition of monensin, thus demonstrating a requirement for intracellular sodium for calcium transport. A second property of both NCK and NCKX is that calcium uptake by reverse exchange is inhibited by high extracellular sodium . This is thought to occur because sodium and calcium compete for a common binding site . We demonstrate that 45 Ca uptake by NCKX30C is strongly inhibited by high extracellular sodium, consistent with its identity as an NCX . A critical distinction between NCKX and NCX is that calcium influx via NCKX requires the presence of potassium, and lithium cannot substitute for potassium . In contrast, calcium influx via reverse exchange in NCX is the same in media containing potassium or lithium . 45 Ca uptake by NCKX30C requires potassium and was not observed in media containing lithium . This result demonstrates that NCKX30C functions as an NCKX. As negative controls, we subjected nontransformed High Five cells to the same 45 Ca uptake experimental protocols described above: 45 Ca uptake in media containing potassium was indistinguishable from that in media containing either sodium or lithium, showing that there is no endogenous NCX or NCKX activity in the cells (data not shown). The above results demonstrate that NCKX30C mediates potassium-dependent sodium/calcium exchange similar to that observed for NCKX1 and NCKX2 . Northern blot analysis revealed an 8-kb transcript that is expressed in embryos and larvae . In the adult, two transcripts of 8 kb and 10 kb were detected. Expression was detected predominately in the head. Transcripts present in the body may represent the thoracic ganglia or other components of the nervous system . The 8-kb and 10-kb transcripts were also detected in eya-1 mutant flies, which lack eyes, indicating that expression is not restricted to the eyes . Previous work has identified a Drosophila NCX, Calx , which encodes a functional ortholog of the NCX-type exchangers . Expression patterns for Calx were reported in Schwarz and Benzer 1997 . In situ hybridization analysis reveals that both Nckx30C and Calx are expressed in the adult nervous system. Fig. 7 A shows that Nckx30C is expressed in the photoreceptor cells as well as in the lamina, medulla, and optic lobes of the brain. Fig. 7 C shows that Calx is also expressed in the photoreceptors as well as in the brain. Analysis of many images did not reveal a convincing difference in expression patterns between Nckx30C and Calx . In situ analysis at higher resolution, on 0.5-μm cryosections, confirmed that expression is detected in the photoreceptor cells (data not shown). However, this does not exclude the possibility that the subcellular distribution of the proteins could be very different. No signal was detected with the sense probes . Beginning at embryonic stage 13–14, Nckx30C transcripts were detected in a discrete pattern of unpaired cells at the ventral midline of the central nervous system . Nckx30C transcripts were not detected in the preblastoderm or blastoderm stages . Unlike Nckx30C , Calx expression is robust in the preblastoderm before and during nuclear migration from the interior to the cortex . Since this is before activation of zygotic transcription, Calx transcripts are likely to be maternally inherited. Calx transcripts disappear in the cellular blastoderm and then reappear in the stage 11 embryo . By stage 12, Calx expression is noted in two cells per hemisegment, one on either side of the ventral midline . In embryonic stage 15, Nckx30C expression was detected in several neurons within the ventral nerve cord . By stage 16, when many neurons within the ventral nerve cord and the embryonic brain express Nckx30C , Calx expression is restricted to a much smaller subset of neurons in the ventral nerve cord . Outside the central nervous system, Nckx30C and Calx transcripts were observed in some cells, which may represent parts of the peripheral nervous system . Drosophila appendages develop from imaginal discs in a highly orchestrated series of events that divide the discs into distinct subregions during patterning. Retinal differentiation begins at the posterior end of the eye disc, which coincides with the dorsal–ventral midline , and proceeds as a wave across the eye disc from posterior to anterior . The morphogenetic furrow is a dorsoventral indentation in the eye disc, with the region anterior to the furrow comprising actively dividing and unpatterned cells, and the region posterior to the furrow containing differentiating photoreceptor cells assembling into ommatidia. . Fig. 9 A shows expression of chaoptin , which encodes a photoreceptor cell surface glycoprotein , in photoreceptor cells located posterior to the furrow. Calx expression was not detected in any of the imaginal discs. Nckx30C transcripts were detected both anterior and posterior to the morphogenetic furrow in a dorsal–ventral pattern with no labeling in the midline . This expression pattern is similar to but broader than that observed for wingless ( wg ), which is known to play an important role in dorsal–ventral patterning in the eye disc . In vertebrates, one of the two apparently distinct Wnt pathways may act through a G protein–mediated phosphatidylinositol signaling cascade that results in an increase in intracellular calcium via inositol 1,4,5 trisphosphate (InsP 3 )-sensitive stores . It is intriguing to hypothesize that NCKX30C may be playing a role in modulating calcium in this patterning pathway. Nckx30C is expressed strongly throughout most of the wing disc and throughout most of the haltere disc . In the leg disc, Nckx30C is expressed in a ventral wedge in the cells that will give rise to the ventral and/or proximal structures . This pattern of expression is similar to that observed for wingless in the leg discs . Given what we know about the role of NCKX in retinal rod and cone photoreceptors, it is tempting to speculate that cells that express Nckx30C may be experiencing large and sustained rises in cytosolic calcium. We have cloned Nckx30C from Drosophila and have shown that it is expressed in the adult nervous system and also during development in the embryo and the imaginal discs. Heterologous expression in cultured insect High Five cells demonstrates that NCKX30C functions as an NCKX. Calx , a member of the NCX gene family , is distantly related to Nckx30C . Both NCX and NCKX function in sodium–calcium exchange in cells experiencing large calcium fluxes. In addition, both Nckx30C and Calx are expressed in Drosophila photoreceptors as well as in other cells, indicating that there may be multiple mechanisms for calcium efflux in these cells. A puzzling finding is that the photoreceptors in Drosophila express both NCKX30C and Calx exchangers. The NCKX exchangers have several unique features that make them well-suited for calcium extrusion during phototransduction. Illumination of Drosophila photoreceptors activates the phototransduction cascade leading to the opening of the cation-selective channels and an increase in the intracellular calcium . In both flies and vertebrate photoreceptor cells, sodium influx contributes substantially to the inward current, potentially leading to elevated [Na + ] inside . As the cytosolic sodium concentration increases and reduces the TM sodium gradient, NCX exchangers, like Calx, are expected to reverse direction at much lower cytosolic sodium concentrations when compared with the potassium-dependent NCKX exchangers . Therefore, the potassium-dependent exchangers will be better able to function in phototransduction under these conditions. The apparent redundancy could also be explained if the subcellular distribution of NCKX30C and Calx are very different, with each fulfilling distinct functions. A combination of immunocytochemistry with mutant analysis will assist in resolving these questions. To date, the central focus of efforts in development of Drosophila has been on the identification of regulatory genes that control development, with the potential contribution of calcium signaling remaining largely unexplored. Notable exceptions include systems responsible for dorsal–ventral positional information, such as the dorsal protein in dorsal–ventral pattern information in the early embryo and the wingless pathway. It has been demonstrated that increased calcium can act as a second messenger in the signal transduction pathway leading to the nuclear localization of dorsal, a maternally inherited transcription factor . Another signaling pathway that may utilize calcium is wingless . wingless , a member of the Wnt gene family, encodes a secreted glycoprotein that is involved in a complex variety of signaling events . In vertebrate embryos, one of the two apparently distinct Wnt pathways can act through a G protein–mediated phosphatidylinositol signaling cascade, resulting in a release of intracellular calcium from inositol 1,4,5 trisphosphate (InsP 3 )-sensitive stores . Here, we demonstrate that Nckx30C is expressed in a pattern similar to, but broader than that observed for wingless . Nckx30C expression would indicate that, most likely, a substantial calcium flux is occurring in these cells. In this study, we show that exchangers are expressed during development, indicating that they may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play critical roles in signaling events during embryogenesis and patterning of imaginal discs. The isolation of Nckx30C in Drosophila permits a genetic analysis of the in vivo role of calcium in modulating signaling pathways in Drosophila . | Study | biomedical | en | 0.999996 |
10545509 | Wild-type φ6 and its host P . syringae pathovar phaseolicola HB10Y were used for NC production. Host cell spheroplasts were prepared from a receptorless phage-resistant derivative of HB10Y, MP0.16 . LB broth was used as the growth medium . Monensin (MN), nigericin (NG), valinomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), polymyxin B (PMB) sulfate, polymyxin B nonapeptide, benzoic acid, apyrase (AP), N -ethylmaleimide, butylated hydroxytoluene (BHT), and Triton X-114 were purchased from Sigma Chemical Co. KCN, N , N ′-dicyclohexylcarbodiimide (DCCD), NaF, and tetraphenylphosphonium (TPP + ) chloride were products of Fluka Chemie AG. NaN 3 was obtained from Riedel-deHaën AG, Na 2 HAsO 4 from Merck Biochemica, and gramicidin D (GD) from Serva. For NC production, bacteriophage φ6 was grown on HB10Y and purified as described previously . 14 C-Labeling of the virus proteins was performed as described by Ojala et al. 1990 . The virus spike protein P3 and the viral membrane were removed by treatments with butylated hydroxytoluene and Triton X-114, respectively . The NC particles in the Triton X-114 water phase were further purified in 5–20% (wt/vol) sucrose gradients in 20 mM Tris, pH 7.4, 150 mM NaCl (Sorvall AH627 rotor; 24,000 rpm, for 55 min, +15°C) and the light-scattering zone containing the NC was collected using a BioComp gradient fractionator. For the experiments where no salt was preferred, NaCl was omitted from the NC purification. EM analysis was carried out with NC particles pelleted through a 20% (wt/vol) sucrose cushion in 20 mM Tris, pH 8.0, 150 mM NaCl (Beckman Ti50 rotor; 40,000 rpm, for 1 h 25 min, +10°C) after Triton X-114 extraction and resuspended in 12% (wt/vol) sucrose, 20 mM Tris, pH 8, 80 mM NaCl. The NC preparations were used either immediately or stored at −80°C. The protein concentrations of the NC preparations were determined by the Bradford 1976 assay using BSA as a standard, and the protein composition was confirmed by SDS-PAGE . The host cell spheroplasts were prepared as previously described , except that a Sorvall GSA rotor (5,000 rpm, for 6 min, +4°C) was used for centrifugation. The NaCl and Tris–sucrose-treated cells were finally resuspended in ice-cold buffer (20 mM Tris, pH 7.4, 3% [wt/vol] lactose, 2% [wt/vol] BSA) to a cell density of ∼10 10 cell/ml, and either used fresh or stored at −80°C in 10% glycerol. The washed cells were treated with lysozyme (5 μg/ml final concentration) and the spheroplasts formed were infected with NCs at a multiplicity of ∼50. The standard infection mixture contained ∼30 mM NaCl derived from the NC preparation. After the desired time of infection (at 23°C), samples were treated either with NC-specific polyclonal antiserum to inactivate the extracellular NCs or with a nonspecific polyclonal antiserum against bacteriophage PRD1. The infected spheroplasts were diluted in ice-cold buffer (20 mM Tris, pH 7.4, 3% [wt/vol] lactose, 2% [wt/vol] BSA) and plated on a lawn of phage-sensitive HB10Y cells on solid LB. Plates were incubated at 23°C overnight and the infective centers (ICs, infected spheroplasts capable of producing infectious progeny viruses and forming plaques) were counted. The production of progeny phages during the incubation in the test tube was studied by assaying infective viruses in the supernatant of the infection mixture after removal of the spheroplasts by centrifugation. Extracellular concentrations of H + , K + , and TPP + ions were monitored at 23°C using ion selective electrodes as previously described . The H + measurements were carried out using spheroplasts resuspended initially in 2% (wt/vol) BSA, 3% (wt/vol) lactose, pH 7.5. Intracellular K + and TPP + concentrations were evaluated after permeation of the spheroplasts by GD (8 μg/ml) and PMB (300 μg/ml). The ΔΨ values were calculated using the Nernst equation . The extracellular ATP content was determined by the luciferin-luciferase method using a 1250 Luminometer (Wallac) and the ATP monitoring reagent from Bio-Orbit. For estimation of intracellular ATP concentration, the spheroplasts were permeated by the ATP releasing reagent (Bio-Orbit) before the ATP measurement. The intracellular volume of the spheroplasts used in the calculations was estimated to be 2% of the total suspension volume. (Based on the cell density and size approximations done in a Bürker chamber). The effects of monovalent salts, ionophores, and energy-depleting agents on NC infection were studied by measuring the formation of plaques using the entry assay and simultaneously assaying the effects of the drugs on ΔΨ and ATP content of the spheroplasts. Spheroplasts were preincubated with drugs for 2 or 6 min at 23°C, and samples were withdrawn for ΔΨ or ATP measurements and for the NC infection assay. In the standard entry assay, a 50-min infection was followed by an additional 10-min or 7.5-min treatment with NC-specific or nonspecific antiserum, respectively. For morphological analysis, NC-infected spheroplasts were subjected either to chemical fixation (CF) or to rapid freezing (RF) and freeze substitution (FS). Spheroplasts were infected with a multiplicity of ∼200. Infected spheroplasts (spheroplasts in 30 mM sodium phosphate, pH 6, 3% [wt/vol] lactose, 2% [wt/vol] BSA, 100 mM NaCl, and 50% LB broth) were conventionally fixed with 3% (wt/vol) glutaraldehyde (in 50 mM sodium phosphate, pH 6, 3% [wt/vol] lactose, 2% [wt/vol] BSA) for 15 min at 23°C, followed by OsO 4 after fixation. Before cryoprocessing (RF and FS), spheroplasts were collected and resuspended in 20 mM potassium phosphate, pH 7.4, 3% (wt/vol) lactose, 8% (wt/vol) sucrose, 10% glycerol, 2% (wt/vol) BSA to a cell density of ∼6 × 10 10 cells/ml. Samples from the infection mixtures were rapidly frozen by slamming onto liquid nitrogen–chilled copper blocks, and then freeze-substituted for 8 h at −90°C. Several substitution protocols were tested: (1) acetone and 1–2% (wt/vol) OsO 4 , (2) acetone and 1–2% (wt/vol) OsO 4 , followed by en-bloc staining with 0.5% (wt/vol) uranyl acetate (for 1 h, +4°C), (3) acetone and 3% (vol/vol) glutaraldehyde, and (4) methanol and 0.5% (wt/vol) uranyl acetate. All samples were embedded in Epon. Thin sections (40–60 nm) were poststained with aqueous 0.5% (wt/vol) uranyl acetate (for 0–40 min at +20–40°C) and lead citrate (for 5 min at +20°C) and viewed at 60–100 kV with a JEOL 1200EX electron microscope. The NC neutralization tests were carried out with purified mAbs against φ6 structural proteins P1, P4, P8, and P3, and with polyclonal antiserum against protein P7 . The NC specimen was preincubated with antibodies at 23°C for 15 min. Spheroplasts were infected with the NC–antibody mixture for 50 min to determine the amount of ICs. Earlier studies with intact host cells have indicated that NC penetration through the host PM is an energy-dependent process . To further analyze the form of energy driving the internalization of the NC, we characterized the energetic state of the uninfected P . syringae spheroplasts. A lipophilic cation TPP + was used to estimate the ΔΨ values. The distribution of this ion between cells (or organelles) and the surrounding medium can be measured by selective electrodes that monitor the change of the TPP + concentration in the incubation medium . Release of accumulated TPP + and cytosolic K + ions after addition of a polycationic membrane–active antibiotic PMB or the channel-forming antibiotic GD showed that the PM of the spheroplasts was able to sustain considerable ion gradients. Typically the ΔΨ of intact spheroplasts was between 120 and 150 mV, and the cytosolic K + level was ∼40 mM. The acidification rate of the spheroplast media was ∼2 × 10 3 H + × spheroplast −1 × s −1 , indicating that the proton pumps were actively extruding H + ions through the PM. Furthermore, the intracellular ATP level of the spheroplasts was ∼600 μM and the extracellular media contained ∼3 μM ATP. Although the spheroplasts were rather well energized, their energetic state was not stable when incubated at 23°C with magnetic stirring, the electrochemical measurement conditions . In these conditions, the spheroplasts became depolarized and the intracellular K + leaked out . Simultaneously, the ability of the spheroplasts to support NC infection was dramatically reduced . However, storage of spheroplasts on ice without stirring for up to 90 min did not reduce their energetic level or capacity to produce mature viruses . The instability of the spheroplasts in conditions for electrochemical measurements was taken into account when the effects of different energy-depleting drugs on NC–spheroplast infection were studied. Despite batch-to-batch variation in the ability of the spheroplasts to support NC infection, the trend of the results was consistent and allowed pooling of the data . During the systematic study of the NC infection, it also became apparent that the maximum virus yield was not affected by the temperature of the infection mixture (10–30°C). However, the rate for achieving the maximum yield was temperature-dependent (not shown). The PM of P . syringae cells was permeable to TPP + and the cells were sensitive to ionophoric antibiotics GD (a channel former) and NG (a K + to H + exchanger) even at +6°C, strongly indicating that the PM stays in a fluid state and, thus, is able to support the formation of invaginations even at low temperatures. Therefore, a low temperature could not be used to dissect the NC binding from the internalization. The original NC infection assay was modified to allow the development of an NC entry assay as follows. The comparison of virus progeny production revealed that both the NC and spheroplasts could be stored frozen without significantly changing the ICs obtained (not shown). This allowed screening of a vast number of conditions using standardized material. The bursting of spheroplasts in the reaction tube at the end of the infection cycle was prevented by the new infection medium containing glycerol but no LB. However, plaques were formed normally when the infected spheroplasts were diluted and plated on a lawn of indicator host on LB plates. Incubation of the infected spheroplasts for up to 4 h at 23°C did not diminish their potential to release infective progeny upon plating. The time course of IC formation on LB plates after different infection times in a test tube is depicted in Fig. 3 a. The maximal yield is reached at ∼45 min postinfection (p.i.). Treatment of the infection mixture with NC-specific polyclonal antiserum allows the dissection of the antibody-inhibitable NC particles (free and PM adsorbed) from those that have already reached an antibody-resistant environment (internalized particles). ICs formed by antibody-resistant NCs as a function of time are shown in Fig. 3 b. In this case, the maximal level is reached at ∼90 min. The total yield is the sum of the ICs formed by spheroplasts infected both by particles that are adsorbed (antibody inhibitable) and internalized (resistant to the antibody) at the time of plating. We set up an NC entry assay based on the following three observations: (1) dilution-resistant NCs, inhibitable by antibodies (cell adsorbed particles) can form ICs on plates ; (2) a 10-min treatment with NC-specific antiserum causes maximal inactivation of these particles, but still allows the antibody-resistant (internalized) NCs to form ICs ; and (3) treatment with the serum did not reduce the ability of spheroplasts to support the viral life cycle . Thus, in this investigation, NC entry was defined as a process where NCs become resistant to NC-specific antiserum. This was analyzed by comparing the IC counts obtained after a 10-min treatment with an NC-specific antiserum to those obtained after treatment with a nonspecific control antiserum. The energy requirements of the NC internalization process were analyzed by using energy-depleting agents. Plaque assays, ΔΨ, and ATP level determinations were simultaneously carried out to monitor the cellular energy status and to correlate it to infection efficiency. As the different agents tested were diluted before plating the infected spheroplasts out, the entered or adsorbed NCs are able to produce progeny on the plates. When a drug inhibits the entry, the number of NCs in the antibody-resistant environment at the time of plating will be lower than in the control infection. However, if the drug does not affect the capability of spheroplasts to produce viruses, or the NC binding to spheroplasts, the antibody-inhibitable NCs (adsorbed to spheroplasts) can enter normally into the spheroplasts after dilution of the drug. Therefore, the number of ICs formed by these NCs will not differ from the control. Extensive screening allowed us to find drug concentrations that had measurable effects on the energetic properties of the spheroplasts, but which did not affect IC production per se. On the basis of the effects on the NC entry, the drugs were categorized into three groups as shown in Fig. 4 . Compounds in the first category did not affect the infection per se , but caused a distinct decrease in plaque numbers if NC-specific antiserum was used, implying an effect on entry . This category includes uncouplers of oxidative phosphorylation, CCCP, and FCCP. These protonophores dissipate the Δp and, accordingly, reduced ΔΨ values (<80 mV) were observed at the concentrations used (25 μg/ml). In spite of the inhibitory effect on entry, the binding of the NC to the spheroplast surface was not reduced because of the decrease in Δp . In the second category , the drug treatment had no significant effect on the production of progeny either in the presence or absence of the NC-specific antiserum. MN and NG, the representatives of this category, reduce the existing H + and K + concentration gradients by exchanging extracellular H + for intracellular K + ions . Consistently, an alkalinization of the spheroplast medium and >90% reduction in the cytosolic K + content was detected. Moreover, a high concentration of sodium benzoate (a salt of a weak acid) had no effect on NC internalization , thus, further supporting the idea that transmembrane ΔpH is not involved . PMB (a membrane-active antibiotic), N -ethylmaleimide (an inhibitor of the SH group–dependent processes), and NaN 3 (an inhibitor of the respiratory chain and membrane H + -ATP synthase) are examples of drugs falling into the third category . Active concentrations of these drugs affected the IC formation both in the presence and absence of the NC-specific antiserum. The inhibition of the total IC production indicates the following: (1) that incubation with these agents has irreversibly lowered the ability of spheroplasts to produce ICs, (2) the drug is irreversibly bound to membranes and cannot be removed by dilution, or (3) dilution-resistant adsorption of the NCs to spheroplasts has been affected in the presence of the agent. Since the distinction between these alternatives could not be made, these drugs were not used in further studies. Concentrations of KCl or NaCl (not shown) up to 50 mM increased the total yield of ICs. At higher concentrations, reduced ΔΨ values were measured , and a decrease in the number of ICs after the NC-specific antiserum treatment was detected . To ensure that the change in the medium osmolarity had not affected the internalization, the osmolarity of the infection mixture was increased with sucrose instead of salt. As shown in Fig. 6 a, the sucrose containing infections did not differ from the control infections. The inhibitory effect of NaCl and KCl on the NC entry was reversed if MN or NG were added . However, the electrogenic K + carrier, valinomycin, could not rescue the effect of KCl (not shown). The ΔΨ measurements showed that NG also induced an increase in the ΔΨ . Although NG works in an electroneutral manner, it can increase ΔΨ if the respiratory chain is active . When a portion of KCl was replaced with an inhibitor of the respiratory chain (KCN), the entry effect detected was similar to that in the presence of KCl only . However, NG could not compensate for the reduction in ΔΨ in the presence of KCN nor was the NC entry rescued . Therefore, it appears that there is a threshold value in ΔΨ between 95 and 120 mV below which the NC internalization is strongly inhibited. Different drug treatments were carried out to decrease the intra- and extracellular ATP content of the spheroplasts. To this end, we analyzed the effects of DCCD (an inhibitor of membrane ATP synthase), NaF (an inhibitor of ATP formation from glycolytic substrates), Na 2 HAsO 4 (an ATP destabilizing agent), as well as AP (an ATP hydrolyzing enzyme). Some decrease in the ATP concentration was observed in the presence of these agents, but the most efficient reduction was achieved if the spheroplasts were washed before the drug treatments . The lowest extracellular ATP concentration measured was 75 nM (2.5% of the ATP level of unwashed spheroplasts) and the lowest intracellular ATP concentration was ∼50 μM (8% of the ATP level of unwashed spheroplasts). Although some decrease in the total IC production was observed after these treatments, the infections treated with NC-specific antiserum remained at the control level, indicating that internalization of the NC was not affected at reduced ATP concentrations. A high concentration of H + ions seems to be crucial for a number of virus entry processes . An increase in the pH of the NC infection medium up to 7.5 did not affect the NC internalization (not shown). However, the pH at the outer surface of a metabolically active bacterium can be considerably lower than in the bulk phase of the medium . The inhibition of respiration by KCN reduced NC internalization, but it also had a profound effect on ΔΨ as shown in Fig. 6 a. The addition of 25 mM KCl or NaCl to the spheroplast suspension caused a clear pulse of acidification of the medium, indicating an exchange of H + to K + or Na + ions at the outer surface of the spheroplasts (not shown). However, this did not considerably affect the IC formation . Neither did the decrease in the surface charge after addition of high concentrations of the polycationic compound, PMB nonapeptide (300 μg/ml), reduce the IC counts (not shown). The density of H + at the outer surface of the PM should also be reduced in the presence of NG, which changes the extracellular H + to cytosolic K + , but no inhibition of NC internalization was detected . These results indicate that low pH at the outer surface of the PM is not crucial for the early stages in NC internalization. In a normal φ6 infection, practically every cell is infected and the specific infectivity of the virus particles is close to one . In the NC–spheroplast infection, NCs enter the cells that lack a specific high affinity receptor and high multiplicity of infection is required. In these conditions, approximately every fifth cell is productively infected . Previous morphological analyses of normal φ6 infections using freeze-fracture EM showed enveloped NC sized particles in the cell interior in early infection . Furthermore, thin section EM analysis of arrested infections has depicted viral NCs entering the cell via a process involving a PM invagination and an intracellular vesicle . These previous morphological observations suggested an NC–PM interaction mechanism similar to that of eukaryotic endocytosis. The NC–spheroplast infection system was used to further characterize the NC–PM interactions. As a complement to conventional chemical fixation (CF) , which may induce artefactual membrane configurations , we also used RF and FS . To optimize the FS for this type of material, several different conditions were applied (see Material and Methods). Adequate preservation of bacteria and NC was achieved with OsO 4 in the substitution media Materials and Methods: transmission electron microscopy protocols (1) and (2), which is consistent with the literature . However, the triple-layered patterns of the PM were not always seen using this method, a common phenomenon in cryoprocessed bacteria . Likewise, in both chemically fixed and cryoprocessed samples, the PM bilayer surrounding the entering viral NCs was not clearly distinguishable from the proteinaceous NC surface shell. However, intracellular enveloped NC particles can be readily distinguished from nonenveloped ones when chemically fixed cells infected with virus mutants producing particles of different composition are analyzed . The envelope appears as an ∼12-nm-thick diffuse halo surrounding the NC . The diffusiveness could be due to the size and density of the particle and the section thickness . The tight membrane envelope around NC in the virion is visible only when purified virus is embedded in thin layers of vitreous water . We carried out the EM analysis at conditions optimized to trap the transient entry event. The infection was arrested by reducing ΔΨ either with CCCP or NaCl. As previously documented , the P . syringae spheroplast appeared spherical and had fragmented OM in loose contact with the cells . Regardless of the fixation method or the ΔΨ reduction, the spheroplasts constantly displayed PM invaginations and/or intracellular vesicles, indicating them to be intrinsic for spheroplasts, not artifacts of the fixation method . The NC-infected spheroplasts showed NC–PM interaction patterns similar to those observed earlier in energy-depleted normal infections : NCs associated with the PM , in PM indentations and invaginations , and inside the cell , presumably within tightly fitting membrane vesicles. In the presence of high ΔΨ, these transient events were difficult to capture, and indentations or invaginations were rare in all samples. We also analyzed the role of different NC proteins in NC internalization. Previous studies have suggested the necessity of the protein P8 shell for the NC infectivity; uncoated NC particles devoid of P8 (the NC cores) were not infectious to host cell spheroplasts , and certain P8-specific mAbs (8D1 and 8Q2) had a neutralizing effect on NC infectivity . We further studied the role of NC proteins P1, P4, P7, and P8 in NC entry using a neutralization assay. A polyclonal P7-specific antiserum and the P1- and P4-specific mAbs that are known to recognize epitopes on the NC surface without aggregating or disrupting the NC were chosen for the assay. A mAb, 3O4, against P3 (the viral spike protein) was used as a negative control. Neither the P7-specific polyclonal antiserum nor any of the selected P1- or P4-specific mAbs could inhibit the NC infection, indicating that these proteins are not directly involved in the NC entry. More detailed investigation using the panel of P8-specific mAbs revealed that most of them had a neutralizing effect on NC infectivity ( Table ). The nonneutralizing mAbs, 8J3, 8Q4, and 8Q7, did not precipitate NCs in an immunoprecipitation assay, indicating that their epitopes are not accessible on the NC surface ( Table ). To confirm that the inhibitory effect was not due to aggregation or disruption of NC particles, the sedimentation assay of NCs was carried out after treatment with several different P8-specific mAbs ( Table ). Although many of the neutralizing antibodies did appear to disrupt the NC particles, four nonaggregating and nondisrupting mAbs (8B1, 8D1, 8K4, and 8L1) still had neutralization activity, thus, confirming P8's role in the NC entry. The endocytotic pathway commonly used by eukaryotic cells to internalize different types of molecules and molecular complexes is not thought to function in prokaryotes. However, phage φ6 has to use an exceptional entry mechanism to deliver its polymerase complex particle (NC carrying the dsRNA genome) into the host cytosol without dissipating the ΔΨ. We have shown morphological evidence here and in our previous studies that this involves association of the NC with the PM, membrane invagination, and release of an enveloped particle into the cytosol. This process resembles the events observed during endocytosis in eukaryotes. The aim of the present study was to analyze the energy requirements of the φ6 endocytic-like NC internalization. Romantschuk and co-workers showed that this process is dependent on the energetic state of the membrane. However, more detailed analysis of the energy requirements, during infection of an intact cell, is difficult as the initial association of the virus particle with the OM is also an energy-dependent process . Therefore, purified NCs and host cell spheroplasts were used. We set up an entry assay that allowed us to dissect the NC–PM interaction into two stages: NC adsorption to PM (dilution-resistant antibody-inhibitable state) and NC internalization (antibody-resistant state). Specific ATP/GTP-dependent cytosolic proteins are needed to direct the normal (clathrin-dependent) endocytic uptake used by many animal viruses . The form of energy driving the clathrin-independent entry of poliovirus, the SV40 entry via caveolae, or the entry of polyoma virus and canine parvovirus via uncoated vesicles is not clear. Phagocytosis as well as macropinocytosis are directed by actin and ATP . However, ATP-independent vesicle formation can be induced in animal cells by exogenous sphingomyelinase treatment . The translocation of the φ6 NC to an antibody-resistant location was not dependent on high extra- or intracellular ATP levels . Therefore, it seems likely that there are no ATP-dependent cytosolic or PM-associated proteins involved, neither is the viral NTPase activity required. Δp is commonly used in gram-negative bacteria to drive the transport of macromolecules into or across the membrane(s) . The translocation of phage T4 genome into the host cytosol depends on phage-induced, Δp-dependent fusion of the OM and PM at the site of phage adsorption . The insertion of channel-forming colicins into the bacterial PM occurs through an electrostatic binding to the membrane surface, spontaneous insertion of hydrophobic hairpin into the membrane, and ΔΨ-driven insertion of amphiphilic helices . The dilution-resistant φ6 NC binding to the spheroplast membrane was not affected at reduced Δp values . However, the subsequent NC transport to the antibody-resistant location was clearly dependent on ΔΨ but not on ΔpH . Simultaneous measurements of ΔΨ and plaque formation allowed us to define a threshold value for the NC internalization . Accordingly, the Δp-dependent processes mentioned above, the T4 phage DNA entry, and the colicin insertion into PM, occur only when the ΔΨ is above a threshold value . Similarly to the colicin insertion, the ΔΨ dependence in NC entry might be associated to polypeptide chain translocation (probably of the NC shell protein P8) into the PM. Alternatively, the ΔΨ might be required as the NC-containing invagination pinches off from the PM. In the bacterial PM, the negatively charged phospholipids, cardiolipin and phospatidylglycerol, are predominantly located in the outer leaflet and phosphatidylethanolamine in the inner leaflet . The transport of phosphatidylethanolamine from one leaflet to another is dependent on Δp . The fusion of an NC-containing invagination might require the transport of phosphatidylethanolamine as only this bacterial phospholipid can support membrane fusion . The NC surface protein P8 was shown to be crucial for the entry. Viral proteins involved in the entry are known to undergo large conformational changes . These changes are triggered by the receptor binding and/or by the low pH during endocytosis. Our preliminary results indicate that acidic conditions change the conformation of P8, suggesting that protonation is also involved in the NC entry process. However, the tight coupling of proton pumping and ΔΨ production makes it difficult to dissect these effects . The NC adsorption and early steps in the formation of the PM invagination were not affected in conditions where the PM surface charge, and thus protonation, was reduced. We consider that the proton-dependent events are crucial later in the entry, in the process of the NC release from the entry vesicle. φ6 entry can now be dissected into a number of stages . The present investigation sheds light on the NC absorption to the PM and subsequent processes. We are actively studying the last step in the infection process, the release of the polymerase complex from the vesicle and its subsequent activation. The accumulated data on the φ6 entry process interestingly highlights common universal mechanisms operating in both prokaryotic and eukaryotic cells, and points out that mechanisms that were thought to operate in eukaryotes only are also used in prokaryotes. | Study | biomedical | en | 0.999997 |
10545510 | Sheep blood was obtained from BioMérieux (Marcy L'Etoile, France). Human blood was obtained from the Etablissement Français de Transfusion Sanguine (Paris, France). Bacteria were grown from overnight precultures diluted 1:100 in aerated flasks of Trypticase Soy Broth (TSB; Becton Dickinson) for 4 h at 37°C to OD 600 = 4. They were collected by centrifugation for 5,000 g for 7 min at 4°C, washed in saline (150 mM NaCl) and resuspended at 10 10 bacteria/ml at 4°C. RBCs were washed 3× in saline by centrifugation at 2,000 g for 5 min at 4°C and resuspended at 5.10 8 /ml. The final resuspension buffer of both bacteria and RBCs contained 30 mM Tris at pH 7.5 (Tris-saline). 100 μl of bacteria were mixed with 100 μl of RBCs in round bottom 96-well plates, centrifuged at 1,500 g for 10 min at 10°C, and incubated at 37°C for 1 h. The cells were resuspended and the samples recentrifuged. 100 μl of supernatant was transferred to a fresh plate where its optical density at 595 nm was measured. The baseline (B) of the assay was set with RBCs incubated with Tris-saline instead of bacteria and total hemolysis (T) was the value obtained when RBCs were incubated with saline containing 0.1% sodium dodecyl sulfate. The percentage of total hemolysis (P) was calculated using the equation P = [(X − B)/(T − B)] × 100, where X is the optical density value of the sample analyzed. All data presented result from at least three independent experiments. Errors given are standard deviations. When AfaE-expressing Shigella were used to lyse human RBCs, bacteria were resuspended at only 10 9 bacteria/ml. Avirulent S . flexneri expressing E . coli hemolysin A are described by Zychlinski et al. 1994 . To obtain the hemolysin containing culture supernatants, bacteria were diluted 1:100 from overnight precultures into 8 ml of TSB and grown for 3 h at 37°C. The bacteria were eliminated by centrifugation and the supernatant used immediately. The ipaA ipgD mutant strain was constructed by integration of pLAC-A carrying an ipaA::LacZ fusion into the large plasmid of the ipgD mutant strain. RBCs were resuspended in a 60-mM solution of sucrose, raffinose (Sigma), or PEG1000 through 3000 (Fluka) made in Tris-saline. After incubation at 37°C, the samples were cooled to stop the reaction, resuspended and immediately recentrifuged. 5 × 10 7 washed RBCs in 100 μl were treated with 0.5 U of neuraminidase, type V from Clostridium perfringens (Sigma) for 60 min on ice. The cells were washed 3× in Tris-saline and resuspended at the same concentration for use in the hemolytic assay. Complete removal of sialic acid was verified by loss of the ability of HA virus, the cell receptor of which is sialic acid, to hemagglutinate the treated RBCs. Bacteria and sheep RBCs were prepared as described above, except that they were resuspended in Tris-saline at 2.10 11 and 1.5.10 10 cells/ml, respectively. Hemolytic reactions were prepared in 50-ml conical tubes with 600 μl RBCs, 120 μl bacteria, 860 μl Tris-saline, and a protease inhibitor cocktail (PIC 1 ; Complete™, Boehringer Mannheim). Samples were centrifuged at 2,000 g at 4°C for 10 min, incubated at 37 or 4°C for 1 h, resuspended by vortexing, and recentrifuged. Hemolysis was assessed spectrometrically as above. 800 μl of distilled water was added to each sample to lyse all RBCs nonspecifically (so that lysed membranes could be isolated even from those samples where no hemolysis had occurred), and these were vortexed and centrifuged again to remove bacteria. 2 ml of supernatant was collected, adjusted to 2.4 ml with Tris-saline and brought to 46% sucrose with 7 ml of 62% sucrose in Tris-saline containing the PIC. The mixtures were deposited at the bottom of SW41 centrifuge tubes (Beckman) and layered with 2 ml of 44% and then 25% sucrose in Tris-saline containing the PIC. Gradients were centrifuged at 15,000 g for 16 h at 4°C. The material at the 44/25% sucrose interface was collected, diluted in Tris-saline and concentrated by centrifugation at 450,000 g for 20 min 4°C in a TLA 100.3 rotor (Beckman). The pellets were resuspended in a minimal volume of buffer. Such samples were checked by transmission electron microscopy but no contaminating bacteria or bacterial ghosts were seen. When 35 S-labeled bacteria were used less than 0.01% of the initial radioactive input was recovered in RBC membranes. The protein content of the samples was estimated using Bradford's assay (Bio Rad) and an equivalent protein amount of each was separated by SDS-PAGE and Western blotted. To assess the strength of association of Ipa proteins with RBC membranes, 100 μl of purified Shigella -lysed RBC membranes were incubated at 4°C for 1 h in Tris-saline or Tris-saline with 5 M NaCl, 8 M urea, or 0.2 M carbonate, pH 11. 300 μl of 62% sucrose in Tris-saline was then mixed with the membranes that were deposited at the bottom of 0.8-ml SW55 tubes (Beckman) and overlaid with 150 and 100 μl of 44 and 25% sucrose in Tris-saline. The gradients were spun overnight at 4°C at 15,000 g . The top 150 μl was collected from the gradients, diluted in Tris-saline, and concentrated by pelleting in a TLA 100.2 rotor for 20 min at 450,000 g at 4°C. The pellets were resuspended in a minimal volume of buffer. 1-ml aliquots of Shigella cultures, grown as above were washed in Tris-saline and resuspended in 500 μl of the same buffer. 200 μM of Congo red was added to the bacteria and the cells were incubated for 10 min at 37°C. The bacteria were pelleted at 14,000 g at 4°C for 15 min. 10 μl of supernatant and of the pellet resuspended in 500 μl of Tris-saline were separated by SDS-PAGE and Western blotted. After the final centrifugation 20 μl of hemolytic assay supernatant and 20 μl of a resuspended reaction containing an equivalent number of bacteria incubated in the absence of any inducer of secretion were separated by SDS-PAGE and Western blotted. mAbs to IpaB and IpaC were a gift from Armelle Phalipon. mAbs against IpaA and IpgD were generated by immunizing female BALB/c mice with Ipa protein containing supernatants obtained after induction of secretion in Shigella flexneri with Congo red. In brief, 10–20 mg of protein was injected subcutaneously into both hind legs (5 injections at 3-d intervals) and the popliteal lymph nodes were fused with X63Ag8 lymphoma cells according to standard protocols. Hybridoma supernatants were screened by ELISA and immunoblotting and clones positive for IpaA and IpgD were subcloned by limiting dilution. Conventional SDS-PAGE was performed and immunoblotting was performed using a semi-dry apparatus onto nitrocellulose or PVDF membranes and developed using the ECL or ECL Plus kits (Amersham) and X-OMAT film (Kodak). Bacteria were grown for 2 h at 37°C from 1:50 dilutions of overnight precultures in 8 ml of TSB. 2 ml of bacteria were collected, washed in filter sterilized PBS, and resuspended in 20 μl of the same buffer. The bacteria were osmotically and mechanically shocked by a 1:4 dilution into a 60-μl drop of distilled water deposited on parafilm. Often lysis of dividing bacteria was favored by incubation of the samples on ice for a few hours before dilution in water. After a few minutes, 200 mesh Formvar-coated copper microscopy grids were placed on top of the drops. After a brief incubation at room temperature, the grids were rinsed with a flow of drops of 2% phosphotungstic acid at pH 7. After drying, the samples were observed in a Philips CM12 electron microscope working in standard conditions. Negative micrographs were digitized on a rotary drum scanner using a 10-μm/pixel square scanning aperture. Windowing and processing of the particle images was performed on digitized micrographs, using SPIDER software and WEB interactive selection program on a Unix Digital Workstation. After contrast normalization, the whole set of selected image was subjected to a reference free alignment process to generate an average projection image of the secreton. The resolution limit estimation was performed using both differential phase-residual and Fourier ring correlation criterion . AfaE-expressing bacteria and human red blood cells were mixed as in for the hemolytic assay but in 500-μl tubes. Hemagglutination occurred and samples were either centrifuged at various g forces for 10 min or left to sediment at 1 g at 10°C. Samples were fixed for 1 h at room temperature with 1.2% glutaraldehyde, and 0.05% ruthenium red (RR) in 0.1 M cacodylate buffer, pH 7.4, to better visualize the carbohydrate surrounding cell membranes . Samples were rinsed with 0.05% RR in cacodylate buffer and post-fixed with 1% OsO4, 0.05% RR in cacodylate buffer for 1 h at room temperature. Cells were rinsed with H 2 O and embedded in 2% agarose (Type VII; Sigma). After gelling on ice samples were dehydrated with an increasing acetone series and embedded in Epon. Thin sections were conventionally stained. To study the interaction of S . flexneri with a simple cell membrane, we reinvestigated its contact hemolytic activity. Bacteria were mixed with RBCs, centrifuged at 1,500 g for 10 min at 10°C and incubated at 37°C for 1 h. Release of haemoglobin was monitored as described in Materials and Methods. As previously reported, contact hemolysis did not occur without centrifugation , and strains carrying mutations in mxiD , ipaB , or ipaD were nonhemolytic, whereas strains carrying mutations in ipaA , ipgD , or in both ipaA and ipgD displayed normal hemolysis . The ipaC mutant had ∼10% hemolytic activity. This low activity had not been detected before because previously the wild-type strain induced only 40% hemolysis , whereas, in the current assay, it caused 75–100% hemolysis. These results confirmed that integrity of the secretion machinery and IpaB, IpaC, and IpaD, but neither IpaA nor IpgD, were required for hemolysis. The residual activity of the ipaC mutant suggested that, in the absence of IpaC, another factor could still destabilize the RBC membrane, albeit inefficiently. To determine whether hemolysis was due to the formation of a pore within the RBC membrane, we performed osmoprotection experiments. Lysis of RBCs by bacterial toxins forming hydrophilic pores in membranes proceeds through osmotic shock . This can be prevented by addition of osmotic protectants at 30 mM to the medium. If the molecule is too large to pass through the pores, it counterbalances the increased intracellular pressure. Osmoprotectants of intermediate sizes induce a size-dependent increase of the half time of hemolysis that can be used to estimate pore size. Molecules larger than PEG1000 allowed significant protection against lysis generated by wild-type bacteria and protection increased with the size of the molecule . A time course analysis of hemolysis in the presence of different osmoprotectants was used to derive a Renkin plot showing the relative permeability of the osmoprotectants versus their size . This allowed estimation of the functional inner radius of the pore at 26 Å (±0.4 Å, standard deviation, n = 3). No osmoprotection was observed in lysis mediated by the ipaC strain . This suggested that hemolysis induced by the ipaC mutant resulted either from formation of a pore larger than 32 Å in diameter or from destabilization of the membrane by another mechanism. To determine the components of the pore formed in the membrane during contact hemolysis, we isolated the lysed RBC membranes by floatation in a sucrose density gradient. The protein content of the membranes was separated by SDS-PAGE, blotted and probed with antibodies against IpaA-D and IpgD. IpaB, IpaC, IpgD, and IpaA were present in membranes recovered after incubation of RBCs with the wild-type at 37°C, but not in fractions recovered after incubation with the mxiD mutant at 37°C or with the wild-type at 4°C . By semiquantitative immunoblotting, the amount of IpaB, IpaC, IpgD, and IpaA recovered in the membrane fraction corresponded to ∼0.1% of the total amount of these proteins present initially in bacteria. IpaD was not detected in the membrane fraction, although the detection procedure was sensitive enough to reveal 0.1% of IpaD in bacterial extracts (not shown). Membranes isolated after contact with the wild-type at 37°C were then incubated in the presence of agents known to release peripheral membrane proteins. The amount of Ipa and Ipg proteins associated with membranes was measured after a second floatation in a sucrose density gradient. After stripping with 5 M NaCl, 0.2 M carbonate, pH 11, or 8 M urea, the majority of IpaB and various amounts of IpaC remained associated with the membranes . In contrast, IpgD and IpaA were lost even in the mock-treated sample (not shown). This indicated that IpaB, and to a lesser extent IpaC, were strongly associated with the membranes, while IpgD and IpaA were located at the membrane periphery. To investigate further the mode of interaction of Ipa and Ipg proteins with RBCs membranes, we examined their presence in this fraction after contact of the ipaC , ipaA , ipgD , and ipaA ipgD mutants with RBCs. Membranes exposed to the ipaA , ipgD , or ipaA ipgD mutants contained relatively high amounts of IpaB and IpaC, and of IpgD or IpaA, or neither of the latter two proteins, respectively. This indicated that IpaA and IpgD were not required for association of IpaB and IpaC with the membranes or for association of each other with membranes. In the ipaC mutant, reduced amounts of IpaB, IpaA and IpgD were associated with the membranes . Although this amount was vastly reduced as compared with that observed with the wild-type, it was significant as incubation of RBCs with the mxiD mutant at 37°C or with the wild-type at 4°C led to undetectable amounts of these proteins in the membranes. The small amount of IpaB associated with membranes after incubation of RBCs with the ipaC mutant could account for the residual hemolysis of this strain. The low amount of IpaA and IpgD in these membranes suggested that these proteins require the presence of IpaC and/or IpaB to associate with membranes. To gain an understanding of how IpaB and IpaC were transferred into target membranes, we studied the relationship between hemolysis and Ipa protein secretion. We investigated the efficiency of hemolysis after incubation for 1 h between 4 and 42°C. No hemolysis was observed below 25°C and the reaction proceeded normally above 30°C . The temperature dependence of lysis correlated with that of Ipa protein secretion after induction of the machinery with Congo red . The spa47 gene encodes a protein related to the β subunit of F1 mitochondrial ATPases which is essential for secretion . This led us to investigate the effect of sodium azide, an inhibitor of oxidative phosphorylation (to which RBCs should be insensitive), on secretion and hemolysis. Addition of increasing amounts of azide progressively inhibited Ipa secretion induced by Congo red , indicating that Ipa secretion required energy. However, the presence of 25 mM azide inhibited Congo red–induced secretion of IpaB ∼20-fold but decreased hemolysis only 4-fold . This suggested that not all of the Ipa proteins that could be secreted were released during hemolysis. Indeed, during a hemolytic assay, only a small amount of Ipa proteins were recovered in the medium, the rest of these proteins remaining within the bacterium . In addition, not all the released Ipa proteins may be required to observe full hemolysis, for which a single pore per RBC is in theory sufficient. In support of this, pretreatment of bacteria with Congo red, which released ∼50% of Ipa proteins , had no effect on the efficiency of a subsequent hemolytic assay, even when novel protein synthesis was blocked (not shown). This may explain why lysed RBC membranes contained <0.1% of the amount of Ipa proteins that were initially present in bacteria. In summary, Ipa secretion was an active process and secretion at the moment of contact was necessary for lysis. To determine whether the proteins responsible for hemolysis ever became exposed to the external medium, we added antibodies to IpaB, IpaC, and/or IpaD to the hemolytic reaction. These reagents had no effect on hemolysis at any concentration tested (up to 100 μg/ml), whether alone or in combination or even upon preincubation of bacteria (not shown). Next we added 1 mg/ml of proteinase K to the assay. This led to only a minor decrease in hemolytic efficiency . This amount of protease instantly degraded E . coli hemolysin A, completely preventing hemolysis mediated by this protein . Interestingly, the inhibitory effects of azide and proteinase K on hemolysis were additive . This suggested that azide was acting by reducing secretion and proteinase K by degrading some of the secreted proteins as they were exiting the bacterium. Individual pretreatment of RBCs and bacteria with azide and proteinase K did not affect hemolysis (not shown), suggesting that any Ipa proteins at the bacterial surface before contact were not involved in hemolysis. These data confirmed that Ipa protein secretion and hemolysis were coupled in time and suggested that the process of Ipa protein transfer into the target membrane was poorly accessible from the surrounding medium. To investigate the requirement for contact between bacteria and RBCs to obtain hemolysis, we used a derivative of S . flexneri carrying the AfaE afimbrial adhesin from enteropathogenic E . coli . This adhesin binds the decay accelerating factor, a ubiquitous surface glycoprotein, and confers upon bacteria the ability to hemagglutinate human RBCs . Despite their close binding to RBCs, these bacteria were unable to lyse RBCs in the absence of centrifugation . However, upon centrifugation, they lysed RBCs efficiently. Since the adhesion mediated by AfaE was not sufficient to induce extensive Ipa protein secretion , AfaE-expressing bacteria were incubated at 37°C in the presence of human RBCs and Congo red. Induction of Ipa protein secretion in the immediate vicinity of RBCs did not lead to lysis but the same procedure in the presence of centrifugation did . This indicated that Congo red did not inhibit hemolysis and suggested that Ipa proteins could not insert into the RBC membrane after release from the bacterium. These data indicated that Ipa secretion was necessary but not sufficient for hemolysis. Given that bacteria adhering to cells via the AfaE adhesin were unable to cause hemolysis without centrifugation at 1,500 g , we examined the consequence of this physical treatment on the cells. Mixtures of RBCs and AfaE-expressing bacteria were exposed to different centrifugation forces at 10°C and then either incubated at 37°C to assay hemolysis or prepared for examination by transmission electron microscopy. Hemolysis did not occur at 160 g but was complete at 630 g ( Table ). In samples centrifuged at 160–630 g , the RBC membrane was deformed to line that of bacteria . In the sample which had not been centrifuged, bacteria associated with RBCs adhered at a tangent rather than over their entire surface . This indicated that centrifugation increased the surface of bacteria in contact with RBCs. Although samples treated at 160 and 630 g looked similar, lysis occurred in the latter but not in the former ( Table ), suggesting that the distance between bacterial and RBC membranes might also be critical for hemolysis. To test this possibility, we shortened this distance artificially, by removing either bacterial lipopolysaccharide (LPS) or sialic acid, a major constituent of the RBC surface . We compared the lysis efficiency of wild-type Shigella and that of an rfe mutant that expressed a complete LPS core but lacked the O-antigen . The rfe mutant was as efficient as the wild-type at lysing RBCs at any g force applied (not shown). Then, we treated RBCs with neuraminidase to remove sialic acid. This treatment did not prevent AfaE carrying Shigella from hemagglutinating RBCs (not shown), as the site on the DAF molecule which is sialylated is distant from the SCR-3 domain to which AfaE binds . This treatment allowed lysis to occur efficiently at 160 g whereas mock-treated RBCs remained intact after exposure to bacteria at 160 g . Surface charge is known to affect the ability of proteins to insert into artificial membranes and the absence of LPS or the removal of sialic acid affect the surface changes of bacteria and RBCs. Thus, both the distance and the charge of the target membrane may be critical for induction of hemolysis. To understand why contact was critical for hemolysis, we searched by electron microscopy for the structure encoded by the mxi and spa genes in osmotically shocked and negatively stained bacteria. Most bacteria were unaffected by osmotic shocking, while some, mostly those undergoing binary fission, were partially lysed and a few appeared as complete membrane ghosts. No structures were visible on intact bacteria. Some structures, resembling the type III secretion apparatus of Salmonella , were visible at the periphery of partially lysed bacteria . In the ghosts, many structures were visible dispersed over the whole bacterial surface (50–100/cell) and they were not identical to that seen in partially lysed bacteria . These structures appeared as composed of three parts: (a) a needle 11 nm in diameter, (b) a neck 10 nm high and 21 nm wide, and (c) a bulb 44 nm wide and 27 nm high . This bulb did not appear to have cylindrical symmetry since we saw several different views of it . Upon alignment and averaging of a gallery of the 22 best preserved structures on the micrograph displayed in Fig. 5 A, we obtained a two-dimensional density map of the bulb at 2.8 nm resolution . This tripartite structure is not identical to that seen by Kubori et al. 1998 . They presented partially lysed bacteria in which secretons were visualized at the cell periphery and lacked the bulb. When examined by this method, intact bacteria and, to a lesser extent, partially lysed ones have a thickness which could explain why secreton structures seemed absent or partial, respectively, at their surface. No structures were found in mxiD , mxiJ , and mxiG mutants (not shown). As these three strains carry mutations within genes coding for components of the type III secretion system, we concluded that the structures observed in the wild-type corresponded to the type III secretons. No obvious differences were detected in the morphology of secretons in ipaB , ipaC , and ipaD mutants relative to the wild-type , nor in their distribution or number (not shown). Likewise, we saw no differences in secreton morphology or distribution of the wild-type which had been induced to secrete with Congo red . This indicated that activation of secretion, as seen in ipaB and ipaD mutants or upon incubation of the wild-type with Congo red, did not result in or from gross alterations of secreton structure or number. We also concluded that the nonhemolytic phenotypes of the ipaB , ipaC , and ipaD mutants did not result from major anomalies in the structure of their secretons. Osmoprotection experiments showed that S . flexneri inserts a hydrophilic pore into RBCs. Amongst the five proteins secreted upon contact with eukaryotic cells, only IpaB and IpaC were tightly associated with RBC membranes isolated after contact-hemolysis. IpaA and IpgD were peripherally associated with RBC membranes while IpaD was not found in the membrane fraction. Since ipaA and ipgD mutants are not impaired in hemolysis, association of IpaA and IpgD with membranes is unrelated to pore formation. As IpaB and IpaC are the only secreted proteins with predicted α-helical hydrophobic regions capable of spanning membrane bilayers, it is likely that they are the sole bacterial components inserted into the host membrane during cell entry. Whether the IpaB/IpaC pore structure contains host components also can not be assessed with our experimental setup. However, studies of the association of Ipa proteins secreted in the presence of Congo red with artificial membranes have yielded only the same four proteins associated with liposomes and these proteins form a nonselective, gated channel of 91 pS in conductance within lipid bilayers (De Geyter, C., F. Homblé, R. Wattiez, P.J. Sansonetti, P. Falmagne, J.M. Ruysschaert, C. Parsot, and V. Cabiaux, manuscript submitted for publication). We also showed that IpaB and IpaC are both required to form the pore. Indeed IpaB alone is unable to form the pore structure since lysis mediated by the ipaC strain was insensitive to osmoprotection yet led to a small amount of IpaB protein becoming associated with lysed RBC membranes . When secretion occurs in the absence of any membrane, IpaB and IpaC are found solely as a complex in the medium (Blocker, A., unpublished observation), suggesting that they associate as they exit the secreton. Hence, to be inserted efficiently into the target membrane IpaB and IpaC must form a complex which may remain associated within lipid bilayers. Although the ipaD mutant was nonhemolytic, we did not recover IpaD in lysed RBC membranes. Given the constitutive secretion phenotype of the ipaD mutant, the requirement of IpaD for hemolysis probably reflects the role of this protein in controlling secretion. IpaA and IpgD were associated peripherally with lysed RBC membranes, yet they were dispensable for hemolysis. As these proteins were found associated with RBC membranes lysed by the wild-type but not the ipaC mutant, they probably associate with the membranes via IpaB and/or IpaC. Evidence is mounting that both IpaA and IpgD proteins are translocated within the host cell at the time of entry when they modulate the actin rearrangements induced by IpaC . This would suggest that IpaA and IpgD associate with the cytoplasmic face of the host membrane. Further work is required to establish the domains in each protein involved in these interactions, their topology relative to the membrane as well as the role of these interactions in cell entry. Electron microscopy studies showed that the type III secretons are assembled before any contact with target cells. In the mxiD , mxiG , and mxiJ mutants, no secretons were detected, which suggests that components of the secreton are degraded when the machinery can not be assembled. This is consistent with evidence that MxiG is unstable in the mxiJ and mxiD mutants (Bahrani, F., and C. Parsot, unpublished observation). The secreton structure is composed of three different parts: (a) an external needle, (b) a neck, and (c) a large proximal bulb. In our images, it is difficult to discern the two bacterial membranes and thus to judge the localization of the bulb. Nevertheless the length of the neck domain is sufficient to traverse both membranes, implying that the bulb lies within bacterial cytosol. This putative cytoplasmic part was not recovered by Kubori et al. 1998 in secreton preparations, which were purified using a protocol derived from that for isolation of flagellar basal bodies . A cytoplasmic structure (other than the C-ring) is known to be associated with the bacterial flagellum and thought to contain the cytoplasmic F1-type ATPase FliI as well as several other components. This structure is not recovered in purified basal body preparations and has never been visualized . Inactivation of spa47 , the homologue of the flagellar biosynthesis gene fliI , abolishes secretion. However, so far, it was unclear whether this ATPase was required for assembly and/or activity of the secretion machinery . We showed that type III secretons were assembled by S . flexneri before host contact and that their structure did not grossly change when secretion was activated (in the presence of Congo red or in the ipaD or ipaB mutants). We also showed that secretion required energy and temperatures above 30°C . Low temperatures did not disassemble secretons (not shown). Thus ATP hydrolysis and conformational change(s) are likely to be required for Ipa secretion, as for flagellin export . We propose that Spa47 is the azide-sensitive element of the secretion machine. Intimate contact between bacteria and RBCs is essential for hemolysis. Indeed, secretion is necessary but not sufficient for hemolysis since inducing protein secretion from AfaE-expressing bacteria bound to RBCs did not provoke hemolysis in the absence of centrifugation. The increased hemolysis which is seen after centrifugation could be due to the greater surface contact which either activates more secretons to secrete and/or allows Ipas to reach the membrane still in active form after release. Both notions are supported by the sharp threshold of g forces required to obtain lysis and by the sialic acid removal experiment ( Table ). Wild-type Shigella do not need centrifugation or the AfaE adhesin to enter HeLa cells, these agents simply make the process more efficient . The shortened glycocalyx, especially on the basal surface of transformed epithelial cells, might explain why AfaE can increase the efficiency of HeLa cell entry but not of hemolysis. Electron microscopy images indicate that the minimal distance required for lysis is ∼100 nm; the secreton needle is 60 nm long. Yet, contact with the needle alone is unable to cause lysis as (a) the diameter of the pore (25 Å) is smaller than that of the needle (80–100 Å), and (b) ipaB , ipaC and ipaD mutants display normal secretons yet no hemolysis. Two hypotheses have been put forward for how Ipas associate with host cells . First, Ipa proteins could be presented at the bacterial surface and inserted from there upon contact. This corresponds to a two-step model. Second, contact may induce secretion and the newly secreted Ipas may insert into the target membrane at this stage. This is a one-step model. We detected only minute amounts of Ipas at the bacterial surface by immunoelectron microscopy and these Ipa proteins never colocalized with secretons revealed by negative staining (Gounon, P., and A. Blocker, unpublished observations). In addition, hemolysis was insensitive to protease pretreatment of bacteria, suggesting that the biologically active Ipa proteins are not at the bacterial surface before contact. This is not in favor of a two-step mechanism for Ipa protein transfer into the target membrane. On the other hand, the temperature and energetic correlation between secretion and lysis and the additive inhibition of hemolysis by azide and proteinase K support a process where secretion or surface presentation upon contact is kinetically coupled to insertion. How might secretion and membrane association of the pore forming Ipas be coupled? The approximate diameter of the canal known to exist in the bacterial flagellum is 20–30 Å . As the secreton structure is evolutionarily derived from flagella there could exist a canal of similar diameter through the needle. The semi-folded state from which IpaB and IpaC would necessarily exit the secreton needle (given the small diameter of the putative canal) might be required for their membrane association. As the role of the Spa47 ATPase is probably to push the secreted proteins into the secreton canal, ATP hydrolysis by this protein could also indirectly serve to allow membrane insertion. Accordingly, the needle may be required both as a tactile sensor for activation of the secreton and to present the Ipas directly at the host membrane surface in a sufficiently unfolded state to allow their insertion. What we can not yet assess is whether the semifolded IpaB/IpaC complex enters the membrane immediately upon exiting the secreton or whether there is a short-lived intermediate in a different conformation which travels <100 Å through the medium to reach the membrane. Nevertheless, as 25 Å is the functional inner diameter of the IpaB/IpaC pore we find in RBCs, we propose that the IpaB/IpaC complex fits around the end of the putative canal within the needle. This linkage is likely to be dynamic in time and/or fragile because otherwise osmoprotection would be difficult to rationalize and bacteria would copurify with floated RBC membranes after hemolysis. The 25 Å IpaB/IpaC pore would then let the other Ipa proteins destined to be translocated through in the semi-folded state generated by Spa47-powered extrusion from a similarly seized secreton canal. | Study | biomedical | en | 0.999997 |
10562274 | Strain YHS2, which expresses red-shifted GFP (F64L, T65C, and I167T) fused to the Cox4p presequence (residues 1–21) under the ADH1 promoter ( ADH1-COX4-GFP ), was constructed as follows. First, pHS1 was constructed by replacing wild-type GFP in pOK29, a HIS3-CEN plasmid which carries ADH1-COX4-GFP (Kerscher, O., unpublished information), with a NcoI-BamHI fragment carrying red-shifted GFP from pQBI25 (Quantum Biotechnologies). The EcoRV-BamHI fragment from pHS1 was inserted into pDH9, which carries 5′ and 3′ untranslated regions of MFA2 (a gift from S. Michaelis), forming pHS2. To integrate ADH1-COX4-GFP at chromosomal MFA2 , a XhoI-SmaI fragment carrying 5′- MFA2-ADH1-COX4-GFP-3 ′- MFA2 from pHS2 was transformed into strain SM1235, which carries mfa2 :: URA3 . Strain YHS2, which contains MAT α mfa2 :: ADH1-COX4-GFP , was selected on 5-fluoro-orotic acid medium . MAT a mfa2 :: ADH1-COX4-GFP strain YHS1 was obtained by crossing YHS2 to SM1227 . Strain 1002 ( MAT a, his3, trp1, ura3, mfa2::ADH1-COX4-GFP ) was constructed by crossing YHS1 to BY4731 . YHS2 was mutagenized with 3% ethane methylsulfonate to ∼30% survival . Mutagenized cells were suspended at ∼9 × 10 5 cells/ml on coverslips and observed using an inverted microscope and the HIQ GFP 41014 filter set (Chroma). Mutants were isolated using micropipettes (10 μm diameter; World Precision Instruments), transferred to a drop of SD on the same coverslip, and then to YPD plates. Micropipettes were handled by an Eppendorf micromanipulator 5171. Crosses to wild-type strain 1002 showed that all class I, II, and III mutations were recessive and caused by a defect in a single gene. Complementation tests revealed that all eight recessive class IV mutants were defective in the same gene. Crosses between class IV mutants and TRP1 strain 194 (a gift from E. Schweizer) or dnm1Δ strain JSY1361 showed the class IV mutation was centromere linked, located on chromosome XII, and allelic to dnm1 . DNM1 -containing plasmid, pRU1-DNM1 , rescued the mitochondrial phenotype of all recessive class IV mutants. All dominant and semidominant class IV mutations also segregated as alleles of dnm1 . Complete disruptions of the DNM1 and FZO1 were constructed by PCR-mediated gene replacement as described into strains BY4733 and BY4744 . For dnm1Δ , we used HIS3 plasmid pRS303 and for fzo1Δ we used kanMX4 plasmid pRS400 . MATa dnm1Δ fzo1Δ strain YHS27 and MATα dnm1Δ fzo1Δ strain YHS23 were constructed by crossing MATa dnm1Δ strain YHS19 to MATα fzo1Δ strain YHS22. Mitochondria in the disruption strains were visualized using pHS12, a CEN-LEU2 plasmid containing ADH1-COX4-GFP . pHS12 was created by inserting the XhoI-NotI fragment from pHS1 into pRS315 . The DNM1 gene with a NotI site immediately preceding its termination codon was PCR amplified from yeast genomic DNA and subcloned into pAA3, a CEN-LEU2 plasmid which contains the HA epitope with a NotI site at its NH 2 terminus (Aiken, A., unpublished data), forming pDNM1-HA (pHS14). DNM1-GFP plasmid pHS20 was constructed as described above except that pAA1, a CEN-LEU2 plasmid which contains GFP with a NotI site at its NH 2 terminus (Aiken, A., unpublished data), was used instead of pAA3. To form pHS15, DNM1-HA coding sequences were PCR amplified from pHS14 with 50 bp of flanking sequences homologous to the GAL1-URA3 promoter in pRS314GU . The DNM1-HA fragment and linearized pRS314GU were cotransformed into yeast and pGAL1-DNM1-HA (pHS15) was formed by homologous recombination . pGAL1-DNM1-GFP (pHS40) was constructed as described for pHS15 except that pHS20 was used instead of pHS14. dnm1Δ fzo1Δ cells carrying pGAL1-DNM1-GFP were incubated in galactose media for 1–2 h, stained with MitoTracker Red CMXRos (Molecular Probes). 12 cells were examined by fluorescence microscopy and the mitochondrially associated Dnm1p-GFP dots (82 total) were assigned to one of two locations: (a) the end of a tubule (50 dots), or (b) the side of a tubule (32 dots). The end of a tubule was defined as when the center of a Dnm1p-GFP dot was located within 0.15 μm from the end. The average length of the mitochondrial tubules was estimated to be 2.7 ± 1.9 μm and the diameter ∼0.3 μm ( n = 44). We calculate that the side of the tubule represents 89% of the mitochondrial surface area and the remainder (11%) represents the end. We screened for yeast mutants defective in mitochondrial shape using a novel strategy in which mitochondria are visualized by the green fluorescent protein (GFP) and mutants were isolated by micromanipulation. GFP was fused to the presequence (residues 1–21) of mitochondrial cytochrome oxidase subunit IV . When expressed in yeast, COX4-GFP targets the mitochondrial matrix, and mitochondria were visible by fluorescence microscopy. We integrated the COX4-GFP gene at the nonessential MFA2 locus , which made fluorescence intensity uniform among cells and enabled efficient screening. After mutagenesis, individual cells with abnormal mitochondrial shape were hand-isolated using micropipettes . This screening procedure allowed us to isolate individual mutant cells with interesting mitochondrial phenotypes from a large total population of cells. Of ∼72,000 cells screened, we isolated 20 mutants, which were classified into four categories . Class I mutants (two isolates) contained one or two large, spherical mitochondria instead of the normal tubules seen in wild-type cells. Genetic crosses showed that both carried mdm10 mutants . The single class II mutant contained one or two oblong mitochondria collapsed to one side of the cell and was found to be defective in SLM1 . slm1 was previously identified as an mmm1 synthetic-lethal mutant . Class III mutants (three isolates) contained numerous mitochondrial fragments and were shown to carry fzo1 mutations. FZO1 encodes a GTPase anchored in the mitochondrial outer membrane that is required for mitochondrial fusion . Class IV mutants (14 isolates) exhibited a novel phenotype consisting of an interconnected network of mitochondrial tubules. In contrast to wild-type, which have 5–10 separate mitochondria per cell, class IV mutants appear to contain a single organelle. Because of their unique networked mitochondrial shape, these mutants were examined further. Genetic crosses showed that our 14 class IV mutants comprised 8 recessive, 5 dominant and 1 semi-dominant mutations. Mapping studies showed that all 14 mutations were centromere linked (1.1 cM) and located on chromosome XII. We noted that DNM1 , a gene related to dynamin GTPase , maps to chromosome XII near the centromere and is required for mitochondrial shape . Using a dnm1Δ strain and a plasmid containing DNM1 (kindly provided by J. Shaw), we found that all 14 class IV mutants carried dnm1 alleles. These results were unexpected since mitochondrial shape in our mutants was strikingly different from previously seen in dnm1 mutants, where mitochondria collapse to one side of the cell and form a single tubule . A complete disruption of DNM1 coding sequences was constructed, and examined for mitochondrial shape . ∼90% of dnm1Δ cells showed a single highly branched mitochondrial network. ∼10% of dnm1Δ cells displayed a single mitochondrial tubule localized to one side of the cell, similar to that seen earlier . The mitochondrial shape was not dramatically altered by growth conditions (not shown). Mitochondria in dnm1 mutants were efficiently segregated during cell division . Small daughter buds often contained a single mitochondrial tubule without branches, while larger buds had small networks. Most mitochondria were continuous from mother cells to buds, and separate mitochondria were only seen after the two cells separated. Our results strongly suggest that dnm1 mutants are defective in mitochondrial division. We speculate that mitochondria in dnm1 mutants may be divided indirectly, perhaps by cytokinesis. The yeast cell division machinery is clearly robust enough for the job, since nuclei are efficiently severed by cytokinesis in S . pombe cut mutants . In yeast, mitochondria are very dynamic, fusing or dividing on average every two minutes . Thus there appears to be an equilibrium between fusion and division. Supporting this idea, when FZO1 , a gene required for mitochondrial fusion, is defective, mitochondria fragment due to continued fission of the organelle . We hypothesized that if mitochondrial division were blocked, cells would have fewer (larger) organelles. Our working model, based on the morphology of dnm1 mutants, is that Dnm1p is required for mitochondrial division. To test this hypothesis, we constructed double mutants containing both dnm1Δ and fzo1Δ by genetic crosses. We found that normal mitochondrial shape was restored . ∼85% of double mutants contained multiply-branched, tubular mitochondria very similar to those seen in wild-type cells ( Table ). This was in marked contrast to dnm1Δ mutants, which usually had a single organelle, and fzo1Δ mutants, with numerous mitochondrial fragments . Mitochondria in dnm1Δ fzo1Δ cells were not always completely normal; the tubules tended to be longer and more curved than in wild-type cells, and occasionally formed bundles. Nonetheless, our observations suggest that excess mitochondrial division in fzo1Δ cells is suppressed by inactivating DNM1 , and that excess mitochondrial fusion in dnm1Δ cells is rescued by fzo1Δ . We propose that division, which requires Dnm1p, and fusion, controlled by Fzo1p, have antagonistic effects on mitochondrial shape and number. Our results also suggest that mitochondrial tubule formation occurs by a mechanism independent of fusion and division. Interestingly, mitochondrial shape and number in dnm1Δ fzo1Δ cells was dependent upon the order of gene disruption. When cells were first disrupted for FZO1 and subsequently for DNM1 ( Table , fzo1Δ → dnm1Δ ), ∼40% of cells carried mitochondrial fragments similar to those seen in fzo1Δ single mutants. In contrast, when cells were first disrupted for DNM1 and then for FZO1 ( Table , dnm1Δ → fzo1Δ ), ∼30% of cells displayed a mitochondrial network like that seen in dnm1 Δ cells. Our results indicate that the mitochondrial networks found in dnm1 Δ mutants persist in the absence of fusion activity, and fragments formed in the fzo1Δ mutant persist in the absence of fission activity. We also found tubular mitochondria in many of the double mutant cells formed by consecutive gene disruption (∼50% for fzo1Δ → dnm1Δ ; ∼60% for dnm1Δ → fzo1Δ ). These results further indicate that mitochondrial tubules form in the absence of division and fusion. It is not clear why dnm1Δ fzo1Δ double mutants generated by crossing a dnm1 Δ cell to a fzo1Δ cell contained mostly (>80%) tubular mitochondria and essentially no mitochondrial networks or fragments ( Table ). During germination and growth of a dnm1Δ fzo1Δ spore, it is possible that cells are simultaneously depleted of Dnm1p and Fzo1p, leading to the formation of tubules, but not networks or fragments. To further test the role of Dnm1p in division, we induced Dnm1p expression in dnm1Δ fzo1Δ cells and observed its effect on mitochondria. Dnm1p was fused to the HA epitope (Dnm1p-HA) and expressed under the galactose-inducible GAL1 promoter . Our pGAL1-DNM1-HA rescued the dnm1 Δ phenotype on galactose medium (not shown). When dnm1Δ fzo1Δ cells containing pGAL1-DNM1-HA were grown in the absence of galactose, no Dnm1p-HA was detected and ∼70% of cells displayed the tubular mitochondria typical of dnm1Δ fzo1Δ mutants . Upon transfer to galactose medium, Dnm1p-HA levels gradually increased, while the level of hexokinase, a control protein, remained constant . Concomitant with the accumulation of Dnm1p-HA, mitochondrial shape changed dramatically . The number of cells with tubular mitochondria decreased, and those with fragmented mitochondria increased. By 5 h, ∼65% of the cells contained completely fragmented mitochondria. At intermediate times (2 h) after inducing Dnm1p-HA, cells contained partially fragmented tubules, and many mitochondrial tubules were adjacent to small fragments. Our results clearly show that the division of mitochondria in dnm1Δ fzo1Δ cells coincides with the expression of Dnm1p. Further supporting a role for Dnm1p in mitochondrial fission, we found that the Dnm1 protein was preferentially localized to sites of mitochondrial division. We constructed a fusion between Dnm1p and the green fluorescent protein (GFP). Consistent with previous results , we found that much of Dnm1p-GFP was associated with mitochondria in punctate structures . In cells that were constitutively expressing Dnm1p-GFP, it was difficult to determine the precise location of Dnm1p because of the complex morphology of the mitochondria and the large number of Dnm1p-GFP dots. To simplify our analyses, we induced the expression of Dnm1p-GFP in the dnm1Δ fzo1Δ mutant and examined cells at early times after induction. We found a tight correlation between the appearance of Dnm1p-GFP and fragmentation of mitochondria. Two representative cells are shown in Fig. 4B and Fig. C ; both cells contained two Dnm1p-GFP dots, one of which was located at the end of a tubule, the other appeared to be reside near a constricted region of the mitochondrion. After analysis of additional cells, we found that Dnm1p-GFP was localized to ends of mitochondrial fragments much more frequently (>60%) than predicted if Dnm1p-GFP was randomly distributed on mitochondria (∼11%). These results suggest that Dnm1p acts at the site of mitochondrial fission. We note that Dnm1p-GFP is not exclusively found at the ends of mitochondria. We surmise that Dnm1p on the sides of the tubules may mark future sites of division, or represent Dnm1p-containing complexes that have diffused away from the end of the tubule. More definitive experiments (e.g., time-lapse videomicroscopy) to determine the role of Dnm1p in fission are in progress. The relatively normal mitochondria seen in dnm1Δ fzo1Δ mutants could be explained by a restoration of fusion activity; for example, if Dnm1p were an inhibitor of mitochondrial fusion. To test this possibility, we monitored mitochondrial fusion during mating . Mitochondria were visualized in one parent, by the galactose-induced expression of a matrix-targeted GFP (CS1-GFP) on plasmid pCLbGFP . MAT a cells containing pCLbGFP were pregrown in galactose medium to induce CS1-GFP expression, then transferred to glucose to inhibit further synthesis. MAT a cells were then mixed with MAT α cells, which did not carry pCLbGFP, and allowed to mate on glucose medium. Mitochondria were visualized in zygotes using MitoTracker. If mitochondrial fusion occurred, GFP and MitoTracker fluorescence would completely overlap, because the matrix-localized CS1-GFP from the MAT a mitochondria diffused into the mitochondrial matrix of the MAT α cell. If no fusion occurred, GFP-labeled mitochondria would be seen in only one half of the zygote. Zygotes formed by two wild-type cells, or two dnm1Δ mutants, exhibited efficient mitochondrial fusion, with GFP fluorescence and MitoTracker overlapping in all mitochondria . In contrast, fusion was defective in fzo1Δ mutants, consistent with previous observations . Mitochondrial fragments tended to aggregate in fzo1Δ cells and individual fragments were difficult to distinguish. Nonetheless, in matings between two fzo1Δ cells, MitoTracker showed clusters of fragmented mitochondria in the zygote and diploid bud, but we detected GFP fluorescence in only half of the mitochondrial clusters. Like fzo1Δ mutants, dnm1Δ fzo1Δ double mutants failed to fuse their mitochondria. Although mitochondria in dnm1Δ fzo1Δ / dnm1Δ fzo1Δ diploid cells had normal shape, only half of the organelles contained GFP. Our results indicate that dnm1Δ fzo1Δ cells are defective in mitochondrial fusion. To eliminate the possibility that low, basal levels of fusion occur in dnm1Δ fzo1Δ cells, we used a more sensitive fusion assay using the matrix markers, CS1-GFP and mitochondrial DNA . 4′,6-diamidino-2-phenylindole (DAPI) stained mtDNA was a more stable probe compared with MitoTracker, allowing us to examine cells for longer times following the initial mating event. MAT a cells, which lacked mtDNA and carried pCLbGFP were mated to MAT α cells, which contained mtDNA, but not the plasmid . The DAPI and GFP fluorescence overlapped in all the mitochondrial tubules in wild-type zygotes (52 zygotes examined). When 500 dnm1Δ fzo1Δ zygotes were examined, we found no overlap between DAPI and GFP. The fusion activity in dnm1Δ fzo1Δ mutants is therefore at least 500-fold less than that in wild-type cells. Even after the zygotes were allowed to grow and divide, we found no fusion in the mutant cells . When 100 dnm1Δ fzo1Δ / dnm1Δ fzo1Δ diploid cells were examined 24 h after mating, none contained an overlap between GFP and DAPI, whereas a complete GFP and DAPI overlap was seen in 43 wild-type diploids. dnm1Δ fzo1Δ cells clearly lack significant mitochondrial fusion activity. Our results above also suggest that dnm1Δ fzo1Δ cells lack fission activity. We therefore propose that in cells lacking Fzo1p and Dnm1p, mitochondrial tubule formation occurs by a mechanism independent of fusion and division, such as growth from the ends of preexisting organelles. Dynamin has been proposed to work as a mechanochemical enzyme that ‘pinches off’ plasma membrane invaginations, forming intracellular vesicles . Supporting this idea, dynamin self-assembles into spiral-like structures which can sever artificial membranes in vitro . It is also possible that dynamin plays a regulatory, instead of an enzymatic, function in membrane scission . Future studies are clearly needed to determine the precise mechanism that Dnm1p plays in mitochondrial division. | Study | biomedical | en | 0.999998 |
10562275 | Numbering of the human PTPα amino acid sequence is according to Krueger et al. 1990 . The pXJ41 vectors expressing PTPα, VSVG-tagged PTPα, fyn, and CD45 have been described . To construct pXJ41-contactin-neo, two contactin cDNA fragments comprising nucleotides 38–1103 and 177–3961 were obtained from pSP72-contactin/F11 and subcloned in two steps into pXJ41-neo to reconstitute the contactin coding sequence. To construct pXJ41-myr-PTPα-neo, the PCR-amplified region of human src cDNA encoding the NH 2 -terminal myristylated sequence MGSNKSKPKDASQ was cloned into pGEM-T (Promega Corp.), and the reverse orientation was selected with the multiple cloning NotI site at the 5′ end of the myristylation signal. A NotI-XhoI fragment was excised and cloned into pXJ41-neo, creating pXJ41-myr-neo. A PCR fragment of PTPα encoding the entire intracellular region (amino acids 148–774), and flanked with engineered SalI sites, was inserted in-frame into XhoI-cut pXJ41-myr-neo. To construct pXJ41-PTPα/CD45-neo encoding the chimeric RPTP, a region of the VSVG–PTPα cDNA encoding the signal peptide and amino acids 1–121 of PTPα was amplified by PCR using forward and reverse primers with engineered NotI and BglII sites, respectively. This NotI-BglII fragment was inserted into a pXJ41-neo intermediate vector. A CD45 cDNA fragment encoding 35 extracellular juxtamembrane amino acids, the transmembrane, and the entire intracellular regions was released from pXJ41-CD45-Hy with BglII and inserted into the intermediate vector. COS-1 cells were maintained and transiently transfected as described . The empty expression plasmid pXJ41-neo was used to normalize the amount of DNA in each transfection. After 24 h of coculture, the cells were harvested and processed as described below. For tunicamycin treatment, 20 μg/ml tunicamycin (Boehringer Mannheim) in DME/10% FCS was added to the cells 6 h after transfection, and the cells were harvested after a further 42 h of culture. COS-7 cells cultured on 1 ml polylysine or alcian blue–coated glass 12-mm-diam coverslips were processed for immunocytochemistry 24–48 h after transfection. Images were taken using a Zeiss Axiovert 100M equipped with a Hamamatsu C5810 3 color CCD cooled camera. Images were processed directly with Adobe Photoshop. Copatching of contactin or VSVG-PTPα was performed on COS-7 cells 24–48 h after transfection as described . Cells were incubated on ice with 10 μg anticontactin (4D1) or anti–VSVG antibodies. Bound antibodies were clustered by incubation with a 1:100 dilution of FITC- or RITC-labeled goat anti–mouse antibodies (Tago), followed by fixation with 4% paraformaldehyde and permeabilization (when appropriate) with 0.2% Triton X-100. Affinity-purified rabbit antibodies to contactin or PTPα (see below) were used complementary to the mAbs, together with goat anti–rabbit antibodies labeled with FITC or RITC (Tago). Six embryonic chick brains (15-d-old) were mechanically suspended in 30 ml of ice-cold PBS buffer, filtered through a nylon mesh, homogenized by 10 strokes in a glass Dounce homogenizer, and washed three times in PBS. Chick brain cells or transfected COS cells were lysed in 10 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Brij-96, 20 μg/ml aprotinin, and 2 mM PMSF, and the lysates were clarified by centrifugation. Antibodies toward VSVG (Sigma Chemical Co.), contactin (4D1) , CD45 or fyn (Transduction Laboratories), PTPα , and NCAM (Chemicon International, Inc.) were used for immunoprecipitation and/or immunoblotting. The phosphatase activity of 5 μl immunoprecipitate toward 2 μM phosphotyrosyl-RR-src peptide was measured at 30°C for 15 min as described . Contactin and PTPα immunoprecipitates from embryonic chick brain lysates were probed for the presence of contactin and PTPα. PTPα was present in anticontactin immunoprecipitates , and contactin was present in PTPα immunoprecipitates, but was not precipitated by preimmune serum . Thus, PTPα and contactin exist in a complex in brain lysates. To check the specificity of the association of PTPα and contactin, we examined the interaction of PTPα with NCAM, another fyn-associated cell adhesion molecule highly expressed in the brain. The 120- and 140-kD NCAM isoforms were detected in anti-NCAM immunoprecipitates from mouse brains, but PTPα was not present . Likewise, NCAM could not be detected in anti-PTPα immunoprecipitates prepared from these lysates using the same anti-PTPα antiserum as employed with the chick brain lysates (raised to the species conserved intracellular D1 region of PTPα). To investigate the molecular basis of the association between PTPα and contactin, we used a transient expression system where the interaction of different forms could be manipulated. PTPα and contactin coimmunoprecipitated with one another from COS cells coexpressing PTPα and contactin . In other experiments, the immunocomplexes were assayed for PTP activity . Anti-VSVG immunoprecipitates from cells expressing PTPα alone or with contactin contained comparable levels of phosphatase activity, whereas virtually no activity was detectable in anti-VSVG immunoprecipitates from cells expressing contactin alone. Anticontactin immunoprecipitates from coexpressing cells contained about a fivefold higher phosphatase activity than those from cells expressing contactin or PTPα alone. These results indicate that the contactin–PTPα complexes are functionally active. The levels of PTPα protein and phosphatase activity were much lower in anticontactin immunoprecipitates than in anti-VSVG immunoprecipitates from the coexpressing cells, likely because only a portion of the expressed PTPα associates with contactin. Similar experiments were carried out with contactin and CD45, a receptor-like PTP with structural similarity to PTPα. No coimmunoprecipitation of contactin and CD45 from coexpressing cells was detected , indicating that the interaction of PTPα and contactin is specific and not merely due to heterologous expression. In situ localization of contactin and PTPα in cotransfected COS cells revealed a similar distribution for both proteins within the plane of the plasma membrane . In contrast, control transfections of contactin together with the RPTP CD45, gave a completely different pattern of distribution to that of contactin (data not shown), indicating that they do not associate in the same cellular complexes. We examined whether an enforced redistribution of either contactin or PTPα, induced by incubating the live cells with antibodies to the extracellular domains of these molecules, would lead to coclustering of the respective partner. Clustering of PTPα caused contactin to redistribute to closely match the PTPα pattern . The close similarity of these two patterns supports the efficacy of clustering via the free-standing VSVG tag on the NH 2 -terminal of PTPα, leading to little or no interference with the interactions between PTPα and contactin. Coclusters of contactin and PTPα could also be induced by 4D1 mAb specific for contactin . Clusters of various sizes were induced in individual cells, with the PTPα localization largely matching the contactin pattern. To identify the region of PTPα involved in the interaction with contactin, we generated a membrane-associated intracellular form of PTPα by replacing its extracellular and transmembrane regions with the myristylation signal of src (myr-PTPα). Expressed myr-PTPα was associated with the membrane fraction (data not shown), however, contactin only associated with wild-type PTPα and not with myr-PTPα . Thus, contactin does not interact with the PTPα lacking the extracellular and transmembrane regions. Since CD45 does not associate with contactin, we created a PTPα/CD45 hybrid molecule where most of the extracellular region of CD45 was replaced with that of PTPα. PTPα or the PTPα-CD45 hybrid was coexpressed with contactin. Anti-VSVG–immunoprecipitated PTPα-CD45 hybrid and PTPα were complexed with contactin , demonstrating that contactin associates with the extracellular region of PTPα. Furthermore, the transmembrane region of PTPα is not specifically involved in the association with contactin since it can be replaced with that of CD45. The mature PTPα protein contains both N- and O-linked oligosaccharides . Chick contactin has nine potential sites for N-linked glycosylation . When COS cells coexpressing PTPα and contactin were cultured with tunicamycin, an inhibitor of N-linked glycosylation, faster migrating, less diffuse forms of PTPα and contactin were detected on SDS-PAGE , which is consistent with a loss of N-linked oligosaccharides. Nevertheless, contactin was present in anti-VSVG immunoprecipitates from cells treated with or without tunicamycin , indicating that the association of contactin and PTPα occurs independently of N-linked glycosylation of either protein. Two experiments were carried out to address the question of whether PTPα and contactin associate in a cis or trans conformation. First, anticontactin precipitates were prepared from lysates of COS cells expressing either PTPα or contactin, or from cells coexpressing both PTPα and contactin , as well as from another sample made by mixing lysates from the cells expressing either contactin or PTPα . Anticontactin immunoprecipitates prepared from coexpressing cells contained PTPα, but those from mixed cell lysates did not . The lack of detectable association of PTPα and contactin in the mixed lysates suggests that interaction cannot take place in a trans conformation. Still, this may require a particular presentation of these cell surface molecules in growing cells that cannot form in solubilized cell lysates. Therefore, contactin-expressing cells were trypsinized 24 h after transfection and replated in dishes containing PTPα-expressing cells (these were not trypsinized for replating because this resulted in a large decrease in PTPα expression). After 24 h of coculture, the cells were lysed and immunoprecipitates were prepared. As a positive control for contactin–PTPα association, PTPα- and contactin-cotransfected cells were cultured for 48 h, harvested, and processed the same way. PTPα and contactin coimmunoprecipitated from cotransfected cells , but not from cocultured cells . Thus, even when cells expressing contactin are cultured together with other cells expressing PTPα, no association in trans of these two receptor proteins occurs. We demonstrate that a receptor protein tyrosine phosphatase forms a membrane-spanning complex with a neuronal GPI-anchored receptor. PTPα and contactin associate with one another in a lateral (cis) forming complex mediated through the extracellular region of PTPα. This complex is sufficiently stable to permit its isolation from brain lysates or transfected cells through immunoprecipitation of either component, and to permit coclustering of PTPα with contactin upon antibody-induced clustering of contactin within cells and vice versa. Our findings that contactin–PTPα complexes form in cotransfected cells, but not upon coculture or in mixed lysates of PTPα-expressing cells and contactin-expressing cells, provides compelling evidence that PTPα and contactin can only associate within the same cell; thus, forming a receptor complex rather than a ligand–receptor pair. This is the first identification of an extracellular partner and potential regulator of PTPα. As contactin lacks any intracellular region and is tethered to the external face of the plasma membrane through a GPI linkage, PTPα could, thus, link an extracellular contactin-mediated signal to an intracellular response. Fyn is complexed with contactin and is transiently activated upon aggregation of contactin . Likewise, fyn is associated with PTPα and is dephosphorylated and activated by PTPα . The association of PTPα and contactin suggests that PTPα might act as an intermediary molecule in a tripartite complex of contactin, PTPα, and fyn, in accord with its proposed role as a transducer. Zisch et al. 1995 reported that antibody-mediated cross-linking of contactin results in the activation of associated fyn, raising the possibility that cross-linking of contactin–PTPα and contactin–fyn complexes brings PTPα into proximity with fyn, allowing the subsequent dephosphorylation and activation of fyn. In view of the constitutive activity of PTPα (and other RPTPs), such a contactin-regulated access of PTPα to its substrate provides an attractive mechanism for mediating RPTP activity and the action of GPI-anchored receptors as modulators in larger signaling complexes. Contactin interacts with multiple ligands and the identification of PTPα as a novel contactin-associated protein extends the number and the nature of possible contactin-containing receptor complexes. Contactin is found in regions of active neuronal migration or outgrowth in the developing brain and in areas of synaptic development and activity . The role of this mobile GPI-anchored receptor may well be to deliver the components of a phosphorylation-competent machinery to receptor complexes mediating neuronal motility or synaptic activity. Evidence for such a role has been obtained for the closely related GPI receptor axonin-1/TAG-1, where neurite fasciculation mediated by complexes of axonin-1 and NgCAM is accompanied by a rapid downregulation of fyn phosphorylation . An intriguing possibility arising from this study is that contactin acts as an adapter to bring together two RPTPs, namely RPTPζ/β and PTPα. The NH 2 -terminal carbonic anhydrase-like region of the transmembrane and secreted extracellular forms of glial cell RPTPζ/β associates in trans with contactin, and in doing so promotes neurite growth . If PTPα functions as a signaling component of a RPTPζ/β–bound contactin–PTPα complex, this would represent a novel mode of RPTP interaction and regulation. The proposed signaling through a contactin–PTPα complex represents a new paradigm of receptor-mediated tyrosine kinase activation. Receptors with intrinsic tyrosine kinase activity or directly associated with active nonreceptor tyrosine kinases have been well documented. Contactin, lacking the intracellular region required for either of these mechanisms, may utilize an associated RPTP, PTPα, to effect intracellular activation of tyrosine kinases. This is reminiscent of the recent finding that the GPI-anchored cell surface receptors GDNFR-α and NTNR-α form functional coreceptor complexes with the transmembrane tyrosine kinase Ret and underlines the concept that GPI-anchored receptor signaling is achieved by modulation of protein tyrosine phosphorylation. Our study unites previous progress in two areas of research: the interaction of extracellular ligands with the neural cell adhesion molecule contactin, and the intracellular signaling events mediated by PTPα. The components of a novel signal transduction pathway, thus, have been identified and can now be tested for function and physiological relevance to aspects of neuronal development. | Study | biomedical | en | 0.999998 |
10562276 | HeLa cells (human epitheloid carcinoma, cervix), Huh7 cells (human hepatoma), and WI-38 cells (human female lung diploid fibroblasts) were grown as monolayers in minimum essential medium supplemented with 2 mM l -glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin, and 10% fetal calf serum (GIBCO BRL). Primary cultures of Schwann cells were prepared as described by Brockes et al. 1979 . In brief, sciatic nerves were dissected from 3-d-old Sprague-Dawley rats, and mixed cultures of Schwann cells and endoneurial fibroblasts were maintained in minimum essential medium supplemented with 10% fetal bovine serum for 1 d. To reduce fibroblast contamination, the culture was then incubated for 2 d in the presence of 10 −5 M cytosine arabinose. On the fourth day, the cells were transferred to polylysine-coated coverslips and maintained for further 3 d in the presence of 10% fetal bovine serum, 2 μM forskolin, and 20 μg/ml bovine pituitary extract. For indirect immunofluorescence cells were grown on 10 × 10-mm glass coverslips. The cells were washed twice in PBS, fixed with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS for 10 min at room temperature, and subsequently permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Alternatively, cells were first permeabilized with 0.5% Triton X-100 in CSK buffer containing 0.1 mM PMSF for 1 min on ice, and subsequently fixed with 3.7% formaldehyde in CSK buffer, for 10 min at room temperature. After fixation and permeabilization, the cells were rinsed in PBS containing 0.05% Tween-20 (PBS-Tw), incubated for 30 min with primary antibodies diluted in PBS, washed in PBS-Tw, and incubated for 30 min with the appropriate secondary antibodies conjugated to fluorescein (FITC), Texas red, or indodicarbocyanine (Cy5) (Jackson ImmunoResearch Laboratories, Inc.). Finally, the coverslips were mounted in VectaShield (Vector Laboratories) and sealed with nail polish. For immunoelectron microscopy, cells were fixed with 3.7% formaldehyde in PBS, ∼20 min. First, the medium was washed twice with PBS and the fixative was added to the culture dishes for 10 min; then, the cells were scraped using a rubber policeman and centrifuged at 7,000 g for 10 min. The cell pellets were sequentially dehydrated with continuous low-speed stirring, in 30% methanol at 4°C for 5 min, 50% methanol at 4°C for 5 min, 75% methanol at −20°C for 5 min, and 90% methanol at −20°C for 30 min. This was followed by mixtures of 90% methanol/Lowicryl K4M (1:1 and 1:2 vol/vol, for 1 h each at −20°C). Finally, the cell pellets were embedded in Lowicryl K4M for 24 h at −20°C and transferred to gelatine capsules. Polymerization was induced by UV irradiation for 5 d at −20°C, followed by one more day at room temperature. Ultrathin sections on gold grids were sequentially incubated with 0.1 M glycine in PBS (15 min), 5% BSA in PBS (15 min), and finally the primary antibody was diluted in 1% BSA, 0.1 M glycine in PBS (1 h at room temperature). After washing, the sections were incubated with appropriate secondary antibody conjugated to either 5 or 10 nm gold particles diluted 1:25 in 0.1% BSA in PBS (45 min at room temperature). Finally, the sections were washed and stained with 10% aqueous uranyl acetate. As controls, sections were treated as described, but omitting the primary antibody. Male rats of the Sprague-Dawley strain were kept on a 12-h day/night lighting regime with lights out at 8:00. All animals were killed by overdose of pentobarbital. For immunofluorescence, the rats were perfused through the ascending aorta with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS, pH 7.4, for 15 min at room temperature. Supraoptic nuclei were dissected out of 500-μm-thick hypothalamic slices and trigeminal ganglia were removed and cut into small fragments. These tissue fragments were washed in PBS for 1 h and individually transferred to a drop of PBS on a siliconized slide. Then, a coverslip was applied on top of the slide and the tissue was squashed by percussion with a histologic needle. The preparation was then frozen in dry ice and the coverslip was removed using a razor blade. The slides with adhered neurons were sequentially dehydrated in 96% and 70% ethanol at 4°C for 10 min and rinsed in PBS. Before immunostaining, the samples were sequentially incubated with 0.5% Triton X-100 in PBS for 10 min, 0.1 M glycine in PBS for 30 min, and 0.01% Tween 20 in PBS for 5 min. For immunoelectron microscopy, animals were perfused with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.12 M phosphate buffer, pH 7.4. The hypothalamic supraoptic nucleus was dissected out from brain slices and left in the same fixative for 3 h at 4°C, dehydrated in increasing concentrations of methanol at −20°C, and embedded in Lowicryl K4M at −20°C. Ultrathin sections were immunostained as described above. Confocal microscopy was performed with either a Zeiss laser scanning microscope LSM 410 or a BioRad MRC-1024, using excitation wavelengths of 488 nm (for FITC), 543 nm (for Texas red), and 633 nm (for Cy5). Each channel was recorded independently and pseudocolor images were generated and superimposed. Ultrathin sections were examined with a JEOL CXII electron microscope operated at 80 Kv. For quantitative analysis, images were obtained from untreated and leptomycin B–treated cells, and double labeled with probes for U2 snRNA (green staining) and coilin (red staining). Each image was decomposed in RGB (y R = red, y G = green) and segmented according to Fwu and Djuriç 1996 into four classes: C 0 = extracellular background, C 1 = intracellular background (i.e., cytoplasm and nucleoli), C 2 = nucleoplasm, C 3 = Cajal bodies. The average intensities for y G ∈ C 3 (m 2 ) and y G ∈ C 2 (m 1 ) were estimated. Finally, the ratio m 2 /m 1 was determined. Immunofluorescence microscopy using anti–SMN antibodies reveals bright intranuclear foci and additional diffuse cytoplasmic labeling , as previously reported by Liu and Dreyfuss 1996 . Labeling of the cytoplasm is more evident when cells are first fixed with formaldehyde, and then permeabilized with Triton X-100, whereas the intranuclear foci are equally stained in cells treated with detergent either before or after fixation. Double-labeling experiments using anti–SMN and anticoilin antibodies show that in some HeLa cells the SMN protein is present in dot-like structures closely related but distinct from Cajal (coiled) bodies , the so-called gemini of coiled bodies or gems . Gems are also often detected independent from Cajal bodies . Most important, gems are only visible in a small proportion of HeLa cells (1–10%). In the vast majority of cells analyzed, SMN colocalizes precisely with coilin . Similar results were observed in both HeLa and Huh7 cells (human cervical carcinoma and hepatoma derived epithelial cell lines, respectively) using either anti–SMN monoclonal antibody 2B1 , or serum 95020 . Triple-labeling experiments using anti–SMN, anti–coilin, and anti–Sm antibodies demonstrate that snRNPs colocalize with SMN in Cajal bodies (arrows), but not in gems. Since colocalization studies based on computer-superimposed images of fluorochrome-coupled antibody labeling are constrained by the resolution limits of light microscopy, immunoelectron microscopy was also performed. Cells were fixed in formaldehyde, embedded in Lowicryl K4M at −20°C, and ultrathin sections were incubated with anti–SMN and anti–coilin antibodies. Fig. 2 depicts thin sections through the nucleus of Huh7 cells. In Fig. 2 A, the section was incubated with anticoilin antibody and appropriate secondary antibody conjugated to 10-nm gold particles. The gold particles decorate the characteristic entangled threads of a Cajal body. A similar labeling pattern (i.e., concentration of gold particles over a Cajal body) is observed in B, which depicts a thin section incubated with anti–SMN antibody. Fig. 2C and Fig. D , shows Cajal bodies simultaneously labeled by anticoilin and anti–SMN antibodies (detected with 5- and 10-nm gold particles, respectively). This demonstrates that SMN is present in the Cajal body of cultured cell lines. We next compared the localization of SMN and coilin in primary tissue cells . The study was focused on neurons from either the hypothalamic supraoptic nucleus or trigeminal ganglia, because neuronal cells contain prominent Cajal bodies . A characteristic feature of neuronal cells is that, in addition to large Cajal bodies , they contain coilin in numerous small cap-like structures that form a ring around the nucleolus . Analysis of the subnuclear distribution of SMN and coilin in >1,000 neurons revealed that SMN localizes precisely in the Cajal body , but it is not detected in the perinucleolar structures . Double-labeling experiments using anticoilin antibodies and monoclonal antibody 4G3 directed against the U2 snRNP protein B″ confirmed that snRNPs are highly enriched in the Cajal bodies , but not in the perinucleolar rings . Thus, SMN and snRNPs colocalize specifically in the Cajal body. Staining of primary Schwann cells kept in culture further revealed localization of SMN in the Cajal body . However, when these cells were stimulated to proliferate by addition of forskolin and pituitary factors , ∼10–15% of the nuclei contained SMN in gems . In conclusion, these results show that SMN may either be present in discrete focal structures that are not enriched in snRNPs (i.e., gems) or colocalize with snRNPs in the Cajal body. The association of SMN with Cajal bodies is observed in both cultured cell lines and primary neurons, whereas gems are only detected in a small proportion (<15%) of rapidly proliferating cells in culture. Although gems tend to be more frequently detected during the G1 stage of the cell cycle (T. Carvalho, unpublished data), the overall proportion of nuclei containing gems is variable depending on cell type and passage number. In agreement with data from earlier studies , remnants of Cajal bodies are visible in the cytoplasm of mitotic HeLa cells . We have previously reported that these mitotic Cajal bodies are labeled with antibodies directed against both snRNP proteins and the m3G cap, and by snRNA-specific antisense probes, indicating that they contain assembled snRNP particles . Here we show that these structures are additionally labeled by anti–SMN antibodies , indicating that the association of SMN and snRNPs with Cajal bodies persists during mitosis. As cells progress from telophase to G1, Cajal bodies reappear in the nucleus , but SMN remains exclusively in the cytoplasm . The presence of SMN in Cajal bodies is detected later in G1, indicating that there is a lag period between assembly of the Cajal body and localization of SMN therein. This suggests that SMN is targeted to preformed Cajal bodies. To further investigate whether the Cajal body plays a role in the intranuclear localization of SMN, microinjection experiments were performed with antibodies that promote a specific disappearance of the Cajal body . HeLa cells were microinjected in the nucleus with purified anticoilin monoclonal antibodies and allowed to incubate for 24 h. The cells were then briefly permeabilized with Triton X-100, fixed with formaldehyde, and sequentially incubated with fluorescein-conjugated anti–mouse IgG (to detect the injected antibody), rabbit polyclonal anti–SMN antibody 95020, and Texas red–conjugated anti–rabbit IgG. As depicted in Fig. 5 A, cells injected with the anticoilin monoclonal antibody 1D4-delta are devoid of Cajal bodies. Staining of these cells with anti–SMN antibodies fails to reveal any intranuclear foci, in clear contrast with the bright staining of Cajal bodies observed in the nucleus of noninjected cells . As expected, Cajal bodies are brightly stained by anti–SMN antibody in the nucleus of cells microinjected with monoclonal antibody 5P10-pi , which does not interfere with the Cajal body structure . To further address the role of the Cajal body in the localization of SMN, primary fibroblasts were also examined because this type of cell is normally devoid of Cajal bodies . As shown in Fig. 5 , several fibroblasts contain SMN localized in nuclear foci (gems), despite the absence of Cajal bodies . As observed in HeLa cells, gems in fibroblasts are not labeled by anti–Sm antibodies . Taken together, these results suggest that Cajal bodies provide a structural framework for the colocalization of SMN and snRNPs in the nucleus. Next, we examined the distribution of SIP1, a novel protein that forms a complex with SMN . Immunolocalization studies were performed using anti–SIP1 monoclonal antibody 2E17 , anti–Sm, and anticoilin antibodies. As depicted in Fig. 6 , both primary neurons and HeLa cells show a precise colocalization of SIP1 and snRNPs in the Cajal body. The presence of SIP1 in Cajal bodies was further confirmed at the electron microscopical level (data not shown). Thus, Cajal bodies represent unique intranuclear sites where snRNPs colocalize with SMN and SIP1. To investigate whether snRNPs accumulated in the Cajal body are “in transit” from the cytoplasm, we treated HeLa cells with leptomycin B, a drug that inhibits U snRNA export to the cytoplasm and, consequently, reimport of assembled snRNPs to the nucleus. HeLa cells were incubated with 10, 20, or 30 nM leptomycin B for 1–10 h. Although these drug concentrations produced similar effects, at the lower doses the time of response was longer and more variable. We therefore decided to work with a concentration of 30 nM. For snRNP detection, the cells were labeled with antibodies directed against Sm proteins , the U2 snRNP-specific B″ protein , and the m3G cap of snRNAs ; in addition, in situ hybridization was performed with a U2 snRNA-specific antisense probe . In nontreated cells, snRNPs are highly enriched in Cajal bodies , whereas after 3 h of drug treatment, snRNPs are no longer concentrated in the Cajal body . In fact, the U2 snRNP is normally approximately threefold more concentrated in Cajal bodies than in the nucleoplasm, but after leptomycin B treatment the average intensity of U2 snRNP labeling in the Cajal body is similar to that in the nucleoplasm . In addition to depleting snRNPs from the Cajal body, leptomycin B causes a decrease in the number of Cajal bodies per nucleus and a redistribution of coilin into the nucleolus . Since Cajal bodies are very dynamic structures with a high turnover rate , leptomycin B could either stimulate the disassembly of preexisting Cajal bodies, or inhibit the assembly of new Cajal bodies. To specifically investigate the effect of leptomycin B on the assembly of new Cajal bodies, we examined the distribution of coilin in cells that have completed mitosis in the presence of the drug. In nontreated samples, Cajal bodies are visible in >80% of early G1 cells . In contrast, the vast majority of early G1 cells incubated with leptomycin B are devoid of Cajal bodies . Instead of Cajal bodies, some of these cells contain perinucleolar patches of coilin reminiscent of what is observed in neurons under physiological conditions . As coilin appears to shuttle from the nucleolus to the Cajal body , these results suggest that leptomycin B interferes with the efflux of coilin from the nucleolus. As a possible consequence, coilin would accumulate within the nucleolus , becoming unavailable for assembly of new Cajal bodies. Due to their high turnover rate , this would lead to a progressive disappearance of Cajal bodies from the nucleoplasm. Staining of leptomycin-treated cells with the 2B1 and 2E17 monoclonal antibodies reveals a progressive increase in the proportion of cells containing gems , most probably due to an effect on the normal trafficking dynamics of SMN-SIP1 proteins in the nucleus. However, both SMN and SIP1 are still highly enriched in Cajal bodies , suggesting that SMN-SIP1 complexes remain associated with Cajal bodies in the absence of newly imported snRNPs from the cytoplasm. This implies that if SMN, SIP1, and snRNPs are part of a common complex in the Cajal body, there must be a mechanism that triggers the release of snRNPs from this structure. This work reports two novel protein components of the Cajal (coiled) body, the Survival Motor Neuron gene product and its associated protein, SIP1. As SMN and SIP1 play an important role in snRNP biogenesis and recycling , this finding provides a direct link between the Cajal body and snRNP metabolism. The SMN and SIP1 proteins were previously immunolocalized in HeLa cells to discrete nuclear foci termed gemini of coiled bodies or gems . The data presented here indicate that gems are observed in a variable (but small) proportion of cells in culture. In the vast majority of cells studied, including primary neurons directly removed from organisms, SMN and SIP1 are present exclusively in the Cajal body and gems were never identified. Notably, the presence of SMN in Cajal bodies was also detected by other groups . A major difference between Cajal bodies and gems concerns the presence or absence of snRNPs. The SMN and SIP1 proteins colocalize with snRNPs in the Cajal body, whereas gems do not concentrate snRNPs. This makes it likely that Cajal bodies contain the SMN-SIP1–snRNP complexes detected in the nuclear fraction of somatic cells . As SMN and SIP1 associate with snRNPs in the cytoplasm, one possibility is that the SMN-SIP1–snRNP complex enters the nucleus and is targeted to the Cajal body. The Cajal body may then represent an assembly platform where snRNPs in transit from the cytoplasm accumulate transiently, until an as yet unidentified stage of their maturation process is completed. Taking into account that 2′- O -methylation and pseudouridylation of U2 snRNA take place in the nucleus and are required for the final assembly of snRNP-specific proteins , it will be important to clarify whether snRNP complexes enriched in the Cajal body contain modified snRNAs. In this regard, it is important to note that the U2-specific proteins collectively termed SF3a/SF3b , which assemble into the splicing-competent U2 snRNP 17S particle, are not detected in Cajal bodies (D. Nesic and A. Krämer, personal communication). This observation further supports the view that snRNPs concentrated in the Cajal body are not yet fully mature. Assuming that both snRNPs and SMN complexes are constantly in motion throughout the cell and accumulate transiently in either Cajal bodies or gems, differences in their trafficking dynamics could account for the differences observed in their steady state nuclear distribution. In fact, expression of a dominant-negative amino-terminal deletion mutant of SMN (SMNΔN27), which is thought to block nuclear snRNP recycling, causes an accumulation of SMN in extremely enlarged Cajal bodies . Moreover, SMN contains a self-oligomerization domain that overlaps with the Sm-interacting domain . This implies that SMN may either form a complex with snRNPs (most probably localized in the Cajal body) or self-oligomerize, giving rise to gems. A prediction from this model is that gems should become more prominent after depletion of snRNPs from the Cajal body, as indeed was observed after treatment of cells with leptomycin B. The observed depletion of snRNPs from Cajal bodies caused by leptomycin B strongly suggests that spliceosomal snRNPs newly imported from the cytoplasm travel through the Cajal body. Leptomycin B is a cytotoxin that interacts directly with the nuclear export receptor CRM1, inhibiting its binding to nuclear export signals and Ran-GTP . As a consequence, this drug blocks the export of substrates recognized by CRM1, including Rev and proteins containing Rev-like leucine-rich nuclear export signals . Although CRM1 mutations in yeast interfere with mRNA export , to date leptomycin B has been found to inhibit export of only one class of RNA in vertebrates, the U snRNAs . As shown in Fig. 7 and Fig. 8 , Fig. 3 h after drug treatment, Cajal bodies are still present but are no longer brightly stained by antibodies directed against Sm proteins, the U2 snRNP-specific B″ protein, and the m3G cap of snRNAs, or by an antisense probe specific for the U2 snRNA. Thus, after exposure to leptomycin B the Cajal body fails to concentrate snRNPs. Since leptomycin B inhibits nuclear export of U snRNAs, the assembly of new snRNP particles in the cytoplasm and their subsequent transport to the nucleus is also blocked. The observed redistribution of snRNPs in the nucleus can therefore be explained if mature snRNPs present in the Cajal body leave the structure while new particles are prevented from reaching it. Direct visual evidence that snRNPs flow through the Cajal body has been recently obtained by microinjection of fluorescently tagged U snRNAs into the cytoplasm of Xenopus oocytes and expression of green fluorescent protein–tagged Sm proteins in HeLa cells . These studies revealed that both snRNAs and snRNP proteins are first detected in Cajal bodies before they start to accumulate at other nuclear locations. In addition to interfering with the concentration of snRNPs in the Cajal body, treatment of cells with leptomycin B also affects the distribution of coilin in the nucleus. Shortly after exposure to the drug, coilin is detected inside the nucleolus while the number of Cajal bodies decreases. Within the nucleolus, coilin colocalizes with fibrillarin in foci that correspond to the dense fibrillar component of the nucleolus. A similar staining pattern was previously observed in HeLa cells labeled with an anticoilin monoclonal antibody (mAb-φ) . Remarkably, this antibody reacts with an epitope adjacent to a critical serine residue (serine 202) that, when mutated to aspartate, induces the formation of Cajal body-like structures inside the nucleolus . The presence of Cajal bodies inside the nucleolus has also been reported in certain rare cases such as breast carcinoma cells, brown adipocytes, and hepatocytes of hibernating dormice . Much more frequently, Cajal bodies are located in the vicinity of nucleoli, even physically attached to them, seemingly emerging from the dense fibrillar component . Supporting this morphological evidence, several molecules are shared by both the Cajal body and the dense fibrillar component of the nucleolus, namely, fibrillarin , NAP57 and Nopp140 . In particular, Nopp140 interacts with coilin and has been proposed to chaperone the transport of molecules from the nucleolus to the Cajal body . It therefore appears that leptomycin B inhibits the flow of molecules between the nucleolus and the Cajal body, causing a retention of coilin in the dense fibrillar component and, consequently, a decrease in the number of Cajal bodies. In summary, the major conclusions from this study are, first, that two proteins essential for snRNP biogenesis, SMN and SIP1, are localized in the Cajal body, and, second, that the nuclear export inhibitor leptomycin B causes a depletion of snRNPs from the Cajal body. The first observation provides a link between Cajal bodies and snRNP biogenesis, whereas the second indicates that the accumulation of snRNPs in the Cajal body is transient. Taken together, these results suggest that snRNPs flow through the Cajal body along their biogenesis pathway. | Study | biomedical | en | 0.999996 |
10562277 | Bacterial media was prepared by standard protocols . Yeast strains were maintained on rich media (YPD) containing 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose, or on synthetic complete media (SC) containing 0.67% yeast nitrogen base without amino acids, 2% glucose, and the appropriate supplements . SC media lacking histidine, leucine, and tryptophan used in the two-hybrid assay contained 2.5 mM 3-aminotriazole. Diploid strains were sporulated at room temperature in liquid media consisting of 1% potassium acetate and 0.02% glucose. Escherichia coli transformations were performed by the method of Hanahan 1983 and yeast transformations were performed by the method of Elble 1992 , except for the yeast genomic library transformation, which was by the method of Schiestl and Gietz 1989 . Plasmids used in this work are described in Table . Plasmid construction was as follows. To generate pSV22, the genomic library plasmid pB4 was digested with PvuII and HindIII, and the resulting 2.7-kb fragment containing YER157w/ SEC34 was ligated into pRS416 that had been digested with SmaI and HindIII. To create pSV24 and pSV25, SEC34 was liberated from the polylinker of pSV22 with either HindIII and BamHI or XhoI and SpeI double-digests and ligated into pRS305 (digested with HindIII and BamHI) or pRS426 (digested with XhoI and SpeI), respectively. The insert for bacterial expression plasmids encoding glutathione S-transferase (GST)-Sec34p and His 6 -Sec34p fusion proteins was generated by PCR including a BamHI site adjacent to the codon for the first amino acid of Sec34p and a SmaI site downstream of the stop codon (5′ primer, 5′ gcc-gga-tcc-atg-gcg-aga-agt-aga-aag 3′; 3′ primer, 5′ tcc-ccc-ggg-gtt-tat-ttc-gtt-atg-gta-tc 3′). The PCR product was digested with BamHI and SmaI, and ligated into similarly digested pGEX4T-1 (Pharmacia Biotech, Inc.) and pQE30 (QIAGEN, Inc.), generating pSV28 and pSV30, respectively. To create the constructs expressing the Gal4p DNA binding domain (Gal4p-BD) or transcriptional activation domain (Gal4p-AD) fused to Sec34p, the SEC34 open reading frame (ORF) was amplified by PCR, placing a BamHI site upstream of the codon for the second amino acid residue of the protein and a PstI site downstream of the stop codon (5′ primer, 5′ cgc-gga-tcc-tgg-cga-gaa-gta-gaa-ag 3′; 3′ primer, 5′ cgc-gct-gca-gtt-tat-ttc-gtt-atg-gta-tc 3′). The resulting product was cleaved with BamHI and PstI, and ligated into a similarly digested pAS2 or pGAD424 (Clonetech, Inc.), yielding COOH-terminal fusions to Gal4p-BD (pSV37) and Gal4p-AD (pSV35), respectively. The constructs expressing the Gal4p-BD or Gal4p-AD fused to Sec35p were constructed in an identical manner, also placing a BamHI site upstream of the codon for the second amino acid residue of the protein and a PstI site downstream of the stop codon (5′ primer, 5′ cgc-gga-tcc-tgg-tca-aca-gtc-ata-g 3′; 3′ primer, 5′ cgc-gct-gca-ggt-ttt-ctc-cca-act-atg 3′), creating COOH-terminal fusions to Gal4p-BD (pSV34) and Gal4p-AD (pSV36), respectively. To delete one copy of SEC34 in a diploid strain by the γ-method , the sec34 Δ plasmid, pSV27, was constructed in two stages. In the first stage, the region 5′ to SEC34 was excised from plasmid pB4 as a PstI/PvuII fragment and ligated into PstI/SmaI-digested pRS305. In the second stage, a HindIII fragment containing the region 3′ to the locus was released from plasmid pB4 and ligated into the plasmid generated from step one, which had been linearized with HindIII; the correct orientation of the HindIII fragment was confirmed by restriction digest. In the resulting construct, the inserts in the polylinker of pRS305 were placed such that the region directly 3′ to the ORF was placed upstream of the region directly 5′ of the ORF, with a unique restriction site, PstI, between the two sequences; digestion of this plasmid with PstI and transformation of the linearized plasmid into a diploid strain results in the replacement of the coding sequences of one allele of SEC34 with the sequences of the integrating vector. To generate pSK81, ORF YOR216c/ RUD3 was isolated from the library plasmid pSOU7 as a 2.2-kb BamHI/SnaBI fragment and ligated into pRS426 that had been digested with BamHI and SmaI. Yeast strains used in this paper are described in Table . Strain construction was as follows. The sec34 Δ ::LEU2/SEC34 strain GWY127 was constructed by transforming GWY30 with PstI-digested pSV27. The presence of the SEC34 deletion in the Leu + transformants was confirmed by PCR amplification of the novel junctions at the deletion locus. The diploid strain heterozygous for both the sec34-2 and sec35-1 alleles was created by mating GWY93 to GWY95 for 6 h on YPD. The diploid was identified by the distinct morphology of the zygote, and was isolated by micromanipulation. To clone SEC34 , the sec34-2 strain GWY95 was transformed with a URA3 YCp50-based library , and temperature-resistant colonies were selected at 38.5°C. Over 100,000 transformants were screened and ∼90 temperature-resistant colonies were isolated. Library plasmids from nine colonies were isolated, amplified in bacteria, and tested for the ability to confer temperature-resistance when retransformed into the sec34-2 strain. Four plasmids restored growth at 38.5°C to wild-type levels, three yielded partial suppression, and the remaining two did not display plasmid-linked suppression. The ends of the inserts of the seven constructs showing plasmid-linked suppression were sequenced with primers YEp24-F and YEp24-R and the resulting sequences were used to search the Saccharomyces Genome Database. To mark the YER157w locus for purposes of integrative mapping, the sec34-2 strain GWY95 was transformed with pSV24 that had been linearized with BglII, an enzyme that cleaves internal to the ORF, such that integration is directed to the YER157w locus. GST-Sec34p and His 6 -Sec34p were expressed in strain XL1-Blue (Stratagene) from plasmids pSV28 and pSV30, respectively, and fusion proteins were purified according to the manufacturers' instructions (Pharmacia Biotech, Inc.; QIAGEN, Inc.). GST-Sec34p was used to immunize rabbits by standard procedures and the resulting serum was affinity-purified against His 6 -Sec34p that had been coupled to cyanogen bromide-activated Sepharose, as per the manufacturer's instruction (Pharmacia Biotech, Inc.). Crude yeast lysates used to characterize the affinity-purified antibody were made as described , and the equivalent of 0.2 OD units of cells was analyzed for each strain. HRP-conjugated secondary antibodies were used at a dilution of 1:3,000. Incubations with both primary and secondary antibodies were for 1 h at room temperature and immunoblots were developed with a chemiluminescent detection kit (Amersham Corp.). A 250-ml culture of wild-type yeast (RSY255) was grown in YPD at 30°C to midlogarithmic phase (OD 595 = 1.4), washed in sterile water, and resuspended at ∼75 OD 595 /ml in Buffer 88 (25 mM Hepes, pH 7.0, 150 mM KOAc, 5 mM MgCl 2 , 1 mM DTT) containing protease inhibitors . 1/2 sample vol of acid-washed glass beads (425–600 μm; Sigma Chemical Co.) was added, and the material was vortexed eight times for 30 s, with 30 s on ice between each burst. The crude yeast lysate was centrifuged at 1,500 g (SA600 rotor, 3,300 rpm, 3 min, 4°C) to generate the S1 lysate. For extraction studies, yeast membranes were isolated on a step gradient in which 2 ml of S1 was layered over a step gradient consisting of 2 ml of 45% sucrose and 8 ml of 10% sucrose, both in Buffer 88. After centrifugation at 200,000 g (SW40 rotor, 40,000 rpm, 2 h, 4°C), membranes were collected from the 10%/45% sucrose interface by piercing the side of the tube with an 18 gauge needle and aspiration of the interface. The membranes (100 μl per reaction) were then mixed with an equal volume of Buffer 88 or a 2× extraction buffer (either 2% Triton X-100 in Buffer 88, 2 M NaCl in Buffer 88, or 0.2 M Na 2 CO 3 , pH 11.5, in water), diluted to 1 ml in Buffer 88 or 1× extraction buffer (either 1% Triton X-100 in Buffer 88, 1 M NaCl in Buffer 88, or 0.1 M Na 2 CO 3 , pH 11.5, in water), and incubated on ice for 45 min. The extraction mixtures (800 μl) were then layered over 200 μl 14% sucrose cushions made in the appropriate 1× extraction buffer and centrifuged at 175,000 g (TLA100.2 rotor, 70,000 rpm, 60 min, 4°C). 900 μl of the supernatant was removed from each sample, and 800 μL of Buffer 88 was added to the pellets (in the remaining 100 μl), which were resuspended with a Dounce homogenizer. Fractions were probed with antibodies against Sec34p, Sec35p , Sed5p , or phosphoglycerate kinase (PGK; Molecular Probes). Subcellular fractionation was completed as previously described . Yeast semi-intact cells from either the wild-type (RSY255) or the sec34-2 strain (GWY95) were prepared from logarithmic phase cultures of strains grown at 23°C and were stored at −70°C . Before assays, an aliquot of cells was quickly thawed and washed in Buffer 88 to remove cytosol, and 35 S-pre-pro-α-factor was posttranslationally translocated into the ER of the semi-intact cells as previously described . Vesicle tethering and transport assays were performed at either 23 or 29°C, as indicated . For tethering assays, vesicles that bud from the ER by the addition of COPII components are freely diffusible and remain in the supernatant fraction after centrifugation at 14,000 rpm; protease-protected glycosylated 35 S-pro-α-factor ( 35 S-gp-α-factor) contained in these vesicles was quantified after solubilization of membranes and precipitation with Concanavalin A–Sepharose. For transport assays, COPII proteins, in addition to purified Uso1p and LMA1, were added as indicated in the figure legends, and the amount of Golgi complex-modified 35 S-gp-α-factor was measured by immunoprecipitation with anti-α1,6-mannose–specific antibodies. The data presented is the average of duplicate determinations and the error bars represent the range. The protease deficient RSY1157 strain was grown to late log phase (OD 600 = 3.4) in 36 liters of YPD at 30°C, after which all manipulations were performed at 0–4°C. The cells were harvested by centrifugation and washed twice with water. The 334 g cell pellet was resuspended in 1 liter of 25 mM Tris-Cl, pH 8.0, 1 M KCl, 2 mM EGTA (lysis buffer) with protease inhibitors [0.5 mM 1:10 phenanthroline, 2 μM pepstatin A, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin, 1 mM PMSF, 200 μM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)] and 1 mM DTT, and lysed in an EmulsiFlex-C5 (Avestin Inc.) at 18,000–20,000 psi. The lysate was centrifuged at 5,000 g , and the supernatant (1.04 liter) was collected and centrifuged at 20,000 g (Sorvall SA600 rotor, 12,000 rpm, 20 min). The supernatant (960 ml) was removed, avoiding the loose pellet, and centrifuged at 175,000 g (Beckman 45Ti rotor, 44,000 rpm, 120 min). The supernatant (S175; 750 ml at 7.3 mg protein/ml) was removed, avoiding the pellets. The S175 was made 1 mM in EDTA, and (NH 4 ) 2 SO 4 was added to 35% saturation, dissolved, and the solution was stirred for 60 min. The (NH 4 ) 2 SO 4 precipitate was collected by centrifugation at 17,000 g , resuspended in enough 25 mM Tris-Cl, pH 8.0, 1 mM DTT to yield a conductivity equivalent to that of 25 mM Tris-Cl, pH 8.0, 100 mM KCl, 1 mM DTT (T8.0/100K/D). This material (1.55 g protein in 368 ml; 4.2 mg/ml) was loaded at 2 ml/min onto a 50 ml DEAE-Sepharose Fast Flow (Pharmacia Biotech, Inc.) column (2.5 cm i.d.) that had been equilibrated in T8.0/100K/D. The column was washed with 200 ml of T8.0/100K/D and eluted with a linear gradient (2 mM/ml) from T8.0/100K/D to T8.0/400K/D, collecting 10 ml fractions throughout. At this and each subsequent chromatographic step, fractions were analyzed for Sec34p and Sec35p content by immunoblotting. The fractions containing Sec34p and Sec35p, which eluted together at ∼160 mM KCl, were pooled (348 mg protein in 60 ml, 5.8 mg/ml). A 200 μl aliquot of this DEAE pool was chromatographed on a 24 ml Superose 6 (HR10/30 Pharmacia Biotech, Inc.) size exclusion column equilibrated in T8.0/150K/D at 0.3 ml/min, collecting 1 ml fractions. The remaining 60 ml of the DEAE pool was then concentrated to a volume of 17.8 ml (284 mg protein at 16 mg/ml) in an Amicon 8050 Ultrafiltration Cell (Millipore Inc.) with a YM100 (MWCO 100 kD) ultrafiltration membrane. The concentrated sample was loaded onto a 700 ml Sephacryl S-300 (Pharmacia Biotech, Inc.) column (2.5 cm i.d.) that had been equilibrated in 25 mM Tris-Cl, pH 7.6, 100 mM KCl, 1 mM DTT (T7.6/100K/D) and chromatographed at 1.5 ml/min in T7.6/100K/D, collecting 10 ml fractions. The fractions containing both Sec34p and Sec35p were pooled (66 mg protein in 47 ml, 1.4 mg/ml) and loaded onto an 8 ml MonoQ (HR 10/10, Pharmacia Biotech, Inc.) anion exchange column equilibrated in T7.6/100K/D at 1.5 ml/min. The column was washed with 36 ml of T7.6/100K/D and eluted with a linear gradient (2.5 mM/ml) from T7.6/100K/D to T7.6/500K/D, collecting 5 ml fractions. The fractions containing both Sec34p and Sec35p, which coeluted at ∼295 mM KCl, were pooled (16 mg protein in 18 ml, 0.89 mg/ml), dialyzed against 40 mM potassium phosphate, pH 6.8, 0 mM KCl, 1 mM DTT (KP/0K/D), and loaded onto a MonoS (HR 5/5, Pharmacia Biotech, Inc.) equilibrated in KP/0K/D at 0.5 ml/min. The column was washed with 4 ml of KP/0K/D and eluted with a linear KCl gradient (10 mM/ml) from KP/0K/D to KP/500K/D, collecting 2 ml fractions throughout. The fractions containing both Sec34p and Sec35p, which coeluted at ∼110 mM KCl, were pooled (1.5 mg protein in 8 ml, 0.19 mg/ml) and concentrated to a volume of 0.1 ml at 9.3 mg/ml with an Ultrafree BIOMAX centrifugal device (Millipore Inc.). The concentrated sample was loaded onto a 24 ml Superose 6 (HR10/30, Pharmacia Biotech, Inc.) size exclusion column that had been equilibrated in T7.6/200K/D, and chromatographed at 0.3 ml/min, collecting 0.5 ml fractions. After this point, protein concentration was no longer determined, to not decrease yield. The fractions containing Sec34p and Sec35p were pooled and loaded onto a Bio-Scale CHT2-I Hydroxyapatite column (Bio-Rad Laboratories). The column was washed with 8 ml 25 mM Tris-Cl, pH 8.0, 200 mM KCl, 0 mM potassium phosphate, 1 mM DTT (T7.6/200K/0Pi/D), and eluted with a linear potassium phosphate gradient (16.6 mM/ml) from T7.6/200K/0Pi/D to T7.6/200K/250Pi/D; Sec34p and Sec35p coeluted at ∼100 mM potassium phosphate. To clone SEC34 , the sec34-2 strain was transformed with a low-copy (centromere, CEN) yeast genomic library and temperature-resistant colonies were selected at 38.5°C. Library plasmids were isolated from these colonies and retested for their ability to confer growth at 38.5°C. Four restored growth at the restrictive temperature to wild-type levels, and the remaining three yielded partial suppression. The ends of the inserts of the seven library plasmids were sequenced and found to contain overlapping regions of the right arm of chromosome V, a portion of which is shown in Fig. 1 a. The only complete ORF contained on each of the plasmids that conferred strong suppression of the temperature-sensitive phenotype was YER157w. Interestingly, the three plasmids that partially suppress the sec34-2 mutation contained identical inserts in which only the 5′ end of YER157w is present; the inability of these plasmids to fully suppress may be due to the absence of the COOH-terminal portion of the protein. The ORF YER157w was isolated from the genomic insert and transferred to a low-copy plasmid. This construct was demonstrated to suppress the temperature sensitivity of the sec34-2 strain, confirming that YER157w is responsible for the suppression conferred by each of the library plasmids. To address the possibility that YER157w was a suppressor of the sec34-2 mutation rather than the gene itself, integrative mapping was performed. A sec34-2 strain in which the YER157w locus was marked with LEU2 was constructed and subsequently mated to a wild-type strain. The resulting diploid strain was subjected to tetrad analysis and, of 38 tetrads examined, the temperature-sensitive phenotype did not segregate away from the marked locus. Thus, integrative mapping strongly suggests that YER157w is SEC34 . SEC34 is predicted to encode an 801-amino acid protein with a molecular weight of 92.5 kD and a pI of 5.2. Sec34p lacks a signal sequence, as well as transmembrane domains or other motifs that could facilitate membrane attachment. Therefore, the protein is predicted to be either cytoplasmic or peripherally associated with membranes. Three putative orthologs of Sec34p have been detected. First, the C . elegans genome encodes a protein designated Y71F9A 290.A that is 25% identical and 35% similar to Sec34p. This 428-amino acid protein is ∼50% the size of Sec34p and is homologous to the NH 2 terminus of Sec34p (spanning amino acid residues 111 to 459). Due to the disparity in size, it is unclear whether this protein is indeed an ortholog of Sec34p. Second, the genome of the fission yeast Schizosaccharomyces pombe contains a 735-amino acid protein that is 26% identical and 40% similar to Sec34p. Finally, several overlapping human expressed sequence tags have been isolated that show high similarity to Sec34p. Sequencing of clones containing the first two ESTs (provided by Genome Systems, Inc., St. Louis, MO) allowed us to analyze additional sequences previously unavailable in GenBank (data not shown). By combining our newly sequenced regions with the overlapping ESTs in the database, we obtained a 263-amino acid portion of the putative human protein. Comparison of this partial protein to Sec34p using the BLAST algorithm revealed a 175-amino acid region of homology (encompassing amino acids 531 to 706 of Sec34p) between the human sequence and Sec34p. Within this region the two proteins are 25% identical and 50% similar. Since the 86-amino acid region 5′ to this homologous region does not display a high degree of similarity to Sec34p, the protein encoded by these ESTs may not be a true Sec34p homolog, but instead may contain a Sec34p-like domain. To evaluate the phenotype of a strain lacking SEC34 , we constructed a diploid strain in which one allele of SEC34 had been deleted and replaced with the gene LEU2 . This strain ( sec34 Δ ::LEU2/SEC34 ) was sporulated and dissected, and the resulting tetrads were incubated on rich media at 30°C. As shown in Fig. 2 (left), a clear 2 + :2 − segregation pattern was observed in which each tetrad contained two large and two very small colonies. The large colonies were without exception Leu − and thus contained the wild-type copy of the gene, while the small colonies were Leu + , indicating the presence of the sec34 Δ locus. Therefore, although SEC34 is not an essential gene, haploid strains lacking SEC34 are at an extreme growth disadvantage. The growth defect of the sec34 Δ strain is complemented by a plasmid bearing SEC34 since the presence of the construct in the diploid sec34 Δ /SEC34 strain resulted in the restoration of a 4 + :0 − segregation pattern . In each tetrad, two segregants were Leu + and Ura + , indicating the presence of both the deletion and the plasmid bearing SEC34 , respectively. Previous analysis of the sec34 mutant strain indicated a role for Sec34p in the docking or fusion of ER-derived vesicles with the cis-Golgi complex . By testing whether SEC34 displayed genetic interactions with other factors required at this stage, as well as at other stages of transport, we sought to confirm this assignment and to further explore the function of Sec34p. We thus tested whether the temperature-sensitive growth defect of the sec34-2 strain could be suppressed by overexpression of other genes required for transport. Two of the best suppressors were found to encode the rab protein Ypt1p and the dominant allele of the t-SNARE–associated protein, SLY1 , termed SLY1-20 . Although neither gene could restore growth to the levels observed when the strain was complemented by SEC34 on a plasmid, both conferred a significant growth advantage to the mutant strain. A second gene, USO1 , which encodes a tethering factor, also suppressed the temperature sensitivity, but to a lesser extent than either YPT1 or SLY1-20 . Weak suppression was also observed upon overexpression of the v-SNAREs Sec22p, Bet1p, and Ykt6p, each of which has been implicated in the ER to Golgi complex stage of transport; no suppression was seen for another v-SNARE involved at this step, Bos1p . Several other genes were tested for their ability to suppress the sec34-2 mutation when overexpressed, but were found to have no effect. These genes encode the tethering factor Sec35p, the TRAPP complex component Bet3p, the cis-Golgi complex t-SNARE Sed5p, and the Golgi complex to plasma membrane rab protein Sec4p. The lack of suppression by the Golgi complex to plasma membrane rab indicates that suppression by the ER to Golgi complex rab is specific. We also tested whether overexpression of SEC34 could suppress temperature-sensitive mutant alleles of several ER to Golgi complex docking factors. Interestingly, overexpression of SEC34 was capable of weakly suppressing the temperature-sensitive growth defect of the sec35-1 strain (data not shown). However, multicopy SEC34 was unable to suppress mutant alleles of all other secretory factors tested, including: the v-SNAREs Sec22p, Bos1p, and Bet1p; the t-SNARE Sed5p; the tethering factor Uso1p; the rab Ypt1p; and the TRAPP complex component Bet3p (data not shown). Therefore, overexpression of either SEC34 or SEC35 is unable to suppress the temperature-sensitive growth defects of the majority of tethering and docking factors, yet multicopy SEC34 can improve the growth defect of a compromised allele of SEC35 , and thus the two genes show a genetic interaction. Since YPT1 and SLY1-20 were efficient suppressors of the temperature-sensitive growth defect of the sec34-2 strain, we explored whether they could also improve the severe growth defect of the sec34 Δ strain. The sec34 Δ/ SEC34 diploid strain was therefore transformed with plasmids expressing either multicopy YPT1 or low-copy SLY1-20 , and the resulting diploids were sporulated and subjected to tetrad analysis. Indeed, the presence of either plasmid significantly improved the growth of the sec34 Δ strain . This result implies that Ypt1p and Sly1p most likely function downstream of, or in a parallel pathway with, Sec34p. Suppression of the sec34-2 temperature-sensitive growth defect was also observed upon overexpression of a gene designated RUD3 . RUD3 was isolated previously in our lab through a screen for genes that, when overexpressed, are able to suppress the temperature-sensitive growth defect of the uso1-1 strain . From a panel of multicopy suppressors, four library plasmids were isolated that contain overlapping regions of chromosome XV that include two hypothetical ORFs and one known gene. The suppressing activity was localized to the hypothetical ORF YOR216c, and this gene was named RUD3 because it relieves the uso1-1 transport defect. RUD3 is an efficient suppressor of the uso1-1 deficiency at 37.5°C, suppressing the growth defect , as well as the ER to Golgi complex transport block, as monitored through movement of the marker protein, carboxypeptidase Y (data not shown). RUD3 is predicted to encode a 484-amino acid protein with a molecular mass of 56.1 kD and a pI of 4.67 . Like Sec34p, this protein lacks any motifs to indicate a localization other than cytosolic. Both the PAIRCOIL and COILS programs indicate a high probability that the protein assumes a coiled-coil secondary structure in a central, ∼215-amino acid stretch . There is a region near the center of this coiled-coil domain, however, that has a decreased coiled-coil probability; this region could potentially be a hinge in the protein . BLAST searches revealed a single putative homolog of Rud3p in the genome of S . pombe . This 401-amino acid protein shares 28% identity and 41% similarity with Rud3p, and thus encodes a Rud3p ortholog that we designate spRud3p. Interestingly, spRud3p also contains a region predicted to form a coiled-coil secondary structure, although the homology between the proteins is not restricted to this motif. Indeed, the COOH termini of the proteins (ranging from amino acid 365 to 484 for Rud3p, and 280 to 401 for spRud3p) are most homologous, displaying 49% identity and 62% similarity. To test whether Rud3p is encoded by an essential gene, a diploid strain was created in which one of the alleles of RUD3 was deleted and marked with LEU2 . Sporulation and tetrad dissection of this strain yielded tetrads with four viable spores, two of which were Leu + . Upon incubation at 25, 30, and 37°C, the haploid rud3 Δ strain did not display a significant growth defect as compared with a wild-type strain (data not shown), and thus RUD3 is not an essential gene. To analyze the Sec34 protein, we generated affinity-purified anti-Sec34p antibody. The antibody recognizes two proteins in crude yeast extracts, the larger of which corresponds to the predicted molecular weight of Sec34p . This protein is absent in a sec34 Δ strain (which expressed SLY1-20 to enhance its propagation) and is overexpressed in a strain containing SEC34 on a multicopy plasmid, and thus represents Sec34p. The smaller protein, which is recognized despite the affinity purification, is not related to the Sec34p locus since it is present in the sec34 Δ strain and its expression level is unaffected by overexpression of SEC34 . With this antibody, we investigated whether Sec34p was capable of associating with membranes, as might be expected for a protein involved in secretion. A crude yeast extract (designated S1) was centrifuged at 175,000 g to separate the organelles of the secretory pathway, which are found in the pellet fraction, from cytosolic proteins, which are contained in the supernatant fraction. As shown in Fig. 5 b (left), the majority of Sec34p is found in the pellet fraction, along with the integral membrane protein Sed5p, while a small amount of Sec34p is contained in the supernatant, as is the cytosolic marker, PGK. This result is consistent with a peripheral membrane association for Sec34p. To explore the basis for the sedimentation of Sec34p, we attempted to extract the protein from an enriched membrane fraction using buffers containing Triton X-100, NaCl, or Na 2 CO 3 , pH 11.5. As expected for an integral membrane protein, Sed5p was released into the supernatant fraction after incubation with buffer containing Triton X-100, but not after treatment with salt or high pH, whereas the peripheral membrane protein Sec35p was released, at least partially, upon incubation with all three buffers. Sec34p was partially shifted into the supernatant fraction upon treatment with Triton X-100, salt, or high pH, and thus behaves as a peripheral membrane protein . To further analyze the membrane association of Sec34p, differential centrifugation was employed. The S1 fraction was centrifuged at 10,000 g , separated into supernatant (S10) and pellet (P10) fractions, and the S10 fraction was further centrifuged at 175,000 g and separated into supernatant (S175) and pellet (P175) fractions. Under these conditions, the ER is contained primarily into the P10 fraction (data not shown), the Golgi complex partitions between the P10 and P175 fractions, as seen with the Golgi protein Sed5p, and cytosolic proteins remain in the supernatant fractions, as observed for PGK . The soluble portion of Sec34p is found in the S175 fraction, while the membrane-associated pool partitions between the P10 and P175, similar to the Golgi complex protein Sed5p. The fractionation pattern of Sec34p is quite similar to that observed for the peripheral membrane protein Sec35p, although Sec34p appears to have a greater proportion of the protein in the membrane fractions. Because the mutant phenotype and suppression profile of sec34-2 is similar to that of other factors required for the tethering stage of ER to Golgi complex transport, we investigated whether Sec34p was required for tethering as well. We thus employed two in vitro assays that, together, are able to distinguish the stages of budding, tethering, and fusion in ER to Golgi complex transport . In the first assay , overall ER to Golgi complex transport is measured in semi-intact cells incubated with a mixture of purified protein components that drive all the stages of transport: vesicle formation from the ER is supported by the addition of COPII proteins, efficient vesicle tethering requires added Uso1p, and vesicle fusion requires a protein complex termed LMA1. Productive transport is monitored by following the addition of α1,6-mannose residues to gp-α-factor, an event that occurs in the cis-Golgi complex. Generation of this transport system from conditional mutants has shown that the system also requires the activities of several peripheral and integral membrane proteins including Ypt1p, Sec35p, and the SNAREs . Although there is no requirement for exogenous Sec34p in the system, Sec34p may be supplied to the assay by peripheral association with membranes of the semi-intact cells. Therefore, to test for a requirement for Sec34p, we generated the in vitro system from the sec34-2 strain. In wild-type or sec34-2 mutant cells at 23°C, movement of gp-α-factor from the ER to the Golgi complex in this in vitro system proceeds with a similar efficiency . In contrast, at 29°C in sec34-2 semi-intact cells, this process is very inefficient relative to wild-type, indicating that sec34-2 is defective for overall ER to Golgi transport in vitro. The second assay we employed examined the functionality of the vesicle budding and vesicle tethering steps in the sec34-2 mutant . In this assay, release of vesicles from semi-intact cells is detected by the appearance of protease-protected gp-α-factor in a low-speed supernatant at the end of the reaction. Vesicles were efficiently generated upon addition of COPII components to wild-type or sec34-2 semi-intact cells at 23 or 29°C . These data indicate that Sec34p is not required for ER-derived vesicle budding. Because addition of Uso1p significantly reduced vesicle release, vesicle tethering was also functional in the semi-intact wild-type cells at 23 or 29°C, as well as in sec34-2 semi-intact cells at 23°C. In contrast, when Uso1p was added to the sec34-2 -derived system at the restrictive temperature of 29°C, vesicle release was only slightly diminished . This result indicates that the sec34-2 mutant cannot efficiently tether ER-derived vesicles to the yeast Golgi complex. The similar genetic interactions of SEC34 and SEC35 with genes involved in the docking stage of vesicular transport, taken together with their genetic interaction with one another, and with the finding that both proteins function in vesicle tethering, lead us to examine whether mutations in the two genes would display a synthetic lethal interaction. To do this, we generated a diploid strain heterozygous for both the sec34-2 and sec35-1 alleles and subjected it to tetrad analysis. Although both the sec34-2 and sec35-1 haploid strains are permissive for growth at both 21 and 30°C, tetrads from the diploid sec34-2 / SEC34 SEC35 / sec35-1 strain yielded numerous inviable colonies at either temperature . After incubation of the segregants for long periods of time, a small proportion of those previously characterized as inviable would form visible microcolonies. Since this phenotype was variable, we hypothesize that the microcolonies result from either the appearance of spontaneous suppressors of the inviability or from background mutations in the strain. Examination of the viable segregants in each tetrad for temperature sensitivity revealed a pattern in which the inviable segregants are predicted to be those containing both the sec34-2 and the sec35-1 alleles. To confirm this prediction, the diploid strain was transformed with low-copy plasmids bearing either SEC34 or SEC35 before tetrad dissection. The presence of either plasmid lead to a greater proportion of viable segregants than was observed for the untransformed strain, concurrent with the appearance of segregants that were sensitive to the drug 5-fluoro-orotic acid, which is toxic to cells that must maintain the plasmid to survive (data not shown). Therefore, the sec34-2 and sec35-1 alleles display a synthetic lethal phenotype, which can be complemented by the presence of either gene on a plasmid. In many cases, synthetic lethality between alleles of two genes involved in secretion indicates that their gene products are involved in the same stage of secretion . Furthermore, such genetic interactions can also be indicative of a physical interaction of the proteins encoded by those genes, as is the case for mutations in the α and β subunits of tubulin . Therefore, we investigated whether Sec34p and Sec35p physically interact using the two-hybrid system . A strain in which transcription of both the ADE2 and HIS3 genes is under the control of the Gal4p transcriptional activator was transformed with plasmids expressing either the Gal4p-BD or Gal4p-AD, either alone or fused to Sec34p or Sec35p. Activation of transcription of the ADE2 and HIS3 genes was assessed by the ability of the strain to grow on media lacking adenine or histidine, respectively. As an example of a positive two-hybrid interaction, coexpression of p53 fused to the Gal4p-BD and the large T-antigen fused to the Gal4p-AD was demonstrated to activate both reporter genes , as has been described previously . Strains expressing either the Sec34p-Gal4p-BD or the Sec35p-Gal4p-AD fusion protein were unable to grow on either media. However, when Sec34p-Gal4p-BD was expressed along with the Sec35p-Gal4p-AD, the strain grew well on media lacking either adenine or histidine , indicating that the interaction of Sec34p with Sec35p was able to localize the Gal4p-AD to the promoters of these genes, activating transcription. When the converse experiment was completed, in which Sec35p was fused to the Gal4p-BD and Sec34p was fused to the Gal4p-AD, expression of the ADE2 and HIS3 genes was also observed only when both fusion proteins were expressed. Thus, Sec34p and Sec35p interact. To characterize the interaction of Sec34p and Sec35p further, we employed immunoblotting to monitor the behavior of these proteins during fractionation of yeast cytosol. Sec34p and Sec35p coprecipitated in 35% saturated ammonium sulfate and cofractionated precisely by DEAE anion exchange chromatography (data not shown). An aliquot of the Sec34p/Sec35p anion exchange pool was then subjected to size exclusion chromatography on Superose 6 , and once again, Sec34p and Sec35p precisely cofractionate. Interestingly, they elute from the column slightly before thyroglobulin, a 669-kD globular protein. These results are consistent with Sec34p and Sec35p existing in a large protein complex with a mass of up to ∼750 kD. A small amount of monomeric Sec35p is also evident upon gel filtration, suggesting either that some Sec35p has dissociated from the complex or that a cytosolic pool of monomeric Sec35p exists. No such monomeric Sec34p has been detected in cytosolic fractions. To further purify the Sec34p/Sec35p complex, the remainder of the DEAE anion exchange pool was subjected to several sequential chromatographic steps (see Materials and Methods), including Sephacryl S-300 gel filtration (data not shown), MonoQ anion exchange , MonoS cation exchange , Superose 6 gel filtration (data not shown), and ceramic hydroxyapatite . Once again, Sec34p and Sec35p precisely comigrate through each step, strongly indicating that Sec34p and Sec35p are present in a large protein complex. Much effort has been extended towards gaining an understanding of the mechanism of transport vesicle docking in the secretory pathway. Several families of proteins are involved in this event, including the rab family of small GTP-binding proteins and the SNARE family of integral membrane proteins. Recently, another class of proteins has been described, the tethering factors. Although these proteins do not display homology with one another and thus do not define a family, they share a similar function in docking, that of connecting the vesicle to the target compartment before the interaction of v- and t-SNAREs . The docking event can therefore be separated into two distinct substages, tethering and SNARE-dependent docking. A recent genetic screen identified temperature-sensitive alleles of two genes, SEC34 and SEC35 , that, when incubated at the restrictive temperature, are defective in ER to Golgi complex transport and accumulate large numbers of vesicles . Mutant alleles of these genes are also able to be suppressed by the dominant allele of SLY1 , SLY1-20 , a trait shared with all previously characterized ER to Golgi complex tethering factors . Based on these data, we hypothesized that SEC34 and SEC35 might be involved in tethering, and this was demonstrated to be the case for SEC35 by the discovery that this gene both displayed a genetic interaction with genes involved in tethering and is required in this process as revealed by an in vitro assay . We therefore investigated whether Sec34p functions in tethering as well. To begin our study of SEC34 , we cloned the gene by complementation of the temperature-sensitive phenotype of a strain bearing the sec34-2 mutation. SEC34 was discovered to be a novel gene encoding a protein with a predicted molecular weight of 93 kD. Deletion of SEC34 in a haploid strain resulted in a severe growth defect, and thus SEC34 is essential for wild-type growth rates, although not for viability. To investigate the genetic interactions of SEC34 we employed multicopy suppressor analysis. The best suppression of the sec34-2 temperature-sensitive growth defect was conferred by overexpression of Ypt1p, the rab required in ER to Golgi complex transport, or by expression of Sly1-20p, the dominant form of the t-SNARE–associated factor, Sly1p. Suppression of the SEC34 deletion strain allowed us to order the action of Sec34p with respect to Ypt1p and Sly1p. Since either YPT1 or SLY1-20 can suppress both mutations in, and a deletion of, SEC34 , yet overexpression of SEC34 cannot suppress mutations in either YPT1 or SLY1 , we hypothesize that Ypt1p and Sly1p function downstream of Sec34p. Weaker suppression of the sec34-2 mutation was observed upon overexpression of the tethering factor Uso1p, or the v-SNAREs Sec22p, Bet1p, or Ykt6p. The suppression of sec34-2 by the v-SNAREs may be through mass action, in which vesicles containing supernumerary v-SNAREs are able to compensate for a deficiency in tethering, albeit with a very low efficiency. This phenomena has been observed previously for mutations in the tethering factors Uso1p and Sec35p , as well as components of the putative tethering complex TRAPP . Interestingly, no suppression of the sec34-2 mutation was observed upon overexpression of either the v-SNARE Bos1p or the cis-Golgi complex t-SNARE Sed5p. Overexpression of Bos1p was also unable to suppress a temperature-sensitive growth defect of the sec35-1 strain or the inviability of the uso1 Δ strain . This lack of suppression could result from inefficient expression of the gene or may indicate a functional difference between Bos1p and the other v-SNAREs that mediate the ER to Golgi complex transport step. The lack of suppression of the sec34-2 mutant strain by high-copy expression of Sed5p was not unexpected, because it has been demonstrated that overexpression of this t-SNARE is toxic to cells . Biochemical analysis of the Sec34 protein reveals that it is a peripheral membrane protein. Although a small amount of the protein is soluble, the remainder partitions between the P10 and P175 fractions, similar to the Golgi protein Sed5p; it is possible, therefore, that Sec34p is associated with the Golgi complex. Due to the association of Sec34p with membranes, we used semi-intact cells made from the sec34-2 strain to test the requirement for Sec34p in tethering through an assay that reconstitutes ER to Golgi complex transport. These semi-intact cells were demonstrated to be able to bud vesicles from the ER, but these vesicles failed to efficiently tether to the Golgi complex at the restrictive temperature, indicating that Sec34p is required for the tethering of ER-derived vesicles to the cis-Golgi complex. Since cytosolic proteins are removed from the sec34-2 semi-intact cells, the membrane-associated pool of Sec34-2p is most likely the source of the tethering defect. In addition, since the membranes involved in tethering are restricted to those of the vesicle and the cis-Golgi complex, Sec34p is most likely associating with one, or both, of these membranes. SEC34 was found to display two interesting genetic interactions with the tethering factor gene SEC35 . First, multicopy SEC34 weakly suppresses a temperature-sensitive allele of SEC35 . Since overexpression of SEC34 cannot suppress the cold-sensitive lethality of the sec35 Δ strain, Sec34p is able to assist a handicapped allele of SEC35 , but cannot replace its function. Second, the sec34-2 and sec35-1 alleles display a synthetic lethal interaction. Although strains bearing either allele alone are permissive for growth at 23 and 30°C, a haploid strain containing both the sec34-2 and sec35-1 alleles is inviable at either temperature. This synthetic phenotype is more severe than the conditional synthetic lethality of the sec35-1 allele in combination with a mutant allele of either YPT1 or USO1 , in which the double mutants are viable at 23°C, but not at 30°C . This finding suggests a close functional interaction of the Sec34 and Sec35 proteins. Based on these results, we investigated whether Sec34p and Sec35p could physically interact through the two-hybrid assay. Indeed, Sec34p and Sec35p were found to interact. The interaction between the two proteins may explain the ability of multicopy SEC34 to suppress the sec35-1 allele, but not the sec35 Δ allele: increased levels of Sec34p could stabilize a defective form of Sec35p but would be ineffectual in the absence of Sec35p, especially if the interaction of the two proteins is essential to their function in tethering. To further explore the interaction of Sec34p and Sec35p we examined the behavior of the soluble pool of these proteins through several chromatographic steps. The proteins cofractionated through ammonium sulfate precipitation and anion exchange, cation exchange, ceramic hydroxyapatite, and size exclusion chromatographic steps, providing strong evidence that the two proteins are in a complex with one another. Intriguingly, the Sec34p/Sec35p complex appears quite large, with an estimated molecular weight (if globular) of ∼750 kD. This size, which is larger than the combined molecular weights of the two proteins (124 kD), suggests several possibilities for the structure of the complex. First, the complex could be homodimeric, containing one molecule of each protein, but highly elongated such that it migrates rapidly through a size exclusion column. We consider this unlikely because the sequences of Sec34p and Sec35p lack motifs (such as coiled-coil domains) that would be indicative of an elongated structure. Second, the complex could contain two or more molecules of at least one protein, resulting in a more massive structure. Finally, the complex could be multimeric, containing heretofore unidentified component(s) in addition to Sec34p and Sec35p. We are currently purifying the Sec34p/Sec35p complex to address this issue and identify any additional components. It appears, however, that Uso1p is unlikely to be a component of the Sec34p/Sec35p complex since immunoblotting fractions from the purification with an antibody against this protein revealed that Uso1p did not comigrate with Sec34p and Sec35p (data not shown). The 750-kD complex containing Sec34p and Sec35p is reminiscent of the TRAPP complex, which migrates at ∼800 kD by size exclusion chromatography . However, two pieces of data indicate that the TRAPP complex is distinct from the Sec34p/Sec35p complex. First, the identities of the low molecular weight members of the TRAPP complex have been elucidated, and none corresponds to Sec35p, whose mobility on SDS-PAGE was within the range of the proteins that have been sequenced . In addition, the known members of the TRAPP complex display genetic interactions with one another , yet no interaction was discerned between the gene encoding the TRAPP component Bet3p and either SEC34 or SEC35 . Since many secretory factors are evolutionarily conserved, we explored whether the components of the Sec34p/Sec35p complex were conserved in higher eukaryotes. The genome of the nematode C . elegans was discovered to contain a protein designated Y71F9A 290.A that is very similar to Sec34p. However, the C . elegans protein is ∼50% the size of Sec34p and therefore may not be a true ortholog. We also discovered a C . elegans protein with moderate homology to Sec35p (22% identical and 33% similar), designated C35A5.6. While the similarity is not high, the proteins are similar in size (C35A5.6 is comprised of 273 amino acid residues, whereas Sec35p is comprised of 275 amino acid residues), and thus, this C . elegans protein is a putative ortholog of Sec35p. Searches of GenBank for additional homologs of these proteins did not reveal additional Sec35p homologs, but several human ESTs were discovered with a high degree of similarity to Sec34p. Interestingly, the sequences contained on these ESTs were homologous to Sec34p over only a portion of the analyzed region of the putative human protein, and thus the protein may contain a Sec34p-like domain and may not be a true Sec34p ortholog. These data indicate that there may be orthologs of the Sec34p/Sec35p complex in higher organisms, but functional experiments will be required to unambiguously address this point. Finally, a putative ortholog of Sec34p was discovered in the genome of S . pombe . No paralogs of either Sec34p or Sec35p exist in S . cerevisiae , and thus these proteins do not define a family of related proteins. Finally, we describe the identification and characterization of a gene designated RUD3 that displays a genetic interaction with SEC34 . RUD3 , which encodes a novel nonessential protein with a predicted molecular weight of 56 kD, was originally identified in a screen for multicopy suppressors of a temperature-sensitive allele of the tethering factor, USO1 , and is also able to suppress the temperature-sensitive growth defect of the sec34-2 strain. Interestingly, RUD3 is unable to suppress mutations in other ER to Golgi complex docking factors such as Sec35p, Ypt1p, Sec22p, Bet1p, or Bos1p, and thus the suppression is specific to mutant alleles of SEC34 and USO1 . Overexpression of RUD3 can weakly suppress the inviability of the uso1 Δ strain (data not shown). Taken together, these data suggest that Rud3p either acts at, or downstream of, the tethering stage of ER to Golgi complex transport. Rud3p does not appear to be a component of the Sec34p/Sec35p complex since the majority of the protein fractionates away from the complex during its purification (data not shown). In summary, we describe the characterization of a novel secretory factor, Sec34p, and its role in tethering of ER-derived vesicles to the cis-Golgi complex. Unlike the SNAREs and rabs, the tethering factors described thus far at different intracellular transport steps are not members of a protein family. Nevertheless, they do share structural similarity, since they are either elongated or present in a large multimeric complex . The large size may be related to the requirement for the tethering factors to span the distance between the vesicle and the target compartment, before trans-SNARE complex formation. Interestingly, three factors meet this criteria in the yeast ER to Golgi complex transport step: the extended homodimer Uso1p, the TRAPP complex, and the Sec34p/Sec35p complex. It will be very exciting to discover in the future how these large protein complexes function to secure a vesicle to its target membrane, and whether their function is more complex than simply connecting vesicle and target membranes. | Study | biomedical | en | 0.999998 |
10562278 | YFP, GFP, and CFP indicate yellow, green, and cyan fluorescent proteins, respectively, and refer to GFP spectral variants based on the 10C (yellow), S65T (green), and W7 (cyan) mutants . The parent vectors for all constructs were pEGFP-N1 or pEGFP-C3 . Yellow variants of these vectors were generated by subcloning an AgeI-BsrGI fragment from the vector pEYFP-C1 (Clontech Laboratories) into the vector backbone of pEGFP-N1 or -C3 digested with AgeI and BsrGI. Cyan variants of these vectors were generated by subcloning an AgeI-BsrGI fragment encoding ECFP from the vector pRSET-ECFP into the vector backbone of pEGFP-N1 or -C3 digested with AgeI and BsrGI. All inserts into these vectors that were generated by PCR were sequenced on both strands using flanking primers. GFP-Rab6 comprises GFP fused to the NH 2 terminus of the corresponding full-length human Rab6 protein. A DNA fragment encoding the Rab6 wild-type protein was generated by PCR to insert an in-frame BsrGI site at the 5′ end, and a stop codon followed by a NotI site at the 3′ end and inserted into pEGFP-N1 to generate pEGFP-Rab6wt. An alternate plasmid that varied slightly in the spacing between GFP and the Rab6 protein was constructed by inserting the rab6 cDNA into the EcoRI-BamHI sites of pEGFP-C3 to generate pEGFP-Rab6wt′. No differences in trafficking or localization between the two constructs were observed. Stable cell lines (below) were generated with pEGFP-Rab6wt. Color variants were generated from pEGFP-Rab6wt′ (pEYFP-Rab6wt′ and pECFP-Rab6wt′). KDELR-GFP comprises GFP fused to the COOH terminus of the full-length human KDEL-receptor (Erd2.1). A cDNA was generated that contained the erd2 . 1 sequence with an EcoRI site and Kozak consensus at the 5′ end, and a BamHI site replacing the erd2 . 1 stop codon at the 3′ end. It was inserted into pEGFP-N1 to yield pKDELR-GFP and subcloned into pEYFP-N1 and pECFP-N1 to generate pKDELR-YFP and pKDELR-CFP. GFP-Sec61β was kindly provided before publication and is described . In brief, it is comprised of GFP fused to the NH 2 terminus of the full-length human Sec61β, with a short linker sequence between, constructed in a derivative of the vector pcDNA3. HeLa (ATCC No. CCL 185) and PtK 2 cells (ATCC CCL 56) were cultured in 5% fetal calf serum as described previously . Stably transfected cell lines were generated by fluorescent cell sorting essentially as described . Lines were resorted as required to maintain the homogeneity of the population. The following antibodies were used: anti-Giantin mouse monoclonal ; anti–β-tubulin mouse monoclonal (1:2,000 for blotting, 1:200 for IF; Boehringer-Mannheim); anti-Rabkinesin-6 rabbit polyclonal ; anti-Paxillin monoclonal (1:500 for IF; Transduction Laboratories); anti-β COP mouse monoclonal E5A3 ; anti-β′ COP mouse monoclonal ; anti-Erd2 ; anti-Rab6 rabbit polyclonal ; anti-GFP rabbit polyclonal . Secondary antibodies were Cy3-conjugated donkey anti–rabbit and Cy3-conjugated donkey anti-mouse (each at 1:1,000 for IF; Dianova). For indirect immunofluorescence, cells grown on glass coverslips were fixed 20 min in PBS + 3% paraformaldehyde (PFA) at room temperature or 2 min in neat methanol at −20°C, and processed using standard protocols described in Louvard et al. 1982 . For immunofluorescence with anti-Rab6 antibody, the staining was performed as described in Martinez et al. 1994 . Coverslips were mounted in Moviol (Hoechst), or viewed under live cell conditions (see below) at room temperature in PBS. Immunoelectron microscopy was performed as described in Röttger et al. 1998 , a modification of the protocol in Nilsson et al. 1993 , using affinity-purified rabbit polyclonal anti-GFP antibody. All transfections used a calcium phosphate protocol as described . Experiments to determine the effect of transient overexpression of native Rab6 wild-type, T27N, Q72L proteins on GFP-Rab6 motility were performed two ways: expression constructs for the native protein (identical to the GFP-Rab6 vector except without the GFP) were (a) cotransfected with GFP-Rab6 at various molar ratios: 0.25, 0.5, 0.75, and 0.90 (native wild-type or mutant:GFP-Rab6) into untransfected HeLa or PtK 2 cells; or (b) cotransfected with a construct expressing soluble BFP as a live cell transfection marker into GFP-Rab6 HeLa or PtK 2 stable cell lines. The degree of cotransfection was monitored by immunofluorescence with anti-Rab6 antibody and by control experiments where the BFP expression construct was cotransfected with pEGFP-Rab6wt. Cotransfected cells were visualized 12, 18, 24, and 36 h after removal of the precipitate. For live cell double-labeling experiments with fluorescent protein spectral variants, cells were transiently cotransfected with CFP and YFP-fusion protein expression constructs mixed in equal amounts by weight before transfection. Cotransfected cells expressing approximately equal levels of CFP and YFP-fusion proteins (based on relative fluorescent intensity) were imaged 18–36 h after removal of the transfection precipitate. FP-Rab6 localization and trafficking were not detectably affected by incubation at the different temperatures used for the STB internalization (see below). Microinjection of anti-EAGE was performed as described . Live cell confocal microscopy was performed on the Compact Confocal Camera (CCC) as described previously . Cells were maintained on the microscope stage using an FCS2 system (Bioptechs) for perfusion experiments, or a slide chamber fitted for 15-mm round coverslips, together with an objective heater (Bioptechs). Cells were imaged in DME (HeLa) or MEM (PtK 2 ) that had been preequilibrated in a 5% CO 2 incubator. All images were taken with a 63× 1.4 NA Plan-Apochromat III DIC objective (Carl Zeiss). For multi-channel imaging, each fluorescent dye was imaged sequentially in either frame-interlace or line-interlace modes to eliminate cross-talk between the channels. EGFP fluorescence was excited with 488 nm argon-ion laser line and imaged using a NT80/20/543 beamsplitter and a 505-nm longpass emission filter. Cy3 fluorescence was excited with a 543-nm HeNe laser and imaged through a 560-nm longpass emission filter. CFP was excited with a 430-nm laser line (Directly Doubled Diode/D 3 , Coherent) and imaged through a combination of 440–505-nm bandpass and 525-nm longpass emission filters. YFP was exited with the 514-nm laser line and imaged through a 525-nm longpass emission filter. Cross-talk in this configuration was quantitated; YFP signal in the CFP channel was below detectable limits. CFP in the YFP channel was 4% of the signal in the CFP channel, adjusted for illumination intensity. All image processing was performed on a Macintosh computer using the public domain NIH Image program version 1.62 (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Eight-bit movies from two channels were overlaid to produce 16- or 24-bit color Quicktime movies in Adobe Premier. NIH Image macros used for data processing are available at http://www.embl-heidelberg.de/~jwhite/. Adobe Photoshop 5.02 and Illustrator 8.0 were used to prepare final figures for publication. Additional image processing procedures and image correlation analysis to quantitate colocalization and motility are described in the online supplemental material. Recombinant wild-type Shiga toxin B-fragment was purified as described . The wild-type fragment contains no KDEL sequence or other known signals for ER retrieval and localization . Purified STB was labeled with Cy3 according to manufacturer's instructions (Amersham Life Science, Inc.) as in previous studies . STB internalization experiments were performed as described . Imaging was initiated by flipping the coverslip into a slide chamber at the imaging temperature (37 or 28°C, as indicated), focusing, and starting the preoptimized time-lapse program on the CCC. All STB experiments were performed with HeLa cells, since PtK 2 cells did not bind the labeled STB. HeLa cells were mock transfected or transfected with mutant Rab6 expression plasmids basically as described . In brief, 10 5 HeLa cells were for 30 min infected with T7 polymerase expressing vaccinia virus and then transfected with 3 μg of vector DNA using DOTAP (Boehringer). After 2 h, serum-free transfection medium was replaced by medium containing 10% fetal calf serum (Life Technology). Transfection efficiency as determined by immunofluorescence analysis was routinely above 80%. Functional assays were performed as described below in the continuous presence of 10 mM hydroxyl urea (Sigma Chemical Co.). Purified Shiga toxin was added to the cells at the indicated concentrations. The cells were incubated for 100 min at 37°C, put on ice, and washed 2 times with leucine-free MEM medium (Life Technology). After addition of 1 μCi/ml of [ 3 H]leucine (151 Ci/mmol; Amersham) in ice-cold medium, the cells were transferred to 37°C for 20 min, washed and lysed in 0.1 M KOH on ice, and proteins were precipitated with TCA. Incorporation of radioactive leucine was determined by liquid scintillation counting after filtration on GF/C filters (Whatman). Scatchard analysis was done as described using B-fragment iodinated with IodoBeads (Pierce) to a specific activity of 5,000 cpm/ng. Movies corresponding to Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 are available online along with instructions for viewing them. Additional Materials and Methods describe how image correlation analysis was used to quantitate colocalization and motility. Available at http://www.jcb.org/cgi/content/full/147/4/743/DC1. To observe the dynamics of Rab6 in live cells we fused fluorescent proteins (cyan, green, or yellow) to the NH 2 terminus of wild-type Rab6 to generate FP-Rab6. Fig. 1 a shows the overall topology of the fusion protein and putative orientation in a membrane bound state. We used FACS ® to generate HeLa and PtK 2 cell lines stably expressing GFP-Rab6. In these cells, GFP-Rab6 associated with compact structures next to the nucleus, consistent with localization to the Golgi. Diffuse fluorescence was observed in brighter cells, and likely represented a cytosolic pool of GFP-Rab6. Western blotting of whole cell extract showed GFP-Rab6 was mildly overexpressed in the stable HeLa cell line . At the ultrastructural level, cryoimmunoelectron microscopy using an antibody against GFP showed labeling of GFP-Rab6 over Golgi stacks and associated tubulovesicular elements, with no significant labeling over other structures . CLSM and immunofluorescence showed that the GFP-Rab6 fluorescence adjacent to the nucleus colocalized with the Golgi protein Giantin . GFP-Rab6 was also present in structures that were devoid of Giantin, in punctate peripheral elements, and at distinct sites in the corners of the cell that were near sites containing Paxillin, a focal adhesion component . The punctate GFP-Rab6 elements were superficially similar in appearance to endosomes, but they did not colocalize with a number of endosomal markers. In fixed cells, transferrin receptor failed to colocalize with GFP-Rab6 peripheral elements (data not shown). In live cells, rhodamine-transferrin internalized for short or long periods did not coincide with FP-Rab6, nor did a fluorescent protein fusion to Rab5, a marker of early endosomes, nor did fluorescently labeled Shiga toxin B-fragment blocked in endosomes at 19.5°C (data not shown). Visualization of the lysosome-specific vital dye LysoTracker in live FP-Rab6 cells showed different dynamics and no colocalization with FP-Rab6 trafficking structures (data not shown). To determine if stable GFP-Rab6 expression altered the endogenous Rab6 compartment, we immunolocalized the Rab6 effector Rabkinesin-6 in GFP-Rab6 HeLa cells . Since anti-Rab6 antibodies recognize GFP-Rab6 , precluding comparison of native and GFP-Rab6, it was not meaningful to localize native Rab6 in GFP-Rab6 cells. Overall Rabkinesin-6 distribution was consistent with its reported localization in untransfected cells . GFP-Rab6 structures at the very periphery did not colocalize with Rabkinesin-6 (arrows). FP-Rab6 localized to the Golgi complex, was consistent with the localization of endogenous Rab6, and did not obviously affect the distribution of a Rab6 effector. These results suggest that the intracellular compartment defined by FP-Rab6 in living cells was relevant to the compartment defined by native Rab6. To observe the dynamics of the intracellular compartment defined by FP-Rab6 in living cells, we observed GFP-Rab6 HeLa and PtK 2 stable cell lines using time-lapse live cell microscopy. Standard imaging conditions used confocal laser scanning microscopy (CLSM) to collect single confocal slices every 1–10 s for periods of 5 to >60 min. The GFP-Rab6 compartment was highly dynamic. It consisted of a stable Golgi region, motile peripheral trafficking elements, a diffuse network with a nebulous appearance, and distinctive accumulations in distal corner regions . Discrete globular and long (>2 μm) tubular structures exited the Golgi and translocated outward along curvilinear paths to the cell periphery. Trafficking elements translocated at ∼0.6–1 μm/s. The primary flux of GFP-Rab6 trafficking was away from the Golgi toward the cell periphery, but some elements translocated from the periphery to the Golgi and entered, some left and reentered, and tubules occasionally extended and then retracted. A brightest-point projection through the movie shows the tracks followed by trafficking structures, and indicates the high probability of elements trafficking through the corner regions. The corner regions were highly dynamic; we observed trafficking elements enter and coalesce there, exit and traffic between sites, and exit and disperse into the diffuse network. Dispersal was not due to trafficking structures simply moving out of the plane of focus, since we observed fluorescence disperse laterally within the focal plane and this dispersion was clearly distinguishable from trafficking structures which did move in and out of focus. The diffuse network had distinct dynamics and structure, and was closely associated with the trafficking elements and corner regions. We observed slight differences in GFP-Rab6 trafficking within a population of cells. Some cells were more active, as judged by the number of globular and tubular GFP-Rab6 trafficking elements and their frequency of translocation. Not all cells in a population showed accumulation in peripheral corner regions, but we always observed significant trafficking to these areas. Occasionally, the tubular structures extending from the Golgi were quite dramatic, extending all the way to a peripheral endpoint without detaching . We observed no substantial differences between trafficking in PtK 2 and HeLa cells, although tubulation was slightly less apparent in PtK 2 cells. Live cell double-labeling experiments with a Golgi marker, T2-CFP and YFP-Rab6 showed FP-Rab6 trafficking did not reflect the dynamics of the Golgi itself, and behavior was specific to the Rab6 fusion, since FP-Rab6 trafficking did not resemble trafficking of an analogous fusion to the Golgi-associated Rab8 protein (data not shown). The level of GFP-Rab6 did not observably affect trafficking, indicating GFP-Rab6 trafficking structures were not induced by GFP-Rab6 expression. High expression levels of GFP-Rab6, indicated by comparing variations in fluorescence intensity within a population, resulted in higher levels of cytosolic fluorescence rather than increased trafficking activity (not shown). Transient overexpression of native Rab6 wild-type or GTP-mutant (Q72L) in GFP-Rab6 cells using a CMV promoter vector also had no noticeable effect on trafficking (see Materials and Methods). Overexpression of native Rab6 wild-type or Q72L using the T7 vaccinia system dispersed GFP-Rab6 into diffuse cytosolic fluorescence (data not shown). To establish whether GFP-Rab6 trafficking elements were relevant to the role of native Rab6, we performed immunofluorescence with anti-Rab6 antibody in untransfected HeLa cells. Careful observation revealed that native Rab6 shares many features with GFP-Rab6. Fig. 2 c shows fixed, untransfected HeLa cells stained with anti-Rab6 polyclonal antibody and examined by CLSM. Peripheral corner regions (1), peripheral tubular and globular elements (2 and 3), and tubules extending from the Golgi (4) are all apparent. These observations suggest that GFP-Rab6 reveals relevant features of the membrane system defined by native Rab6, and further indicate that GFP-Rab6 expression does not induce abnormal trafficking structures. Since native Rab6 associates with a microtubule-dependent kinesin, Rabkinesin-6, we determined whether GFP-Rab6 trafficking elements translocated along microtubules. To establish a requirement for microtubules, we treated GFP-Rab6 HeLa or PtK 2 cells with nocodazole, which prevents dynamic microtubules from repolymerizing, and observed the effects directly in live cells. During the first 10 min of nocodazole exposure, the flux of GFP-Rab6 trafficking concentrated along a few linear tracks, which would be consistent with a flux directed along remaining stable microtubules. After this period, long-range GFP-Rab6 trafficking ceased, showing microtubules were required for long-range GFP-Rab6 translocation (not shown). To directly show FP-Rab6 trafficking along microtubules, we performed live cell double-labeling experiments with CFP- or YFP-tubulin and YFP- or CFP-Rab6. FP-Rab6 trafficking elements translocated along fluorescent microtubules; corner regions and peripheral sites corresponded to the ends of labeled microtubules . Together these two results show GFP-Rab6 elements traffic along microtubules. To ascertain whether the trafficking that we detected at the acquisition frequency of the laser-scanning microscope (1–10 s/frame) was a complete representation of GFP-Rab6 trafficking, we imaged GFP-Rab6 in live cells with conventional fluorescence and fast CCD detection to acquire images every 100–500 ms. GFP-Rab6 trafficking observed at rapid acquisition rates was substantially the same, considering the lower spatial resolution of this approach and the out-of-focus haze that obscured the objects in focus . Together with direct observation by eye, the fast CCD acquisition makes it highly unlikely that we have missed detecting significant fast-moving GFP-Rab6 structures. Thus, our movies are an accurate representation of GFP-Rab6 trafficking. Since earlier studies provided evidence that Rab6 plays a role in retrograde transport, and FP-Rab6 trafficking structures appeared to be transport carriers translocating from the Golgi to the cell periphery, we determined whether they contained retrograde cargo. As a well characterized retrograde cargo, we used the wild-type B-fragment of Shiga toxin . STB retrograde transport can be synchronized by binding the purified fragment to the cell surface at 4°C and initiating transport by shifting cells to 37°C . We examined the retrograde transport of STB fluorescently labeled with Cy3 in live GFP-Rab6 HeLa cells. The overall pattern of STB transport was consistent with that previously described in untransfected HeLa cells . STB trafficking structures coincided with GFP-Rab6 trafficking during the time when STB undergoes transport from the Golgi to the ER . Coincidence was first apparent at a time after STB had started to accumulate in the Golgi, 20–40 min after the shift to 37°C, and was most apparent until 60–90 min at 37°C. Tubular and globular structures labeled by both STB and GFP-Rab6 left the Golgi and traveled outward to peripheral sites. STB accumulated together with GFP-Rab6 in corner regions and seemed also to disperse outwards with GFP-Rab6 from these endpoints . STB in corner regions became brighter, persisted for 60–90 min of internalization, and then gradually disappeared as ER fluorescence increased . STB and GFP-Rab6 were not found in the same element at concentrations that gave comparable fluorescence intensities, making conventional colocalization difficult. Movies circumvented this problem by showing structures labeled by both proteins moving in the same manner over time. To further compare STB and GFP-Rab6 trafficking we varied the temperature shift protocols. To compare STB endosome→Golgi transport with GFP-Rab6, STB was blocked in early endosomes at 19.5°C and then cells were shifted to 37°C to initiate transport . At 19.5°C or directly after the shift, STB in early endosomes did not coincide with GFP-Rab6. Only after STB coalesced from early endosomes and began to accumulate in the Golgi and in peripheral corner regions did we observe coincidence of STB and GFP-Rab6 trafficking structures. To extend initial exit from the Golgi apparatus over a longer period of time, we bound STB at 4°C, pulsed it into the Golgi at 37°C for 15–25 min, and then shifted to 28–30°C . Coincidence of GFP-Rab6 and STB trafficking was best under these conditions, with fewer extraneous structures that did not contain both proteins. These results are consistent with the hypothesis that GFP-Rab6 TCs are used by STB during the initial phase of transport from the Golgi to the ER. STB trafficking structures also coincided with GFP-Rab6 trafficking at later times of STB internalization . Coincident trafficking after 5 h of internalization was more difficult to discern because the high level of STB fluorescence in the ER obscured visualization of discrete trafficking structures, and not all GFP-Rab6 trafficking structures contained visible levels of STB. However, coincident trafficking was apparent in regions at the edge of the cell, where STB ER fluorescence was less intense . Since at this time STB cycles between the ER and the Golgi , this result is consistent with the idea that STB traffics extensively in GFP-Rab6 elements during the initial pulse of STB transport from the Golgi to the ER, and then continues to traffic in GFP-Rab6-positive structures during the retrograde stage of Golgi↔ER cycling. To verify that FP-Rab6 trafficking structures specifically contained retrograde cargo, we observed a number of different anterograde cargo markers in live cell double-labeling experiments. In no case did we observe coincidence of these markers with peripheral FP-Rab6 trafficking elements (not shown). To verify that stable expression of FP-Rab6 did not induce STB trafficking structures, we observed STB transport live in untransfected HeLa cells using the same temperature shifts under the same imaging conditions. STB trafficking appeared the same as in the GFP-Rab6 stable cell line (not shown). Since STB and GFP-Rab6 trafficking structures were observed independently, it is unlikely that they are induced both by STB and by GFP-Rab6 expression. Rather, the coincident trafficking of the two proteins is consistent with a preexisting pathway. Since our results suggested GFP-Rab6 trafficking structures were retrograde transport carriers along a pathway from the Golgi to the ER, we determined whether they coincided with the Golgi→ER pathway used for the retrieval of ER residents and for the cycling of the KDELR and p24 proteins. As a marker for this pathway, we used a fluorescent protein fusion to the KDELR, KDELR-FP . In stably transfected HeLa and PtK 2 cells, KDELR-FP distributed between a compact juxtanuclear region and the network of the ER, indicating that it localized properly (see supplemental movies at http://www.jcb.org/cgi/content/full/147/4/743/DC1). KDELR-FP showed characteristics of native KDELR; it accumulated in peripheral punctate structures upon incubation at 15°C and after treatment with BFA. Fluorescence loss in photobleaching (FLIP) experiments showed that the Golgi can be depleted of fluorescence by repeatedly photobleaching a region of the ER, indicating KDELR-FP exchanged from the Golgi to the ER (not shown). KDELR-FP thus reveals features of early Golgi↔ER cycling in live cells. KDELR-GFP trafficking in live cells differed markedly from GFP-Rab6 trafficking . Tubulation was much less extensive, and globular elements translocated over shorter distances in a saltatory fashion, switching direction often. KDELR-GFP did not accumulate in corner regions, unlike both GFP-Rab6 and STB . Unlike KDELR-GFP, GFP-Rab6 did not accumulate in peripheral punctate structures upon prolonged (2–4 h) incubation at 15°C (not shown). To test whether these differences represented a separation of peripheral trafficking elements, we performed live cell double-labeling experiments with KDELR-CFP and YFP-Rab6. In transiently cotransfected cells, KDELR-CFP and YFP-Rab6 peripheral trafficking elements did not coincide, and exhibited different dynamics in the same living cell . Consistent with these results in live cells, immunofluorescence in fixed cells showed that endogenous KDELR peripheral elements did not overlap with GFP-Rab6 in the stably transfected cell line (not shown). These results suggest that FP-Rab6 trafficking is distinct from the KDELR-FP trafficking, and thus morphologically separate from the transport pathways used by the endogenous KDELR and KDELR-FP. To further establish these differences, we compared the distribution of two different COPI coatomer subunits, β and β′, to peripheral GFP-Rab6 trafficking structures in the stably transfected HeLa cells. Immunofluorescence with antibodies against either β- or β′-COP showed that peripheral COPI structures were separate from peripheral GFP-Rab6 elements, and indeed seemed to exclude one another . This result indicates that GFP-Rab6 trafficking elements are not COPI-coated. FP-Rab6 trafficking structures accumulated wild-type Shiga toxin B-fragment, but excluded KDELR and KDELR-FP. To determine whether STB trafficked in KDELR-GFP-positive as well as in GFP-Rab6-positive structures during transport to the ER, we observed STB transport in live KDELR-GFP HeLa cells . At all times of transport, STB and KDELR-GFP tended to segregate from one another outside of the Golgi. At early times (15–120 min at 37°C), we observed little or no coincidence of STB and KDELR-GFP peripheral trafficking structures, and KDELR-GFP did not coincide with STB in corner regions . At later times (2–5 h at 37°C), we observed partial coincidence of STB and KDELR-GFP globular trafficking structures, especially in cells that had internalized high levels of STB, but never coaccumulation in distinctive corner regions. After 5 h of internalization, after STB was readily visible in the ER network, STB partitioned to subdomains of the ER network that were depleted for KDELR-GFP fluorescence ; STB in the ER extended further to the cell periphery. These results are consistent with the observation that FP-Rab6 trafficking structures coincide with STB but not KDELR-FP, and provide morphological evidence that the bulk of STB is not transported to the ER via the pathway defined by KDELR-GFP. The partial coincidence may represent a minor proportion of STB that is transported via this pathway at later times, or it may represent mis-sorting into this pathway at high levels of STB. To determine whether the dynamic morphological differences we observed between FP-Rab6 and KDELR-FP represented functional differences between the two pathways, we inhibited COPI function in KDELR-GFP or GFP-Rab6 HeLa cells and observed the effect on trafficking. We microinjected anti-EAGE antibody, a potent inhibitor of COPI function in vivo , and compared the dynamics of injected cells to uninjected cells in the same microscope field. In injected cells, the overall motility of KDELR-GFP was inhibited to 60% of an uninjected cell in the same movie, but GFP-Rab6 motility was not significantly inhibited . These results indicate a functional difference underlies the segregation of FP-Rab6 or STB from KDELR-FP, endogenous KDELR, and COPI components, and agrees with those of Girod and coworkers . Since the ER is the final destination of STB retrograde transport, and extends in a network throughout the cell periphery, where active FP-Rab6 trafficking takes place, we observed ER dynamics together with FP-Rab6 or STB in live cells. PtK 2 cells were cotransfected with expression constructs for the ER resident protein CFP-Sec61β, and YFP-Rab6. FP-Sec61β is a fluorescent protein fusion to a component of the machinery that translocates nascent polypeptides into the lumen of the ER . FP-Rab6 TCs translocated along the network of the peripheral ER . ER and FP-Rab6 dynamics were intimately related—often an ER tubule extends with FP-Rab6 TC at its tip, or pulses of brighter intensity within the ER network fluctuated synchronously, following behind a moving FP-Rab6 TC . Stable domains of FP-Rab6 remained associated with the tips of ER tubules. These associations appear specific to FP-Rab6 TCs, since other microtubule-associated trafficking structures, such as those defined by KDELR-FP, do not display such associations . FP-Rab6 transport carriers are thus closely associated with the final destination of their cargo. Corner regions were a distinctive feature of both GFP-Rab6 and STB Golgi→ER transport. The appearance and disappearance of STB accumulation in these regions correlated with the period of maximal initial transport from the Golgi to the ER; they appeared after ∼15 min of transport and disappeared by 5 h . This correlation prompted us to further define the relationship between the corner regions and the ER during STB retrograde transport, so we observed STB dynamics in live HeLa cells transiently transfected with GFP-Sec61β. Fig. 8 b shows STB dispersal from a peripheral corner region into the network of the ER. Quantitative image correlation analysis shows that STB and GFP-Sec61β are initially separate, and increasingly colocalize as STB adopts the pattern of the ER network over time . Relative STB levels in the ER increase 22-fold comparing the first and last frame of the movie , and the general trend shows a fivefold increase over time . The correlation between STB and the ER images at the later times are comparable to images of two different antibodies staining the same structure (not shown). Peripheral corner regions are thus sites of concentrated ER entry. Shiga holotoxin transfers from the ER to the cytosol where it inactivates ribosomes through a specific modification of 28S rRNA . Toxicity is thus dependent on ER arrival of the toxin, presumably mediated by STB. To ascertain whether Rab6 regulates Golgi→ER transport, we examined the effect of the Rab6 T27N mutant on toxicity of Shiga toxin. Rab6T27N has a strongly reduced affinity for GTP and is thus at steady state preferentially in the GDP-bound conformation. Corresponding Rab protein mutants have dominant negative effect in several experimental systems . Overexpression of Rab6T27N with the T7 vaccinia system significantly reduced Shiga toxin-induced inhibition of protein biosynthesis in HeLa cells . Overexpression of a Rab6 mutant with a mutation in its effector loop (Rab6I46E) had no effect on toxicity . Scatchard analysis on cells that were transfected for 6 h showed that compared with mock transfected cells, Rab6T27N overexpressing cells had the same number of binding sites for STB (0.79 × 10 6 versus 0.78× 10 6 sites/cell, respectively), and that STB affinity for its receptor globotriaosyl ceramide was the same in both conditions (0.135 and 0.103 μM, respectively). STB transport to the Golgi was not significantly altered in cells overexpressing Rab6T27N for 6 (not shown) or even 12 h , and costaining for the Golgi marker CTR433 shows overall Golgi morphology is intact, indicating that inhibition of toxicity is due to specific impairment of a Golgi→ER transport step. Together, these results suggest that native Rab6 regulates the FP-Rab6/STB Golgi→ER transport pathway. Our results are consistent with the idea that the FP-Rab6 membrane system defines a separate compartment that corresponds to a Golgi→ER transport pathway containing specific retrograde cargo and exhibiting distinct functional requirements. The FP-Rab6 compartment comprises all FP-Rab6 elements, the Golgi-associated pool, tubular and globular trafficking elements, and peripheral corner regions. It is separate from some of the traditionally defined morphological compartments (endosomes, lysosomes, and Golgi→plasma membrane transport carriers), and partly overlaps with others (Golgi, ER). FP-Rab6 dynamics in live cells emphasized elements that were neglected in static images, thus revealing morphological features of endogenous Rab6 that had previously been overlooked . Since endogenous Rab6 and FP-Rab6 have common features , FP-Rab6 dynamics are most likely not induced by overexpression of the FP-Rab6 fusion protein. During the period when STB underwent transport from the Golgi to the ER, it accumulated in FP-Rab6 trafficking structures and together with FP-Rab6 in distinctive corner regions. At later times, when the STB has been shown to cycle between the ER and Golgi , it continued to traffic in FP-Rab6 elements, although to a lesser extent. These observations indicate that FP-Rab6 trafficking elements are retrograde TCs, and are consistent with the idea that the initial vectorial pulse of STB transport concentrates it in FP-Rab6 retrograde carriers, resulting in strong coincidence at early times. At later times, as STB partitions between the Golgi and ER and begins cycling, only a proportion undergoes Golgi→ER retrograde transport during a given period, resulting in weaker coincidence. Since STB and FP-Rab6 trafficking behavior can be observed independently, it is unlikely that one or the other induces the transport carriers, and further support the ideas that FP-Rab6 does not induce trafficking elements, and that STB uses a preexisting cellular pathway. The coincidence of FP-Rab6 and STB in distinctive peripheral corner regions prompted us to investigate these regions further. STB dissipated into the ER network directly from these regions, indicating that they are ER entrance sites . We cannot exclude that STB entered the ER at other locations or along the entire ER network; we conclude only that ER entrance from the corner regions was particularly pronounced. Other transport steps are also concentrated at specialized sites: ER exit occurs in specialized regions that recruit COPII coat proteins to the membrane to concentrate and sort anterograde cargo from the ER , and delivery of secretory cargo also appears to be directed to a specialized area of the plasma membrane, a targeting patch, in both yeast and polarized mammalian cells . STB traverses endosomes before it arrives in the Golgi , so it remained possible that the STB which coincided with FP-Rab6 trafficking elements was in endosomes. This is, however, highly unlikely since we observed STB in FP-Rab6 trafficking structures long after the bulk of STB had exited endosomal structures (after 5 h at 37°C), and since STB blocked in the endosomes at 19.5°C did not coincide with FP-Rab6 (not shown). Consistent with this, FP-Rab6 peripheral elements never colocalized with a number of different endosomal markers, including transferrin receptor and FP-Rab5 in fixed cells, and labeled transferrin in live cells (continuous uptake; not shown). It is also highly unlikely that FP-Rab6 trafficking elements represent TCs containing post-Golgi anterograde cargo, despite superficial similarities . When imaged in live cells, Golgi-to-plasma membrane TCs, revealed by four separate FP markers, exhibited different behavior, trafficked to different regions of the cell, and did not coincide in live cell double-labeling experiments (White, J., unpublished observations). Since FP-Rab6 coincided with a retrograde cargo during Golgi→ER transport, we expected FP-Rab6 TCs to coincide with proteins involved in retrieval of ER residents. This was, however, not the case. FP-Rab6 was separate from KDELR-FP in live cells, and COPI components and endogenous KDELR in fixed cells. Consistent with this, STB segregated away from KDELR-FP in live cells; we observed no coincidence at early times of STB transport, and only partial coincidence from 2 to 5 h. Even when both proteins were in the ER network, they excluded one another, appearing as separate domains within the ER network. These results indicate that STB traffics predominantly in the FP-Rab6 pathway. The partial coincidence of STB and KDELR-FP-positive structures may be due to specific retrograde transport in these structures, or may be mis-sorting into this pathway at higher STB levels. Since KDELR-FP cycles between the ER and the Golgi, a subset of KDELR-FP trafficking may represent an anterograde transport step, leading to the additional possibility that STB cycles to the Golgi in anterograde structures that contain KDELR-FP, and to the ER in FP-Rab6 structures. The differences between ER retrieval and FP-Rab6/STB retrograde transport extend to the functional level. Previous studies indicate toxin proteins define two functionally different retrograde pathways to the ER. Jackson and coworkers have shown some toxin proteins require the KDEL-retrieval system for efficient transport to the ER, while others do not . Girod and coworkers have recently shown that microinjection of anti-EAGE antibodies, potent inhibitors of COPI function in live cells inhibits Golgi→ER transport of toxin proteins which contain KDEL-like motifs. In contrast, transport of STB, and likewise the holotoxin, is unaffected (Girod, A., B. Storrie, J.C. Simpson, J.M. Lord, T. Nilsson, and R. Pepperkok, manuscript submitted for publication). Here, we show that FP-Rab6 trafficking is not inhibited by anti-EAGE microinjection, but KDELR-FP trafficking is reduced to 60% of control . These results are consistent with the observation that modified STB reach the ER equally well with or without a KDEL retrieval signal. They also imply that trafficking is linked to transport of KDELR (and presumably other components of the ER retrieval system), a relationship that is often assumed but which has not been shown. It was previously unclear whether the toxin protein pathway was preexisting or a consequence of the toxin itself, but the coincidence of FP-Rab6 and STB in trafficking structures with the same functional characteristic strongly argues that STB uses a COPI-independent cellular pathway defined by FP-Rab6. Our results suggest native Rab6 regulates this alternate Golgi→ER transport pathway. In previous studies, high overexpression of active forms of Rab6 (Q72L mutant or wild-type) stimulated retrograde transport from the Golgi to the ER, progressively relocating Golgi residents to the ER and indirectly inhibiting intra-Golgi anterograde transport . This interpretation fits well with the general idea that Rab proteins are active in their GTP-bound form . Consistent with these observations, overexpression of Rab6:GDP (T27N) reduces toxicity of the holotoxin by inhibiting a Golgi→ER transport step , without altering earlier transport steps or overall Golgi morphology. We note that overexpression of active forms of Rab6 does not in fact stimulate ER arrival of STB, because STB must be transported to the ER via an intact Golgi structure , and upon overexpression of active Rab6 the Golgi redistributes to the ER and is no longer intact . Taken together, these results imply that this pathway is activated by GTP-Rab6 and inhibited by GDP-Rab6. Why would an alternate Golgi→ER pathway exist? Presumably it would be used by a subset of cellular components, not only by toxin proteins. Supporting this, verotoxin and Shiga-like toxins have homology to cellular proteins that are transported from the cell surface to the nuclear envelope, a subdomain of the ER . These components do not have a recognizable ER retrieval motif, yet appear to reach the ER. Other cellular components that return to the ER also lack a retrieval motif. Resident Golgi proteins slowly but continuously cycle through the ER , apparently via COPI-independent mechanisms . A transport pathway to the ER could be used for degradation of transmembrane proteins: certain transmembrane ER proteins may be extracted from the membrane by retrotranslocation to be targeted for proteasome-mediated degradation in the cytosol . Transmembrane proteins from other intracellular compartments could also undergo ER-based degradation. Another rationale for an alternate pathway would be a mechanism to recycle lipid components to the ER. Intracellular lipid trafficking is not yet well characterized, but it is notable that STB binds a glycolipid receptor, globotriaosyl ceramide, at the cell surface . It remains to be shown whether STB follows its glycolipid receptor as far as the ER. The idea that Rab proteins define trafficking routes within the cell was proposed based primarily on the apparent specificity of their localization , but until this study has never been directly tested. Observation of Rab protein dynamics provides even further insight than simple localization: Previous studies localized native Rab6 to the Golgi , but its cellular function there was unclear . Observation of FP-Rab6 TCs and the accumulation of a specific retrograde cargo within them during Golgi→ER transport directly indicate a role for Rab6 in transport to the ER. Furthermore, microtubule-dependent translocation of FP-Rab6 TCs rationalizes the direct association of Rab6:GTP with Rabkinesin-6 . Our studies of FP-Rab6 dynamics in live cells provide the cellular context in which to interpret the function of Rab6 at the molecular level. | Study | biomedical | en | 0.999999 |
10562279 | Skin fibroblast cell lines from patients with peroxisome biogenesis disorders are referred to by their PBD numbers and were cultured in complete medium (DME supplemented with 10% FBS and penicillin/streptomycin). Transfections were performed by electroporation using the protocol outlined by Chang et al. 1997 and were processed 2 d later for immunofluorescence. Immunofluorescence experiments were performed essentially as described . In brief, cells were grown on cover glasses, fixed, permeabilized, washed, incubated with the primary antibodies, washed extensively, incubated with the secondary antibodies, washed extensively, and then mounted on glass slides. Standard permeabilization was for 5 min with 1% Triton X-100, which permeabilizes both plasma and peroxisome membranes. Differential permeabilization was for 2–4 min with 25 μg/ml digitonin, which permeabilizes the plasma membrane but does not permeabilize the peroxisome membrane. Hence, only cytoplasmically exposed antigens can be detected under these conditions. Differential permeabilization experiments were generally performed with additional controls to ensure that the incubation in digitonin had not permeabilized any intracellular membranes. Rabbit polyclonal antibodies against the PTS1 tripeptide ser-lys-leu-COOH have been described and the anti-human PEX5 antibodies were generated against bacterially expressed forms of the protein. Mouse mAbs against the c-myc epitope were obtained from the tissue culture supernatant of the hybridoma 1-9E10 . Rabbit polyclonal antibodies against c-myc, sheep anti-human catalase antibodies, and fluorescently labeled secondary antibodies were obtained from commercial sources. We previously described a mutation detection strategy for human PEX12 . In brief, the entire 2.3-kb PEX12 gene was amplified from the total human genomic DNA using the primers Chang-21 and Chang-20 ( Table ). The coding regions and intron/exon junctions were sequenced directly from the PCR product using the primers used for amplification as well as several additional gene specific primers ( Table , Chang-17, Chang-24, and Chang-30). Total genomic DNA was isolated from PBD054 cells using the PureGene kit (Gentra Systems, Inc.). Most of the plasmids used for yeast two-hybrid studies were based on pPC62, a LEU2 -based GAL4 DNA–binding domain fusion protein expression vector and pPC86, a TRP1 -based GAL4 transcriptional activation domain (AD) fusion protein expression vector . These plasmids contain two PvuI sites in symmetric positions, and the PvuI fragments of these plasmids were switched to create pJL59 and pPC86/L2. The plasmid pJL59 is identical to pPC62 except that it contains the TRP1 gene in place of the LEU2 gene, and pPC86/L2 is identical to pPC86 except that it contains the LEU2 gene in place of the TRP1 gene. The plasmid pGAD424 (CLONTECH Laboratories) was also used for expression of some GAL4-AD fusion proteins. The plasmid pJL59- PEX12C was created by amplifying a subregion of PEX12 using the primers 12.2HC5′ and 12.2H3′ ( Table ) and pcDNA3- PEX12 as the template , cleaving the resulting 370-bp product with SalI and NotI, and inserting this fragment between the SalI and NotI sites of pJL59. This plasmid is designed to express a fusion between the GAL4 DNA–binding domain and amino acids 260–359 of human PEX12. The plasmid pJL59- PEX12C /S320F was created using the same cloning strategy except that pcDNA3- PEX12 /S320F (see below) was used as the template. The plasmid pPC86/L2- PEX10C was created by amplifying a subregion of PEX10 using the primers 10.2HC5′ and 10.2H3′ ( Table ) and pcDNA3- PEX10 as the template, cleaving the resulting 327-bp product with SalI and NotI, and inserting this fragment between the SalI and NotI sites of pJL59. This plasmid is designed to express a fusion between the COOH-terminal 87 amino acids of human PEX10 and the GAL4 activation domain. The plasmid pPC86/L2- PEX14 encodes a fusion between the GAL4 activation domain and full-length PEX14 and was created by amplifying the entire PEX14 open reading frame (ORF) using the primers 14.2H5′ and 14.2H3′ ( Table ) and total human cDNA as a template, cleaving the resulting product with SalI and NotI, and inserting this fragment between the SalI and NotI sites of pPC86/L2. The PEX5 two-hybrid plasmids were created as follows. The entire PEX5 S ORF was excised from plasmid pGD100 by cleavage with NcoI, after which the ends were made blunt with the Klenow fragment of DNA PolI and dNTPs, and then cleavage with BglII. The resulting 2-kb PEX5 S fragment was isolated and inserted between the SmaI and BglII sites of pGAD424, downstream of and in-frame with the GAL4 transcription activation domain. The resulting plasmid, pGAD424-PEX5S, encodes a fusion between the GAL4 activation domain and full-length PEX5S. The plasmid pGAD424- PEX5 L was created by cleaving pPEX5L with NcoI, making the ends blunt with the Klenow fragment of DNA PolI and dNTPs, cleaving this DNA with BglII, isolating the resulting 2-kb PEX5 L fragment, and inserting it between the SmaI and BglII sites of pGAD424. The plasmid pGAD424- PEX5S /ΔN encodes a fusion protein between the GAL4 transcriptional activation domain and the COOH-terminal 317 amino acids of PEX5. It was created by excising the NH 2 terminally truncated PEX5 fragment from pGD105 with NcoI (after which the ends were made blunt with the Klenow fragment of DNA PolI and dNTPs) and BglII and inserting the resulting 1-kb PEX5 fragment between the SmaI and BglII sites of pGAD424. Bacterial expression vectors were based on a derivative of pMAL-c2 (New England Biolabs Inc.), pMBP differs from pMAL-c2 in that it contains a SalI site downstream of the EcoRI site ( GAATTC AA GTCGAC , EcoRI and SalI sites underlined), and a NotI site upstream of the HindIII site ( GCGGCCGCAAGCTT , NotI and HindII sites underlined). pMBP-PEX12C was created by excising the PEX12 SalI-NotI fragment from pJL59- PEX12C and inserting it between the SalI and NotI sites of pMBP. pMBP- PEX12C /S320F was created by excising the PEX12 SalI-NotI fragment from pJL59- PEX12C /S320F and inserting it between the SalI and NotI sites of pMBP. All mammalian expression vectors are based on pcDNA3 (Invitrogen Corp.). We have previously described the expression vectors pcDNA3- PEX5 S , pcDNA3- PEX5 L , pcDNA3- PEX10 , and pcDNA3- PEX12 . To create pcDNA3- PEX12 /3xmyc, the PEX12 ORF was amplified using the oligonucleotides Chang-21 and Chang-10 ( Table ) and pcDNA3- PEX12 as a template. These primers append an Asp718 site upstream of the ORF and replace the stop codon with a BamHI site. The resulting PCR fragment was cleaved with Asp718 and BamHI and cloned upstream of the triple c-myc tag in pcDNA3-3xmyc . To create the plasmid pcDNA3- PEX12 /S320F, we first amplified the PEX12 ORF from PBD054 cDNA. Total RNA was extracted from PBD054 cells using the PureScript kit (Gentra Systems, Inc.) and PEX12 cDNA was synthesized as described . The PEX12 ORF was amplified from the first strand PBD054 PEX12 cDNA using the primers Chang-21 and Chang-20 ( Table ), cleaved with Asp718 and BamHI, and cloned between the Asp718 and BamHI sites of pcDNA3, generating pcDNA3- PEX12 /S320F. The sequence of the final plasmid was confirmed to ensure the presence of the S320F mutation and the absence of any undesired mutations. The plasmid pcDNA3-3xHA has a 114-bp DNA insert between the Asp718 and XbaI sites of pcDNA3, which contains a BglII site (AGATCT) immediately upstream of short ORF encoding three repeats of the HA epitope tag (GRIF YPYDVPDYA G YPYDVPDYA GS YPYDVPDYA L STOP , the HA epitopes are underlined). To create pcDNA3- PEX10 /3xHA, we excised the PEX10 ORF (lacking its stop codon) from pcDNA- PEX10 myc using the restriction enzymes Asp718 and BamHI, excised the 3xHA tag from pcDNA3-3xHA by cleavage with BglII and XbaI, and inserted these fragments in tandem between the Asp718 and XbaI sites of pcDNA3. The regions of all plasmids that were generated by PCR were sequenced to confirm the absence of any unintended mutations. Any plasmids that did contain undesired mutations were discarded and additional clones were characterized until one with the desired sequence was obtained. The Saccharomyces cerevisiae two-hybrid reporter strain BY3168 was used for all experiments . All strains were grown overnight on a nitrocellulose filter membrane (Schleicher & Schuell, Inc.) that was placed on a plate with minimal medium lacking tryptophan and leucine (Sc-W-L). The cells were lysed by submersion in liquid nitrogen, and activity of the two-hybrid reporter gene β-galactosidase was assessed by placing the filter membrane onto a filter paper saturated with 0.1% 5-bromo-4-chloro-3-indoyl β- d -galactopyranoside (X-gal) in 100 mM potassium phosphate buffer, pH 7.0. The filters were photographed after color development. Protein extracts from BY3168 carrying either pJL59- PEX12C or pJL59- PEX12C /S320F were prepared according to established protocols . In brief, the yeast were grown in 4 ml Sc-W-L medium to an OD 600 of 2. The cells were transferred to a solution containing 50 mM Tris, pH 7.5, and 10 mM sodium azide, and then pelleted by centrifugation (5,000 g ) for 10 min. The pellets were resuspended in 30 μl of ESB (80 mM Tris, pH 6.8, 1.5% DTT, 2% SDS, 10% glycerol, and 0.1 mg/ml bromophenol blue), and immediately boiled for 3 min. The tubes were cooled on ice, and then mixed vigorously with 0.1 g of 425–600-micron glass beads (Sigma Chemical Co.) for 2 min to lyse the cells. The resulting lysates were added to an additional 70 μl of ESB and boiled for 1 min. Equal amounts of each sample were separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and probed with antibodies raised against the COOH-terminal 17 amino acids of human PEX12 (a segment that is not present in yeast PEX12). A 1-liter culture of DH10B cells carrying pMBP- PEX12C was grown to an OD 600 of 0.4, induced with 1 mM isopropyl-β- d -thiogalactopyranoside, and grown overnight at 18°C. These cells were harvested and resuspended in column buffer (20 mM Tris, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10 mM β-mercaptoethanol) plus 0.5 mg/ml lysozyme. The cells were frozen in liquid nitrogen, thawed, and lysed by sonication. The lysate was centrifuged for 20 min at 14,000 g , and the supernatant was loaded onto a column containing 10 ml of amylose resin. The column was washed with 12-bed volumes of column buffer and the protein was eluted with 10 mM maltose. The eluant was collected in 1.5-ml fractions and the purity was assessed using SDS-PAGE. MBP-PEX12C/S320F and MBP-LacZα fusion proteins were purified following the same protocol. Filter binding experiments were performed as follows. 10 μg of purified MBP-PEX12C and 10 μg of MBP-LacZα were spotted separately onto Immobilon-P membranes (Millipore). Two identical membranes were prepared, one for overlay with HsPEX5S and one for HsPEX5L. The membranes were allowed to dry for 15 min at room temperature, and then washed in methanol for 30 s followed by Milli-Q H 2 O for 1 min. The membranes were transferred to buffer A and incubated with shaking for 1 h at room temperature. Radiolabled HsPEX5S and HsPEX5L were made using the TNT T7-Quick Coupled in vitro transcription/translation kit (Promega Corp.) and [ 35 S]methionine (NEN Life Science Products) according to the manufacturer's protocols. 20 μl of the in vitro transcription/translation reaction was mixed with 5 ml of buffer A plus 10 μg/ml BSA and incubated with the membrane for 1 h at 37°C with shaking. The membranes were washed twice for 5 min at room temperature with buffer A, dried, and placed on film. For assessing the PEX10-PEX12 interaction, 10 μg of purified MBP-PEX12C and 10 μg of MBP-LacZα were separated by SDS-PAGE and transferred to Immobilon-P membranes. After transfer, the proteins were renatured for 2 h at 4°C in buffer A. Radiolabeled PEX10 was synthesized using the TNT T7-Quick Coupled system and [ 35 S]methionine as described above. The membranes were incubated overnight at 4°C with shaking in 5 ml of buffer A containing 25 μl of the in vitro transcription/translation reaction. After washing twice with buffer A at room temperature, the membranes were dried, and bound [ 35 S]PEX10 was detected by autoradiography. Normal human skin fibroblasts were transfected with pcDNA3- PEX12 and pcDNA3- PEX12 /3xmyc. 2 d after transfection, cell lysates were prepared from each of the transfected populations by scraping the cells into TBSN buffer (10 mM Tris, pH 7.8, 150 mM NaCl, 1% NP-40, 5 mM benzamidine, 0.2 mg/ml NaF, 25 μg/ml aprotinin, and 62.5 μg/ml leupeptin). Each cell lysate was mixed with 1 μg of rabbit polyclonal antibodies against c-myc (Santa Cruz Biotechnology) for 1 h at 4°C. Protein A agarose beads were preincubated with 1% BSA, and then incubated with the lysate–antibody mixture for 1 h at 4°C with gentle agitation. The agarose beads were collected by centrifugation (1,000 g ), washed four times with 1 ml of TBSN buffer, and resuspended in 30 μl SDS-PAGE sample buffer. Equal amounts of each sample were immunoblotted using anti–PEX5 antibodies. For assessing the coimmunoprecipitation between PEX10 and PEX12, cells were cotransfected with pcDNA3- PEX12 and pcDNA3- PEX10 /3xHA or pcDNA3- PEX12 /3xmyc and pcDNA3- PEX10 /3xHA. After preparation of lysates and immunoprecipitation with anti-myc polyclonal antibodies, levels of PEX10/3xHA were determined by immunoblot using the 12CA5 monoclonal anti–HA antibody (Boehringer Mannheim Corp.). Equivalent levels of PEX5 and PEX10/3xHA in the crude lysates were confirmed by standard immunoblotting techniques. Skin fibroblast cells were grown to 90% confluency in 100-mm dishes, removed from the plate by trypsinization, washed, resuspended in lysis buffer, and lysed in a ball bearing homogenizer as previously described . Postnuclear supernatants were prepared by successive 1,500 g spins, and then separated into organelle pellets and cytosolic supernatants by centrifugation at 15,000 g for 10 min. To determine the relative levels of PEX5 in the cytoplasm and peroxisome of human fibroblasts, organelle pellets and cytosolic supernatants were prepared as above, transferred to membranes, and probed with polyclonal anti–PEX5 antibodies. For protease protection experiments, organelle preparations were derived from PBD006 and PBD054 cells in the same manner and were each split into eight equal fractions of 8 μg of protein. Triton X-100 was added to four of the eight tubes to a final concentration of 1%. We placed the samples on ice and added 0, 15, 30, or 60 μg of a trypsin preparation (Calbiochem-Novabiochem) to the four samples lacking detergent and the four samples containing detergent. These mixtures were incubated on ice for 20 min. Reactions were terminated by adding a twofold excess of bovine trypsin inhibitor (Sigma Chemical Co.). Equal amounts of each sample were processed for immunoblot with anti–PEX5 antibodies. We have identified an array of PEX12 mutations that cause Zellweger syndrome and the milder phenotypic variants of neonatal adrenoleukodystrophy and infantile Refsum disease . To better understand the regions of PEX12 that are important for its role in peroxisome biogenesis, we compared the deduced products of the PEX12 alleles in severely and mildly affected patients . Severe defects in PEX12 activity were associated with mutations that truncated PEX12 upstream of the cytoplasmically exposed zinc ring domain. Furthermore, all moderately and mildly affected patients expressed at least one PEX12 allele capable of encoding a protein that contained the COOH-terminal zinc ring domain. This phenotype–genotype correlation suggested that the COOH-terminal zinc-binding domain is critical for PEX12 function. This hypothesis is also supported by the results from directed mutagenesis experiments on PEX12 in both yeast and mammalian cells . Previous studies have suggested that zinc ring domains may mediate protein–protein interactions , and the important role of this domain in PEX12 suggested that it may mediate interactions between PEX12 and other proteins that are involved in peroxisome biogenesis. We employed the yeast two-hybrid system to search for such proteins. A fusion between the GAL4 DNA–binding domain and the COOH-terminal 100 amino acids of PEX12 was used as bait to screen a library of fusions between the GAL4-activating domain and all known human peroxins. We detected a strong interaction between the zinc-binding domain of PEX12 and PEX5, the PTS1 receptor . Recent studies have established that two isoforms of PEX5, PEX5S and PEX5L, are synthesized in roughly equivalent levels in human cells, but we did not observe any difference in the interaction between PEX12 and PEX5S or PEX5L. Next, we used a protein binding assay to independently assess the interaction between PEX12 and PEX5. We generated a recombinant fusion protein between maltose-binding protein (MBP) and the COOH-terminal 100 amino acids of PEX12, which includes its zinc-binding domain. The resulting protein, MBP-PEX12, was tested for its ability to bind PEX5 using a filter binding assay. Equal amounts of purified MBP-PEX12 and an MBP-LacZα fusion protein were spotted onto membranes and subsequently probed with 35 S-labeled PEX5 that had been synthesized in vitro in a rabbit reticulocyte lysate. PEX5 was bound by MBP-PEX12 but not by MBP-LacZα, indicating that PEX12 was capable of binding PEX5 . To determine whether the physical interaction between PEX5 and the zinc-binding domain of PEX12 reflected an association between these proteins in vivo, we tested whether PEX12 and PEX5 formed a complex of sufficient stability to withstand coimmunoprecipitation from cell lysates. Human fibroblasts were transfected with either of two plasmids, pcDNA3- PEX12 or pcDNA3- PEX12 /3xmyc. 2 d after transfection the cells were lysed, the lysates were subjected to immunoprecipitation with anti-myc polyclonal antibodies, and the immunoprecipitates were separated by SDS-PAGE and probed with anti–PEX5 antibodies. Equal amounts of PEX5 were present in both crude lysates, but PEX5 was immunoprecipitated only from the lysate of cells expressing PEX12/3xmyc . We next mapped the PEX12-binding domain of PEX5 by expressing different regions of PEX5 in the yeast two-hybrid system and assaying their interaction with the PEX12 zinc-binding domain. These fragments of PEX5 were also assayed for their ability to interact with PEX14, a known docking factor for PEX5 . The PTS1 binding site of PEX5 is contained within its COOH-terminal half, a region that contains seven tetratricopeptide repeats . A fragment of PEX5 containing little more than the PTS1-binding domain of PEX5 retained full binding to PEX12. However, it was unable to bind PEX14, as expected from the recent study by Schliebs et al. 1999 in which the PEX14 binding sites were localized to the NH 2 -terminal half of PEX5. Additional truncation mutants failed to define a smaller PEX12-binding site within PEX5 (data not shown). In addition to the interaction between PEX12 and PEX5, the yeast two-hybrid screen also revealed an interaction between PEX12 and PEX10, an integral PMP that is required for peroxisomal matrix protein import . Like PEX12, human PEX10 contains a cytoplasmically exposed zinc ring domain and the interaction we detected between these two proteins was mediated through their COOH-terminal zinc ring domains (amino acids 240–326 of PEX10 and 260–359 of PEX12). Independent biochemical evidence for physical interaction between PEX12 and PEX10 was obtained using blot overlay experiments. Equal amounts of purified, recombinant MBP-PEX12 and MBP-LacZα were resolved by SDS-PAGE, immobilized on membranes, and probed with 35 S-labeled PEX10 that had been synthesized in vitro in rabbit reticulocytes . PEX10 was bound by the MBP-PEX12 fusion protein but not by MBP-LacZα, suggesting specific binding between PEX12 and PEX10. To assess whether these proteins were present in a complex in vivo, we transfected normal human fibroblasts with plasmids designed to express tagged forms of these proteins, PEX12/3xmyc and PEX10/HA, or PEX10/HA and an untagged version of PEX12. 2 d after transfection, lysates were prepared from the two sets of transfected cells, subjected to immunoprecipitation using an anti-myc polyclonal antibody, and then blotted with a monoclonal anti–HA antibody. Equivalent amounts of PEX10/HA were detected in both crude lysates, but PEX10/HA was only detected in the immunoprecipitate from cells expressing PEX12/3xmyc. Thus, PEX12 and PEX10 do appear to be present in a complex in vivo . Control experiments revealed that these tagged forms of PEX12 and PEX10 have normal activity in vivo. This region of PEX12 failed to interact with any of the remaining 12 human peroxins in the yeast two-hybrid assay. The simplest explanation for the physical association of PEX12 with both PEX5 and PEX10 is that these interactions contribute to the biogenesis of peroxisomes. In such an instance, we might expect that high dosage, allele-specific extragenic suppression could be observed among the corresponding three genes. Therefore, we tested whether overexpression of any one of these genes could suppress mutations in either of the other two genes. In brief, fibroblast cell lines with mild or severe mutations in PEX12 , PEX10 , or PEX5 were transfected separately with expression vectors designed to express these genes, as well as a vector control. 2 d later, each cell population was processed for indirect immunofluorescence using antibodies specific for a peroxisomal matrix protein marker, catalase. The relative rescue activity of each gene in each cell line was calculated by comparing the frequency of cells importing catalase in each set of transfected cells. All three patients from complementation group 2 of the PBDs have mutations in PEX5 . Two of these patients (PBD018 and PBD093) are homozygous for a PEX5 -N489K/N526K mutation (N489/N586 refers to the position of this asparagine in PEX5S and PEX5L, respectively). The other PEX5 -deficient patient, PBD005, is homozygous for a PEX5 nonsense mutation, R390ter/R427ter, which inactivates PEX5 . PBD018 and PBD005 cells were transfected with the plasmids pcDNA3, pcDNA3- PEX5 , pcDNA3- PEX12 , and pcDNA3- PEX10 , incubated for 2 d, and then processed for immunofluorescence using antibodies specific for peroxisomal catalase. Although expression of PEX5 efficiently rescued catalase import in both cell lines, PEX12 and PEX10 were unable to restore catalase import in these lines (data not shown). We have also characterized eight complementation group 3 PBD patients, all of whom are mutated in PEX12 . Fibroblast cell lines from each of these patients were transfected with the above vectors, and the import of peroxisomal matrix proteins was determined in each population of transfected cells. As expected, pcDNA3- PEX12 efficiently rescued the peroxisomal protein import defects in all of these cell lines. However, we failed to observe any evidence for extragenic suppression in the CG3 cells that were transfected with the PEX10 or PEX5 expression vectors (data not shown). Our previous work has established that PEX10 is the gene defective in two patients from complementation group 7 (CG7), PBD052, and PBD100 . Although we have yet to identify the PEX10 mutations in four other CG7 patients, we used fibroblasts from all six available CG7 patients for our studies. Expression of PEX10 rescued peroxisomal matrix protein import in all six CG7 cell lines, whereas PEX12 and PEX5 failed to have any effect on the cytosolic catalase distribution in five of these cell lines. However, expression of PEX12 clearly led to the restoration of catalase import in PBD054 cells . Expression of PEX5 also rescued peroxisomal matrix protein import in this cell line, though to a lesser extent ( Table ). To better understand the molecular basis of the apparent suppression of PEX10 allele(s) by overexpression of PEX12 or PEX5 , we sequenced the PEX10 gene from PBD054 cells. Surprisingly, we failed to detect any alteration to the gene in this patient. This result, combined with the fact that PEX12 was more effective than PEX10 at rescuing peroxisomal protein import in PBD054 cells ( Table ), led us to consider whether PEX12 , rather than PEX10 , might be mutated in PBD054. The PEX12 gene was amplified from PBD054 genomic DNA and all coding portions of the gene were sequenced directly from the PCR products. We detected a missense mutation, S320F, in the PEX12 gene from this patient and no evidence of the wild-type sequence, suggesting that this patient was homozygous for this mutation in PEX12 . Although S320 is a conserved residue of PEX12 from yeast to humans (always a serine or threonine), missense mutations may be silent. We engineered the S320F mutation into the PEX12 expression vector and used a functional complementation assay to assess the effects of this mutation. PEX12 -deficient cells were transfected with pcDNA3, pcDNA3- PEX12 , or pcDNA3- PEX12 /S320F, and 2 d after transfection the percentage of cells importing matrix proteins into peroxisomes was determined by immunofluorescence using antibodies to the peroxisomal matrix protein catalase. In each of two trials the PEX12 /S320F cDNA displayed 10–15% of the rescue activity of the wild-type PEX12 cDNA (data not shown). The fact that the S320F mutation reduced but did not eliminate PEX12 function was consistent with the relatively mild cellular and clinical phenotypes of this patient . The ability of PEX10 and PEX5 to suppress the PEX12 /S320F mutation was a clear example of allele-specific suppression rather than bypass suppression since neither PEX10 nor PEX5 were capable of rescuing peroxisomal protein import in any cells with severe mutations in PEX12 . This finding, together with the fact that the PEX12 -S320F mutation lies within the zinc-binding domain of PEX12, suggested that this mutation might reduce the interaction between the zinc ring domain of PEX12 and either PEX10 or PEX5. Using the two-hybrid assay and the blot overlay assay, we observed that the S320F mutation led to a marked reduction in the PEX12–PEX10 interaction . Similarly, the PEX12/S320F mutation appeared to reduce the interaction between PEX12 and PEX5 in the yeast two-hybrid assay . It is interesting to note that the placement of PBD054 cells into CG7 of the PBDs was based upon its noncomplementation with PBD052 cells. PBD052 cells are mutated in PEX10 and express one allele with a missense mutation (H290Q) in the PEX10 zinc-binding domain . These results indicate that the combination of the PEX12 /S320F and PEX10 /H290Q alleles may have a deleterious effect on peroxisomal matrix protein import even in the presence of normal alleles of each gene. A variety of earlier studies have established that loss of PEX12 or PEX10 results in a severe defect in peroxisomal matrix protein import . It is also known that PEX12 and PEX10 are not required for synthesis of peroxisome membranes or import of peroxisomal membrane proteins . Furthermore, the morphological abnormalities that have been reported for peroxisomes in cells lacking PEX12 or PEX10 are indistinguishable from those of peroxisomes in PEX5 -deficient cells and appear to be a secondary effect of the metabolic deficiencies that are caused by the matrix protein import defects in these cells . These results, together with our observation that PEX12 interacts with both PEX5 and PEX10, suggest that PEX12 and PEX10 participate in peroxisomal matrix protein import. Current models suggest that there may be several steps of peroxisomal matrix protein import that are limited to the peroxisome membrane and could, therefore, involve PEX12 or PEX10. These include docking of receptor–ligand (matrix protein) complexes to the peroxisome, matrix protein translocation across the peroxisome membrane, and receptor recycling . To distinguish between these different possibilities, we first tested whether PEX12 or PEX10 were required for docking of the PTS1 receptor, PEX5, to peroxisomes. Postnuclear supernatants were prepared from normal human fibroblasts and from a fibroblast cell line which appears to lack PEX12 activity altogether . Peroxisomes were pelleted from each postnuclear supernatant by centrifugation, and the relative amount of PEX5 in the cytosolic supernatant and organelle pellet was determined by immunoblot. Levels of peroxisome-associated PEX5 were not reduced in the absence of PEX12 or PEX10 and actually appeared to be slightly elevated in the pex10 and pex12 mutants . This was also evident in immunofluorescence experiments in which the staining for peroxisome-associated PEX5 appeared to be greater in the pex12 and pex10 mutants than in wild-type cells . These results argue against roles for PEX12 and PEX10 in PEX5 docking and suggest that they participate in a downstream step of peroxisomal matrix protein import. Taken together, the properties of PEX12 indicate that it may participate in peroxisomal matrix protein translocation rather than receptor docking or recycling (as described in Discussion, the phenotypes of PEX12 -deficient cells are not consistent with a role for PEX12 in receptor recycling). One prediction of this hypothesis is that mild mutations in PEX12 might alter the properties of the peroxisomal matrix protein translocation apparatus without eliminating translocation altogether. Before our discovery that PBD054 cells are mutated in PEX12 , we reported that PBD054 cells import PEX5 into the peroxisome lumen and also import small amounts of some peroxisomal matrix proteins into peroxisomes . We revisited the issue of PEX5 distribution in PBD054 cells and also compared it to the distribution of PEX5 in PBD006 cells. By immunofluorescence studies in which all cellular membranes are permeabilized, both PBD006 and PBD054 cells contain detectable levels of peroxisome-associated PEX5 . However, differential permeabilization experiments (in which antibodies only have access to cytoplasmically exposed antigens) revealed that cytoplasmically exposed PEX5 could still be detected in PBD006 cells but not in PBD054 cells . Similar results were observed in each of three trials. These differential permeabilization experiments indicated that PBD006 cells contained more cytoplasmically exposed PEX5 than PBD054 cells and that PBD054 cells imported PEX5 into the peroxisome lumen. However, there are two potential caveats to these experiments. First, immunofluorescence experiments can be influenced greatly by conformational changes in the antigen, in this case PEX5. Second, they do not address the question of whether some of the peroxisomal PEX5 in PBD006 cells may also be protected from antibodies in the differential permeabilization experiments. Therefore, we performed protease protection experiments on organelle preparations from PBD006 and PBD054 cells. Postnuclear supernatants were prepared from each cell line, and peroxisomes and other large organelles were recovered by differential centrifugation. These organelle pellets were resuspended and incubated with various amounts of protease in the presence or absence of detergent, the reactions were quenched, and each sample was assayed for levels of PEX5 by immunoblot . PBD054 cells appeared to contain more protease-resistant PEX5 than PBD006 cells, which may reflect import of PEX5 into the peroxisome lumen of these cells. Similar results were obtained in each of three trials. If PBD054 cells actually do import PEX5 into the peroxisome lumen, we might expect that peroxisomes of these cells would contain more PEX5 than those of PBD006 cells. We tested this hypothesis by cell fractionation studies. Postnuclear supernatants were prepared from wild-type, PBD006 and PBD054 fibroblasts, peroxisomes were separated from cytosol by differential centrifugation, and the levels of PEX5 in the cytosolic supernatant and organelle pellet fractions were determined by immunoblot. PBD054 cells contained more peroxisome-associated PEX5, as predicted . In this paper we investigated the role of PEX12 in peroxisome biogenesis by examining the phenotypes of PEX12 -deficient cells and identifying peroxins that physically and genetically interact with PEX12. Previous studies have established that loss of PEX12 results in the absence of detectable peroxisomal matrix protein import, but has virtually no effect on the synthesis of peroxisomes or the import of peroxisomal membrane proteins . Such a phenotype alone points to a role for PEX12 in peroxisomal matrix protein import. However, the data presented in this report advance this hypothesis by demonstrating physical and genetic interaction between PEX12 and PEX5, the receptor for newly synthesized peroxisomal matrix proteins. We detected interactions between PEX12 and PEX5 using the yeast two-hybrid system, by filter binding assay, in coimmunoprecipitation experiments, and by genetic suppression studies. It is generally accepted that PEX5 is the receptor for newly synthesized PTS1-containing proteins and is a predominantly cytoplasmic protein in mammalian cells and several fungal species . Studies in human cells have suggested that PEX5 shuttles between the cytoplasm and peroxisome , and several models predict that PEX5 moves through a variety of steps as it catalyzes peroxisomal matrix protein import. The proportion of PEX5 that resides in the cytoplasm at steady state probably reflects the needs of each cell to efficiently capture newly synthesized peroxisomal matrix proteins (ligands) from the cytoplasm. However, once these ligands are bound by PEX5 many additional events must occur. These may be grouped into the general processes of (1) the transport to and docking of PEX5–ligand complexes with the peroxisome membrane, (2) the translocation of ligands into the peroxisome lumen, and (3) the recycling of receptors back to the cytoplasm . To distinguish which of these events may involve PEX12, we considered the phenotypes that are expected for a mutant in each process and compared them to the phenotypes of PEX12 -deficient cells. Several studies have implicated the integral peroxisomal membrane proteins PEX13 and PEX14 in docking of PTS receptors to the peroxisome membrane . The defining features of these docking factors are as follows: (1) their ability to bind PEX5 and PEX7, either directly or indirectly, and (2) the fact that loss of PEX13 or PEX14 results in a significant reduction in the amount of peroxisome-associated PEX5. The fact that loss-of-function mutations in PEX12 do not result in any detectable reduction in the levels of peroxisome-associated PEX5 argues strongly against the hypothesis that PEX12 participates in receptor docking. In fact, our data indicate that the loss of PEX12 may actually increase the levels of peroxisome-associated PEX5. This observation, together with the fact that receptor docking is the first peroxisome-localized step of peroxisomal matrix protein import, demonstrates that PEX12 acts downstream of the docking event. Thus, PEX12 appears to be the first known PEX5-binding protein that is not required for docking the PTS1 receptor to the peroxisome. Of the two remaining aspects of peroxisomal matrix protein import, protein translocation and receptor recycling, there is no report of a bona fide protein translocation factor, but there is one report that proposes a role for PEX4 in receptor recycling . This conclusion was based in part on the phenotypes of pex4 mutants, which display a very mild defect in peroxisomal matrix protein import and can be suppressed by overexpression of PEX5 . However, we observed that cells lacking human PEX12 display a severe defect in peroxisomal matrix protein import that cannot be suppressed by overexpression of PEX5 . Thus, PEX12 does not have the properties we might expect of a factor that is required for receptor recycling. The remaining aspect of peroxisomal matrix protein import to consider is the protein translocation process. Actually, a role for PEX12 in peroxisomal matrix protein translocation would fit well with the known properties of this protein. First, PEX12 has the appropriate physical characteristics for such a role: it is an integral peroxisomal membrane protein that spans the membrane twice and extends its NH 2 and COOH termini toward the cytoplasm where they may interact with other protein import factors . Second, it utilizes its COOH-terminal zinc-binding domain to interact with PEX5, the PTS1 receptor. Third, cells with inactivating mutations in PEX12 are unable to import peroxisomal matrix proteins but do synthesize peroxisomes and import integral peroxisomal membrane proteins . Fourth, PEX12 interacts with PEX10, another integral peroxisomal membrane protein that displays a specific defect in the import of peroxisomal matrix proteins and yet does not appear to participate in receptor docking or recycling. Fifth, a missense mutation in PEX12 , S320F, appears to affect the specificity of the translocation apparatus, resulting in the import of PEX5 into the peroxisome lumen. Although there is no established in vitro protein translocation assay that can be used to test this hypothesis directly, it is useful to consider possible roles for PEX12 in the translocation process. The main function of a matrix protein translocon would be to move matrix proteins from the cytoplasmic side of the peroxisome membrane to the lumenal side. Given that most of these proteins arrive at the peroxisomes in a complex with PEX5 and that studies of Yarrowia lipolytica PEX5 strongly suggest that PEX5 participates in the matrix protein translocation process , we favor a model for matrix protein translocation that includes PEX5. Such a model may involve: (1) acceptance of PEX5–ligand complexes from PEX14, the primary PEX5 docking site; (2) retention of PEX5 at the translocation apparatus; (3) opening of the matrix protein translocation pathway; (4) PEX5–ligand dissociation and ligand translocation; (5) closure of the translocation pathway; and (6) release of the unoccupied receptor from the translocation apparatus. The ability of PEX12 to bind PEX5 suggests that PEX12 may contribute to retaining PEX5 at the translocation apparatus. Some additional support for this hypothesis comes from the fact that PEX5 appears to be imported into the peroxisome as a result of the PEX12 /S320F mutation, which reduces the interaction between PEX12 and PEX5. This model predicts that PEX5 enters the peroxisomal compartment during the normal course of peroxisomal matrix protein import. However, it also predicts that PEX5 should normally be retained at the translocation apparatus rather than being released to move freely through the peroxisome lumen. A low rate of PEX5 release from the translocation apparatus into the lumen could explain the detection of intraperoxisomal PEX5 in Hansenula polymorpha . Y . lipolytica PEX5, which is detected only on or in the peroxisome, could also function within such a model, provided that it participates in just the peroxisome-limited steps of matrix protein import . | Study | biomedical | en | 0.999997 |
10562280 | The CatS −/− and Ii −/− mice used here have been described; C57BL/6 mice (Jackson Laboratories) were used as wild-type (wt) controls. All animals were maintained under pathogen-free conditions at the animal facilities of Harvard Medical School in compliance with institutional guidelines. Spleens were enriched in vivo with DC by stimulation with flt3 ligand . CatS −/− , Ii −/− , and C57BL/6 mice were subcutaneously injected with 4 × 10 6 flt3 ligand–secreting B16 melanoma cells (C57BL/6 background) prepared by transfection of murine flt3 ligand cDNA with the retroviral vector MFG . Spleen cells were harvested after the tumors reached 2–3 cm in diameter, at which time the spleens were 5–10-fold enlarged over those of untreated mice. A single-cell suspension of whole splenocytes was resuspended in a high-density BSA solution (4 ml per spleen) containing 10.6 g BSA (Intergen), 18.6 ml PBS, 2.9 ml 1 N NaOH, and 6.5 ml H 2 O as described . After overlaying 2 ml of ice-cold RPMI, the splenocyte/BSA solution was centrifuged for 20 min at 9,500 g . About 5 × 10 7 DC per spleen were recovered from the interface and resuspended into RPMI. The cells were characterized by FACS using anti-B220, CD11c, CD11b, CD3, CD4, CD8, CD86, CD80, GR-1, and I-A b antibodies. The surface phenotype of these cells, as revealed by extensive FACS analysis, did not differ significantly from the phenotype reported for DC generated by injection of the recombinant cytokine . Although this DC population is by no means homogenous with regard to its myeloid and/or lymphoid phenotype, it displays a uniform phenotype with regard to MHC class II surface expression, shows a robust antigen-presenting capacity in vitro , and therefore is considered as functionally mature DC . Incubation of cells in either granulocyte/macrophage colony-stimulating factor (GM-CSF) or lipopolysaccharide (LPS) or both for up to 8 d did not lead to an increase in surface class II expression, and confirms the mature stage. Neither by FACS analysis nor light microscopy did we find any evidence for a maturation defect in CatS −/− DC. To assess the purity and confirm the phenotype of individual DC preparations, surface expression of I-A b , B220, and CD11c was analyzed routinely by FACS for each experiment. For comparison of morphology and subcellular distribution of β-hexosaminidase, bone marrow–derived DC were generated by culturing bone marrow cells in GM-CSF for 6 d as described . On day six, semiadherent clusters were isolated, purified on a 50% serum cushion, and allowed to mature in vitro by culturing them for 48 h. N22, a hamster mAb that recognizes mouse MHC class II molecules , was a gift from Dr. R.M. Steinman (Rockefeller University, New York, NY). The rabbit antiserum raised against the NH 2 -terminal and the COOH-terminal regions of Ii (JV5: anti–NH 2 terminus, Ii 1–29; JV11: anti–COOH terminus, Ii 156–190) as well as the rabbit antiserum against murine protein-disulfide isomerase (PDI) were generated in our laboratory using standard techniques. Murine MHC class I molecules were retrieved with the p8 antiserum. Antibodies against murine transferrin receptor (TfR), LAMP-1 as well as fluorochrome-labeled antibodies against murine I-A b , B220, CD80, CD86, CD11b, CD11c, CD8, and GR-1 were purchased from PharMingen. The rabbit antiserum against mannose-6-phosphate receptor (M6PR) was a gift from K.V. Figura (Max Planck Institute, Gottingen, Germany), the H2-DM antiserum was kindly provided by P. Pierre and I. Mellman (Yale University, New Haven, CT). Single-cell suspensions were incubated for 30 min at 4°C with the appropriate conjugated antibodies in the presence of Fc Block (PharMingen), washed, and analyzed immediately on a FACScan (Becton Dickinson) using Cell Quest software. Freshly isolated cells (5 × 10 5 ) were plated in each well of glass chamber slides (Nalge Nunc International Laboratories) in complete RPMI medium supplemented with 20% FCS, and incubated at 37°C for 1 h to allow the cells to attach to the slide. All subsequent steps were performed at room temperature. Cells were washed once in PBS, and fixed for 20 min in a 3.7% solution of paraformaldehyde. After four washes in PBS, cells were permeabilized in RPMI medium containing 10% goat serum (GIBCO-BRL) and 0.05% saponin for 15 min. The primary and secondary antibody solutions were prepared in the same medium. Cells were incubated with the antibodies for 30 min and then washed three times. Slides were mounted in Aquapoly/Mount solution (Polysciences Laboratories) and analyzed in a Bio-Rad MRC 1024 confocal laser scanning microscope. The merged images were analyzed for the presence of class II–Ii molecules in LAMP-1 or DM positive structures using the colocalization program from Bio-Rad. I-A b molecules were detected using the Y3P antibody that recognizes mature αβ complexes and αβl, mouse Ii with the rabbit antisera JV5 and JV11. Secondary antibodies labeled with FITC were used for I-A b and Ii detection. LAMP-1 was detected using a rat mAb (PharMingen) and a secondary antibody coupled to CY3. H2-DM was detected by the combination of rabbit anti-DM antiserum and an anti–rabbit antibody labeled with CY3. All secondary antibodies were made in goat and purchased from Jackson ImmunoResearch Laboratories. Equal numbers of freshly prepared DC were incubated in 1 ml methionine/cysteine–free medium, supplemented with 10% FCS, 2 mM l -glutamine, penicillin (1:1,000 dilution U/ml), and 100 mg/ml streptomycin for 30 min. Cells were either continuously labeled with 0.5 mCi/ml [ 35 S]methionine/cysteine (80/20) (Dupont New England Nuclear) for 5 h or pulsed for 30 min and chased in 15 vol of complete RPMI supplemented with FCS, l -glutamine, penicillin, and streptomycin as above for the times indicated. LHVS, synthesized as published , was added to the pulse medium 20 min before the addition of [ 35 S]methionine/cysteine and the chase medium at a final concentration of 3 nM. Subcellular fractions were prepared essentially as described for murine B-lymphoblasts ; all steps were performed at 4°C. At each timepoint, cells were washed with PBS and 0.25 M sucrose, taken up in 2.2 ml homogenization buffer (0.25 M sucrose, 1 mM EDTA, pH 7.4, 1 mM PMSF), homogenized by six passes in a ball-bearing homogenizer (10-μm gap), and spun at 1,000 g for 10 min to obtain postnuclear supernatants (PNS). The amounts of incorporated [ 35 S]methionine/cysteine of the individual homogenates, as assessed by TCA precipitation, were adjusted to identical levels of radioactivity concentration with homogenization buffer. 9 ml of a 27% Percoll (Amersham Pharmacia Biotech) solution in 0.25 mM sucrose was layered on top of a 1-ml cushion of 2.5 M sucrose and overlaid with 2 ml of PNS (containing equal amounts of total incorporated radioactivity for each individual sample as described). After 1 h centrifugation at 34,000 g (4°C), 1-ml fractions were collected from the bottom of the tube. Fractions containing the low density peak of β-hexosaminidase activity (fractions 9 and 10) were pooled, applied to a 10% Percoll gradient, and fractionated by centrifugation as described for the 27% gradient. For both wt or CatS −/− mice, the first gradient (27% Percoll) yielded two peaks of activity of the endocytic marker enzyme β-hexosaminidase (high density peak: fractions 1+2, referred to as peak A). To distinguish between early and late endosomal compartments, the low density peak of the β-hexosaminidase activity in the 27% Percoll gradient was applied to a subsequent 10% Percoll density gradient. This separation resulted in a predominant intermediate density peak of β-hexosaminidase activity at the bottom of the gradient, peak B (fractions 1+2 of the 10% gradient). Peak C (fractions 11+12) was defined based on distribution of radioactivity, although a small amount of β-hexosaminidase was detected reproducibly. This fractionation pattern, as assayed by the endocytic marker β-hexosaminidase, was not affected by the lack of CatS. The marker profile showed that peak A contains mature lysosomes based on its density of 1.09 g/ml, its β-hexosaminidase activity, and the presence of the late endosomal and lysosomal marker LAMP-1. Nonlysosomal characteristics (the endosomal marker TfR and M6PR, the Golgi apparatus and ER markers galactosyl transferase and PDI, respectively, and MHC class I, which serves as a marker for surface expression) were absent from peak A. The intermediate density peak of the 10% Percoll gradient (peak B) represents late endosomes (density 1.05 g/ml, positive for β-hexosaminidase, M6PR, and LAMP-1, but negative for TfR as well as for galactosyl transferase and PDI and MHC class I). The low density peak (peak C) consists of a mixture of compartments, namely ER–Golgi apparatus and early endosomes–plasma membrane (PM), in agreement with published observations . For every fraction of each individual experiment, the distribution of the endocytic marker β-hexosaminidase activity , was assayed as described . Similarly, quantification of total incorporated radioactivity in each fraction was routinely performed from TCA-precipitated material. Galactosyl transferase was measured as published . The distributions of TFR, LAMP-1, M6PR, and PDI were visualized by Western blot using 100 μl of each fraction with the appropriate primary antibody and secondary antibody conjugated to peroxidase (Santa Cruz Biotechnology). Immunoprecipitation experiments were performed as described . DC labeled in a pulse–chase experiment were washed three times in ice-cold PBS and incubated for 30 min at room temperature in PBS containing 0.5 mg/ml NHS-Sulfo-Biotin (Pierce). For each timepoint, the biotinylated cells were lysed in NP-40 lysis buffer, and class II molecules were immunoprecipitated with the N22 antibody. Staph A pellets were resuspended in 50 μl of PBS containing 1% SDS and boiled for 10 min. 1/10 of each precipitate was retained for later comparison with reimmunoprecipitates in SDS-PAGE. 1 ml lysis buffer containing 0.1% BSA was added to the remaining 9/10 of the precipitate. After centrifugation, the supernatant was precleared once, reimmunoprecipitated with streptavidin agarose beads as above, and analyzed by SDS-PAGE and autoradiography. For quantitative evaluation, autoradiographs were analyzed using a MultiImage Scanning Densitometer (Alpha Innotech) and the software provided by the manufacturer. Values for MHC class I, MHC class II α-chain, SDS-stable dimers, and Ii breakdown intermediates were obtained by summing the respective background-corrected autoradiography signal corresponding to peak A, B, or C, either relative to each other or in relation to the total amount of the particular species in the entire gradient . LHVS-phenol (PhOH) was synthesized following a scheme modified from that described . 125 I–LHVS-PhOH (obtained by an Iodogen-catalyzed reaction) was purified further by reverse-phase HPLC. The peak fractions of radioactivity were dried down under reduced pressure and resuspended in DMSO. DC were washed three times with ice cold Hepes/RPMI. 1 × 10 6 cells were lysed for 2 h at 4°C in 100 μl lysis buffer (50 mM Tris, 5 mM MgCl 2 , 0.5% NP-40, pH 7.4). Lysates and subcellular fraction samples (100 μl) were incubated with 10 nM 125 I–LHVS-PhOH at 37°C for 1 h (final DMSO concentration of 1.7–3%). Labeling was terminated by addition of 4× SDS sample buffer. The samples were boiled before analysis by 12.5% SDS-PAGE and visualization by fluorography. DC from CatS −/− mice and wt controls were generated by inoculation of the animals with flt3 ligand–secreting melanoma cells. Purified splenic DC were analyzed by FACS and confocal microscopy. Cells isolated from both types of animal consisted of ∼80% B220 low , CD11c high , MHC class II high cells and therefore were judged to be DC, whereas the remainder were B cells and B220 low CD11c low cells in roughly similar numbers. These cells were used for the experiments described here, and will be referred to simply as DC. Of note, significant morphological differences were detected when comparing flt3-induced splenic DC with DC generated from bone marrow precursors using GM-CSF . Flt3-induced DC are smaller in size and allow poorer resolution of intracellular compartments by microscopy. Furthermore, they show a different distribution profile of the endocytic marker β-hexosaminidase when compared with bone marrow–derived DC (see below). Although class II surface expression was remarkably similar in CatS −/− and wt DC , the intracellular distribution of class II was distinct, whereas no other gross morphological differences were detected . We examined the steady-state distribution of class II molecules, Ii, and the late endocytic marker LAMP-1 by indirect immunofluorescence on freshly isolated and permeabilized DC. To detect class II molecules, the Y3P antibody was used; for detection of Ii, polyclonal antisera directed against the cytoplasmic tail of Ii (NH 2 terminus, JV5 antibody) or its lumenal region (COOH terminus, JV11 antibody) were used . The staining for total class II molecules observed in CatS −/− DC is considerably enhanced compared with wt cells, suggesting that CatS activity influences the level of intracellular class II . Colocalization of class II and LAMP-1 molecules showed that the fraction of class II in late endocytic structures is enhanced in CatS −/− DC. A similar observation was made when analyzing the colocalization of class II with H2-DM molecules , confirming the increased fraction of class II antigens in late endocytic compartments from CatS −/− DC. Similar results were obtained when using the JV5 antiserum directed against the NH 2 -terminal portion of Ii , suggesting that class II molecules in late endocytic compartments of CatS −/− DC are bound largely to Ii fragments that have retained an intact NH 2 terminus. To determine if these class II–Ii complexes contain intact Ii molecules, an antiserum that exclusively recognizes the p31 and p41 Ii forms (raised against the COOH-terminal part of Ii) was employed. No difference was seen in the extent of colocalization of LAMP-1 and Ii between wt and CatS −/− DC . Thus, the MHC class II–Ii complexes that accumulate in late endocytic structures of CatS −/− DC must lack the COOH-terminal portion of Ii. As shown below, results from subcellular fractionation experiments are in agreement with this conclusion. In all experiments, CatS −/− DC showed increased staining with Ii antibodies as compared with wt cells, indicating that the total intracellular amount of Ii is enhanced when CatS activity is absent . Cytofluorometric analysis of CatS −/− and wt DC using the JV11 antiserum also revealed a significant increase of intact Ii at the surface of CatS −/− cells (data not shown). Finally, a stronger signal for LAMP-1 and H2-DM staining in CatS −/− compared with wt DC was consistently observed . In conclusion, the CatS −/− mutation leads to an accumulation of class II molecules bound to Ii with an intact NH 2 terminus in late endocytic compartments of mature DC. This observation is consistent with those obtained in LHVS-treated bone marrow–derived DC, resembles the intracellular distribution of class II in immature DC, and is most likely the result of incomplete cleavage of Ii in the absence of CatS. To verify this hypothesis and to determine the nature of the MHC class II–Ii complexes that accumulate in late endocytic compartments, subcellular fractionation experiments on CatS −/− and wt DC were performed. Immunoelectron microscopy has shown that the majority of intracellular class II molecules in professional APC are found in conventional late endocytic structures . To resolve the major class II–containing compartments biochemically, DC from CatS −/− and wt animals were labeled with [ 35 S]methionine and subjected to subcellular fractionation. In brief, a homogenate was prepared using a ball-bearing–type homogenizer and the PNS was fractionated by two successive Percoll gradients. The first gradient (27% Percoll) allowed resolution of lysosomal fractions. This lysosomal peak will be referred to as peak A . The unresolved material at the top of the 27% gradient was applied to a second gradient (10% Percoll), which yielded a high-density fraction enriched for late endosomes (peak B), and at low density, an unresolved peak comprising PMs, Golgi, early endosomes, and ER (peak C). No attempt was made to further resolve fraction C into its individual constituents. This fractionation scheme was used to characterize the subcellular distribution of CatS activity and to dissect trafficking and maturation of class II molecules in mature DC of wt and CatS −/− mice. The modest activity of β-hexosaminidase in the lysosomal peak A appears to be a cell-specific trait. A direct comparison of the distribution profile of β-hexosaminidase activity between wt flt3-derived splenic DC and bone marrow–derived DC generated in GM-CSF reveals differences in the profile of the endocytic compartments for these two cell types. While the majority of β-hexosaminidase activity is found in the lysosomal compartment (peak A) in bone marrow–derived DC, this activity is distributed more towards the late endosomal compartment (peak B) with a relatively lesser amount in lysosomes in flt3-derived DC . Immunoprecipitations were performed with antibodies capable of recognizing class II molecules assembled with intact Ii as well as Ii intermediates. Assignment of Ii fragments was based on their reactivity with antipeptide antisera directed either against the cytoplasmic NH 2 terminus or against the COOH-terminal trimerization domain (antiserum JV11) in conjunction with estimates of their molecular size and the presence or absence of N-linked glycans. For reference purposes, we performed subcellular fractionation on cells obtained from Ii −/− animals. In the absence of Ii, class II molecules assemble poorly and fail to be delivered efficiently to their normal destination. For all subcellular fractionations, the distribution of MHC class I molecules (H-2K b ) was examined as a representative protein largely excluded from endocytic compartments and unlikely to be affected in its intracellular trafficking by the absence of CatS. Fractionation experiments were conducted either on cells labeled continuously for 5 h or in conjunction with pulse–chase labeling . Analysis of the 27% Percoll gradients revealed that class II molecules in DC from Cat S −/− mice are more pronounced in their distribution over the lysosomal fraction and the denser regions of the gradient than in wt DC . Furthermore, the overall recovery of class II molecules appears greater for the CatS −/− DC than for the wt DC, in agreement with the qualitative assessment by immunofluorescence . Class I products are recovered in approximately equal amounts and are distributed similarly in CatS −/− and wt DC . As expected, in Ii −/− animals few if any class II molecules are recovered from the dense lysosomal fraction A . Analysis of fractions 9 and 10 obtained from the 27% Percoll gradient on a subsequent 10% Percoll gradient showed that in Cat S −/− DC, class II molecules accumulate in the late endosomal fraction with a very similar distribution to wt DC in the remaining portions of the gradient . The distribution and recovery of MHC class I molecules from Cat S −/− DC is indistinguishable from wt cells . As expected, class II molecules were not recovered from late endosomal fractions of Ii −/− DC (compare with both wt and CatS −/− DC). The immunofluorescence in conjunction with the subcellular fractionation experiments showed that class II molecules accumulate intracellularly in DC from CatS −/− animals. The sites of intracellular accumulation are predominantly the late endosomes, and to a lesser extent, the lysosomes. Therefore, CatS regulates either the access of class II molecules to, or the egress from, these compartments in flt3-induced splenic DC. Next, we performed pulse–chase experiments in conjunction with the subcellular fractionation scheme outlined above . The results from these experiments not only confirmed those obtained for continuously labeled cells, but also allowed important additional conclusions. By analysis of each of the immunoprecipitates under fully denaturing conditions (B; boiling in SDS sample buffer) as well as under more mildly denaturing conditions (NB; SDS sample buffer at room temperature), processing intermediates of Ii were recovered in a stable complex with the αβ heterodimer from different subcellular fractions. The sequence of events by which Ii is removed from class MHC class II–Ii complexes in wt and CatS −/− DC can thus be established. Recovery and distribution of MHC class I molecules was relatively constant for the different chase points and did not differ between wt and CatS −/− DC . To quantitate the class II molecules themselves, the relative distribution of the class II α chain was analyzed. This parameter is valid because the N22 antibody used to recover class II molecules recognizes an epitope on the class II β subunit. The pattern of reactivity of N22 is generally assumed to be conformationally sensitive and dependent on proper heterodimer formation . After 30 min of labeling, class II molecules were visualized in CatS −/− and wt DC in peak C as SDS-stable complexes of ∼100 kD, which represent the nonameric structure (αβIi) 3 referred to as αβp . At this timepoint, almost all of the α chain carried high mannose-type oligosaccharides as indicated by its higher mobility compared with the mature form, which suggested that these molecules were still located in the ER–Golgi. This αβ–Ii complex appeared in peak B in both cell types at later timepoints, but the α chain was in its mature, fully glycosylated form. After 1 h of chase, class II molecules reached endocytic compartments, and were transformed into ∼70 kD αβl complexes by COOH-terminal degradation of Ii. No differences in either the kinetics or the subcellular distribution of αβl complexes were seen between wt and CatS −/− DC. This suggests direct trafficking from the ER–Golgi to early as well as late endosomes upon maturation of class II molecules, and demonstrates that access of class II complexes to late endosomes is independent of CatS activity. Comparison of the 1 and 3 h chase points immediately revealed the progression of class II molecules from endocytic compartments to the peak that includes the PM (peak C) in wt DC, whereas in CatS −/− DC a greater fraction of class II molecules was arrested in late endocytic fractions . As expected, in wt DC the accumulation of mature, peptide-loaded class II molecules increased with time, and was most pronounced in lysosomes (peak A) and at the cell surface . The difference in intensity between the signal retrieved for mature class II complexes at the cell surface and its adjoining intracellular compartments (late endosomes) was striking, and suggested that none of these compartments could solely account for the total amount of class II complexes that finally reach the cell surface. In contradistinction, no fully mature αβ–peptide complexes can be detected after a 3-h chase in CatS −/− DC . Of note, the αβl isoform of class II, which is converted into class II–CLIP complexes by CatS, was detected along the entire endocytic route in wt DC. This raised the possibility that the activity of CatS might show a similar subcellular distribution. We addressed this aspect by active site–labeling of CatS, shown below. In wt DC only the Iip10 form accumulated to easily detectable levels, whereas the p22 and p18 forms of Ii were not readily detected . In CatS −/− DC, Ii intermediates were more prominent at 3 h of chase than after 1 h. In absolute amounts, late endosomes contained the largest quantities of Ii breakdown intermediates . Interestingly, in late endosomes the p22 intermediate was more abundant than p18, whereas in lysosomes p22 was less abundant than p18 . In peak C, which based on the presence of TfR includes early endosomes, the p22 to p18 ratio was even higher than in peaks A (lysosomes) and B (late endosomes). These findings are consistent with a precursor–product relationship for p22 and p18, where the most likely sites of conversion are late endosomes and lysosomes. In contrast to CatS −/− DC, only accumulation of Iip10, but not of p22 and p18, was observed when the CatS activity in wt DC was pharmacologically inhibited by incubation with LHVS , although conversion from αβl to αβm is efficiently blocked by this inhibitor. This suggests that LHVS, even at 3 nM, might not completely block CatS activity, i.e., p22 and p18 were still degraded by CatS in the presence of LHVS, although Iip10 did accumulate. Combined, these data suggest that in CatS −/− DC, the maturation of class II molecules is severely compromised due to a failure to process Ii properly. Breakdown intermediates of Ii remain associated with class II molecules, which accumulate intracellularly in late endosomes and lysosomes. In view of the similar steady-state levels of class II molecules at the cell surface of wt and CatS −/− DC, the flux of class II molecules from the endocytic pathway to the cell surface must be strongly inhibited in CatS −/− DC. The surface deposition of class II molecules was measured more directly for wt and CatS −/− DC by performing surface biotinylation in conjunction with pulse–chase analysis . Only surface-disposed radiolabeled class II molecules that arrive over the course of a pulse–chase will become a substrate for surface biotinylation and allow their recovery on a streptavidin–agarose matrix. In wt DC, we observed extensive maturation of class II molecules over a 3-h and overnight chase, as inferred from the accumulation of SDS-stable, fully mature αβ–peptide complexes. Of these molecules, a fraction can be surface biotinylated, and this fraction increased very little between the 3-h and overnight chase timepoints. Although surface biotinylation is not quantitative, the data suggest that deposition of class II molecules on the cell surface reached a plateau at or shortly after 3 h of chase in wt DC. In CatS −/− DC, we observed the accumulation of the αβl complex at 3 h and after overnight chase, whereas very little material was accessible to surface biotinylation. Therefore, the flux of class II molecules from intracellular compartments to the cell surface is dramatically reduced in CatS −/− DC. The wide subcellular distribution of substrates for CatS (p10 in wt DC, p22, p18, and p10 in CatS −/− DC) suggested a corresponding distribution of the active enzyme in mature DC. For direct visualization of CatS activity, we synthesized a derivative of LHVS that carries a phenolic substituent on the vinylsulfone moiety to allow the introduction of 125 I . This compound, LHVS-PhOH, was radiolabeled in an Iodogen-catalyzed reaction, and purified by reverse-phase HPLC. The peak fractions of radioactivity were used for labeling experiments. Extracts were prepared from DC obtained either from wt or CatS −/− animals and labeled with 125 I–LHVS-PhOH. This comparison immediately revealed the labeled polypeptide of ∼28 kD that corresponds to CatS . LHVS-PhOH behaved similarly to LHVS with respect to substrate specificity and affinity, as revealed by competition experiments (data not shown). Using 125 I–LHVS-PhOH significant CatS activity was demonstrated in all three subcellular fractions (peaks A, B, and C) of wt DC, while CatS −/− DC showed no detectable signal for active CatS . Therefore, CatS activity is not restricted to late endocytic compartments, but is found along the entire endocytic route in mature DC, in good agreement with the wide pH range of CatS activity in vitro . This result is consistent with the presence of substrates of CatS and conversion of αβl into αβm in all endocytic compartments examined (see above). The additional polypeptides seen in Fig. 9 C most likely represent other cysteine proteases, as the vinyl sulfone functionality of LHVS-PhOH is reactive toward active site cysteines. Furthermore, not only are the mature cathepsins labeled with these probes, but some of their proforms can also be decorated (Bryant R.A.R., and H.L. Ploegh, manuscript in preparation). Some of the higher molecular weight polypeptides are absent in the CatS −/− sample and may indeed correspond to proCatS, which would be in good agreement with a localization in the Golgi–ER compartment represented in peak C. The pattern of labeling appeared highly pH-dependent, such that at near-neutral pH, excellent selectivity of labeling was obtained. At more acidic pH, a newly labeled species became prominent that we could identify as CatB, based on comparison with DC obtained from CatB −/− mice (data not shown). We conclude that LHVS-PhOH is a selective, but by no means uniquely specific probe for CatS, and infer from this result that LHVS can inhibit not only CatS, but also other thiol proteases. The identification of proteases involved in the degradation of Ii in living cells has relied largely on the use of class-specific protease inhibitors, and more recently, on the use of mice genetically deficient in the lysosomal proteases CatD, CatB, CatL, and CatS. Earlier work that implicated CatB and CatD in both Ii degradation and generation of antigenic peptides has been superseded by analyses of CatB- and CatD-deficient mice . In living cells, neither protease is essential for maturation of class II molecules or antigen presentation. Results obtained with the inhibitor LHVS, rather specific for CatS at low concentrations, implicated CatS in removal of Ii . In the presence of LHVS, proteolytic processing of Ii is arrested at intermediate stages . As expected, LHVS severely compromises peptide loading, as assessed in pulse–chase experiments. Our data obtained with a radiolabeled version of the LHVS analogue LHVS-PhOH used as an active site–directed probe allowed the identification of enzymatically active CatS, demonstrated its presence throughout the endocytic pathway, and indicated that CatS could act on class II–associated Ii at these different locations. Although LHVS is quite specific for CatS at low concentrations in vitro, higher concentrations of LHVS will readily target other thiol proteases (Bryant, R.A.R., G.-P. Shi, H.A. Chapman, and H.L. Ploegh, unpublished observations), not all of which have been identified to date. CatS −/− –deficient mice allow the analysis of the contribution of CatS to maturation and intracellular trafficking of MHC class II independently of the use of pharmacological inhibitors. Whereas class II maturation has been analyzed biochemically in CatS −/− mice, the contribution of CatS to proper trafficking of class II molecules had not been addressed in this model . However, a role for CatS in such control was made plausible in experiments in which the trafficking of class II products was examined in DC in the presence and absence of LHVS . DC are the most potent antigen-presenting cells described to date and represent a rather heterogeneous set of professional APCs that share certain characteristic features . However, bone marrow–derived DC, as generated by incubating bone marrow cells with GM-CSF in vitro, and flt3 ligand–induced splenic DC used in this study, differ in size and relative distribution of marker enzymes. Immature DC, upon encounter with antigen, efficiently internalize and convert the foreign material into class II–peptide complexes. In response to inflammatory stimuli, presumably coincident with acquisition of antigenic material, these DC undergo maturation, in essence externalizing and freezing class II–peptide complexes at the surface for maximal exposure to T cells . These processes, proposed to be at least in part under the control of CatS, march in lockstep with migration of DC from the periphery to lymph nodes, where contact with T cells occurs. However, the low levels of class II expression on the surface of immature wt DC , versus the high class II expression on mature DC from CatS −/− mice is a discrepancy that shows differential activity of CatS to be not the only major regulating factor for surface expression of class II during DC maturation. Application of the inhibitor LHVS to mature DC resulted in an intracellular distribution of class II molecules highly reminiscent of that seen in immature DC. What is the actual route taken by class II molecules in DC and what is the step controlled by the activity of CatS? Class II molecules reach the endocytic compartment either by internalization from the cell surface or by targeting from the TGN, probably to the early endosomal–late endosomal junction. The intracellular localization of CatS activity and the site of conversion of its substrate (αβl into αβm) in DC can provide important information to resolve these questions. Using a less direct assay, CatS activity could be demonstrated only in lysosomal compartments of bone marrow–derived DC . This contrasts with the broad distribution of CatS activity observed here. CatS is active throughout the endocytic pathway, as demonstrated both by active site–labeling of CatS and analysis of degradation intermediates of Ii in the presence or absence of CatS. NH 2 -terminal processing of Ii by CatS occurred throughout the entire endocytic route in mature DC, whereas COOH-terminal degradation by proteases other than CatS appears to be restricted to nonlysosomal compartments. In the absence of CatS, the NH 2 terminus of Ii retains a sizable fraction of class II molecules in endocytic compartments until it reaches lysosomes. This situation applies to DC as well as splenocytes (Driessen, C., unpublished observations). The sorting event that directs class II molecules to the cell surface in mature DC was suggested to be localized to an early endocytic compartment, where active CatS would cleave Ii and release the NH 2 -terminal sorting signal of Ii from the class II–Ii complexes . Class II molecules from that location would reach the cell surface, bypassing later endocytic structures. Alternatively, and at the other end of the spectrum, class II molecules may reach the lysosomal compartment largely independently from the activity of CatS. CatS would then control the egress of class II molecules from lysosomes. We envision that these two extreme possibilities would result in major differences in terms of peptide loading, because of the distinct characteristics of the subcellular compartments encountered by class II molecules before reaching the cell surface in either model. While our data are compatible with either model, they suggest that there are major factors in addition to CatS activity that control the trafficking of class II molecules in DC. After 3 h of chase, we detect a substantial amount of fully mature class II molecules in lysosomal compartments of wt DC, indicating that conversion of Iip 10 into CLIP is probably not the rate-limiting step in directing class II to the PM. Since αβ–peptide complexes can thus be generated along the entire endocytic route in mature DC, they might leave the endocytic compartment from these many locations independently rather than following a single, shared track. The amount of mature αβ–peptide material retrieved from the cell surface and/or early endosome fraction after 3 h of chase by far exceeds that of any of its precursor forms in a single compartment at a given time. This would support the hypothesis that class II molecules at the surface are recruited from several subcellular compartments. The sizable amount of αβm retrieved from the lysosomal compartment after 3 h of chase contrasts with the smaller amount retrieved under steady-state conditions. We suggest it represents a kinetic intermediate and similarly contributes to the fraction that reaches the surface. Delivery of class II molecules from lysosomes directly to the cell surface has been suggested . The ensuing exchange of CLIP for antigenic peptides necessitates an interaction with H2-DM. Furthermore, peptide cargo directly affects intracellular transport of class II molecules . Alternatively, all αβm may traffic to late endocytic or lysosomal compartments before an additional signal is required to release class II to the surface. Again, the impact of interactions with accessory molecules (H2-DM, H2-DO) remains to be assessed. Breakdown intermediates of Ii are abundant in CatS −/− DC. These intermediates are detected first in early endocytic compartments but are particularly prominent in late endocytic compartments and lysosomes. Based on their molecular mass and the epitopes present, as well as the temporal relationships in the occurrence of these intermediates, a precursor-product relationship was established that could be related to their intracellular location. Iip10, Iip22, and Iip18 accumulate in CatS −/− DC, and therefore are dependent on CatS activity for further processing. They were present in different relative amounts in early and late endocytic compartments. Whereas p22 preferentially was found in early and late endosomes, the p18 form was favored in lysosomal compartments. Therefore, the nature of the CatS substrate(s) changes gradually along the endocytic route. The enzyme(s) involved in conversion of Ii into p22 and p18 have not been identified. In contrast to CatS −/− DC, no accumulation of Iip22 and Iip18 was observed when CatS activity was inhibited by treatment with LHVS in wt DC (making the limitations of the use of this pharmacological inhibitor evident). The incomplete block of CatS activity by LHVS, as demonstrated by this comparison, could be explained by a possible loss of LHVS specificity under the acidic conditions that prevail in late endocytic compartments. Indeed, results obtained with the radiolabeled derivative of LHVS, LHVS-PhOH, showed excellent selectivity of label under neutral conditions rapidly lost at lower pH. The predominant localization of p18 and p22 in late endocytic compartments would be in line with this explanation. Under normal circumstances, the activity of CatS presumably coincides with that of other enzymes that attack Ii, but at present their individual contributions cannot be distinguished. There is of course a paradox: how can the intracellular retention of MHC class II molecules be reconciled with unaltered levels of surface-expressed class II complexes observed in CatS −/− DC? This puzzle could be resolved if the following conditions apply. First, the half-life of class II at the cell surface must exceed by far the normal rate of surface deposition of class II molecules. There is ample support for this suggestion; peptide-loaded class II complexes are extraordinarily stable . Second, a homeostatic mechanism that limits the number of class II molecules that can be inserted at the cell surface might be operative. Given sufficient time, even a slow rate of surface deposition, coupled to an upper limit for the number of class II molecules allowed at the cell surface, would result in comparable levels of surface-expressed MHC class II. Relative to their CatS −/− counterparts, in normal DC any surplus of class II molecules might be destroyed, for example in lysosomes, rather than reach the cell surface. This suggestion is consistent with the reproducibly higher recovery of total class II molecules from CatS −/− DC, whereas the recovery of class I molecules is comparable. Proteolysis of Ii is a compound reaction that controls intracellular transport of MHC class II molecules, and ultimately, their loading with peptide and display at the cell surface. Therefore, class II molecules must function not only at the cell surface, but also in a lysosomal environment. There, the class II–associated Ii chain represents the equivalent of a propiece that keeps MHC class II molecules in an inactive form until Ii is released by proteolysis and its remnants have been cleared from the class II peptide-binding cleft by accessory proteins such as H2-DM. Removal of Ii is a process of interest not only for its immunological consequences but also because proteolytic destruction of Ii is one of the few examples in which proteolysis controls trafficking of a surface glycoprotein. Such controlled proteolysis appears to regulate molecular trafficking and maturation of at least three other transmembrane proteins, the Notch receptor , the sterol regulatory element binding protein SREBP , and β-amyloid precursor protein APP . For all three of these proteins, two or three consecutive proteolytical steps are suggested to occur in a sequential fashion, culminating in the release of an effector molecule (in these cases a protein fragment) that traffics from the PM to either the nucleus (Notch, SREBP) or the extracellular environment (APP) . The proteases that perform this final cleavage and therefore largely control the biological activity of the cleaved substrates have not been identified. The regulation of MHC class II trafficking by sequential degradation of Ii, as unraveled using DC from CatS −/− mice in this study, is not unlike these models: at least two proteolytic steps, one of which is performed by CatS, occur in a sequential fashion in the course of Ii degradation along the endocytic route. This late step releases the αβ–CLIP complex and initiates loading with antigenic peptide to generate the biologically active entity. Unlike the known examples for the control of trafficking and maturation of transmembrane proteins by sequential proteolysis, degradation of Ii does not occur in the cytosol but in late endocytic compartments where multiple proteolytic enzymes with partly overlapping functions and specificities are concentrated. Even in the lysosomal environment, rich in a large selection of proteases, proteolytic enzymes afford sufficient specificity to tightly regulate essential cellular properties. | Study | biomedical | en | 0.999997 |
10562281 | Table lists the yeast strains and Table the plasmids used. Sherman 1991 and Ausubel et al. 1996 describe the methods used for growth, maintenance, and genetic manipulation of yeast, and Gietz and Schiestl 1995 describe the method used to transform yeast. Temperature-sensitive myo2 strains were generated by replacing the 3′ region of the endogenous genomic MYO2 gene with mutagenized plasmid DNA. To construct a plasmid capable of integrating 3′ to MYO2 , pJP10-2B (YCp50 containing MYO2 ) was cut with SpeI and the resulting fragments circularized using T4 DNA ligase. The ligated DNA served as template for five cycles of amplification by PCR using Taq DNA polymerase and the oligonucleotide primers GATAATGAAATCGATATTATGGAAGA and CGGGATCCATTATCATACTATACTATTGACAAATACTTC. The ClaI-BamHI segment of the 1.7-kb product was inserted into pRS303 . Destruction of the pRS303 SpeI site by cutting with SpeI (partial digest) and XbaI and religation produced the plasmid pRS303MYO2, containing the 201-bp ClaI-SpeI fragment of the MYO2 3′ untranslated region, followed by the 1,587-bp segment of MYO2 from the SpeI site at +3271 on, fused to the sequence GCACTAGA and the NotI site of pRS303. pRS303MYO2 served as a template for PCR-based mutagenesis , modified to reduce the mutation frequency. The reaction mix (1 mM dCTP, 1 mM dTTP, 0.2 mM dATP, 0.2 mM dGTP, 40 ng pRS303MYO2 cut with PvuII, 7 mM MgCl 2 , 0.5 mM primers, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 10 mg BSA, and 0.05% Tween 20; and after the mix had warmed up to 80°C, 0.3 mM MnCl 2 and 5u Taq DNA polymerase) was subjected to six cycles of amplification and the EcoRI-NotI fragment of the PCR product was subcloned into the corresponding region of pRS303MYO2 to make libraries of mutagenized MYO2 . When cut with SpeI and transformed into yeast, the library plasmids insert pRS303 (containing HIS3 ) into the ClaI site of the MYO2 3′ untranslated region, and replace the EcoRI-ClaI region of MYO2 with mutagenized sequence encoding amino acid residues 1119 to the COOH terminus. Roughly 500 transformants at 30°C were replica-plated to 14 and 37°C to detect conditional-lethal mutants. Phloxine B included in plates at 10 mg/liter allowed easier identification of growth-arrested yeast . Because dead cells absorb this red dye, red marks correspond to the positions of temperature-sensitive colonies. Genomic DNA from each of the temperature sensitives was cut with SpeI and circularized to reconstitute versions of pRS303MYO2 containing mutations. Transformation of these plasmids relinearized with SpeI into the yeast strain PSY316 yielded the temperature-sensitive strains ABY532, 533, 534, 536, 537, 538, and 530. The temperature sensitivity could be complemented by transformation with pJP10-2B ( MYO2 on a low copy number plasmid) or by mating with a MYO2 strain, but could not be complemented by mating with JP7B, a myo2-66 strain. ABY531, an isogenic MYO2 strain, was made by transformation of PSY316 with pRS303MYO2. An analogous series of plasmids made from pRS306 (containing URA3 ) allowed the construction of yeast strains containing myo2 alleles tagged with URA3 . To make an isogenic myo2-66 strain, genomic DNA from JP7B transformed with pRS303MYO2 was cut with NcoI and religated, resulting in an integrating plasmid analogous to pRS303MYO2, but containing the full-length myo2-66 gene instead of the MYO2 fragment. This plasmid was cut with NcoI and transformed into PSY316 to construct ABY535. Diploid versions of these strains were made by transiently transforming with YCp50HO, a plasmid bearing HO . Genetic interactions of myo2-13 with sec1-1 , sec2-56 , sec5-24 , sec6-4 , and sec9-4 mutations were tested by mating ABY533 with MATa sec ts strains ( MATa strains being generated from MAT α by standard genetic methods), sporulating, and dissecting tetrads onto synthetic minimal dextrose medium (SD) 1 and yeast extract peptone-dextrose medium (YPD) at 30°C. Crosses producing a high frequency of tetrads with four viable spores are considered not to show synthetic lethality. P < 0.05 in all cases reported and P < 0.0005 in most cases, based on a binomial distribution, assuming meiotic gene conversion does not exceed 20%. Nonsynthetic lethals were confirmed by testing temperature sensitivity to show that some viable spores are likely to be myo2-13 sec ts double mutants. We suspected that myo2-13 is synthetically lethal with sec4-8 but could not prove it by the method described above, because even MYO2 + strains have low spore viability when crossed with the sec4-8 strain NY405. Therefore, we determined synthetic lethality of sec4-8 with myo2 ts mutations by constructing sec4-8 myo2 ts strains bearing pJP10-2B, a CEN plasmid containing MYO2 and URA3 . Strains with synthetic lethal combinations are unable to grow at 22°C on minimal medium containing 5-fluoro-orotic acid, which selects against URA3 . To determine synthetic growth defects of nonlethal combinations, restrictive temperatures with and without pJP10-2B were compared. At least three different strains from the sec4-8 × myo2 ts cross were tested for each combination of alleles and conditions. To screen for overexpression suppressors of myo2 ts , myo2-17 , myo2-18 , and myo2-20 strains were transformed with a multicopy (2μ) genomic library in YEp351 , and 287,000 colonies replica-plated onto rich media at 37°C. SMY1 was isolated from one clone by subcloning the PstI-StuI fragment of the insert into PstI-SmaI of YEp351. YEpSMY1, the resulting plasmid, contains the SMY1 open reading frame flanked by the 5′ 651 bp and 3′ 266 bp of untranslated sequence. To produce anti-Myo2p antibodies, the NsiI-EcoRI fragment of pJP10-2B, encoding amino acid residues 784–1118 of Myo2p (encompassing the IQ repeat and coiled–coil domains), was subcloned into pRG32D and the glutathione S -transferase (GST)–Myo2p fusion protein was purified as described (Amersham Pharmacia Biotech). Rabbit antisera against the fusion protein were affinity-purified using fusion protein coupled to activated CH-Sepharose 4B (Amersham Pharmacia Biotech). To block antiyeast antibodies in the rabbit anti-LexAp antiserum , the antiserum was mixed with an equal volume of yeast, fixed, zymolyase digested, methanol/acetone extracted as for immunofluorescence , gently mixed overnight at 6°C, and centrifuged to remove cells and debris. Immunofluorescence was performed as described using 1:50 anti-Myo2p and 1:100 anti-LexAp. To measure the proportion of cells containing polarized Sec4p and Myo2p, we identified a random small-budded cell by differential interference contrast (DIC), switched to viewing by fluorescence to score whether the bud or bud tip is brighter than the mother cell, then switched back to DIC and moved to a new field, repeating until we had scored 200, or in some experiments, 100 cells. For experiments where small-budded cells become scarce (75-min temperature shift of myo2 ts mutants or expression of LexA fusion protein) we counted all cells regardless of the cell cycle stage. Calcofluor staining , vacuole inheritance , and endocytosis of the water-soluble dye Lucifer yellow were observed by the methods described. As negative controls, we tested DBY2000 ( act1-1 ) and NY405 ( sec4-8 ) for endocytosis: ∼10 and 1%, respectively, take up Lucifer yellow at 37°C, compared with 50–75% of wild-type and myo2 ts cells. To express high levels of a GST fusion protein containing the Myo2p COOH-terminal region, pEGM was constructed by subcloning the EcoRI-ClaI fragment of pJP10-2B into pRS303, and then inserting the BglII-SalI fragment of the resulting plasmid into the BamHI and SalI sites of pEG, a 2μ plasmid with GST under GAL control (pEG[KG]) . A CEN plasmid, pCGM, was made by replacing the smaller NcoI-SalI fragment of pRS316 with the smaller NcoI-SalI fragment of pEGM. Similarly, pCG was made by replacing the smaller NcoI-XbaI fragment of pRS316 with the smaller NcoI-XbaI fragment of pEG. pCLM was made by replacing the SacI-NruI fragment of pCGM encoding GST with a PCR product encoding LexA protein plus one COOH-terminal glycine. This construct contains three silent mutations in LexA. pCL was constructed by cutting pCLM with ClaI, filling in the 5′ overhang, cutting with NruI, and recircularizing, thereby removing the entire MYO2 sequence. Western blotting for GST and LexAp confirmed inducible expression of fusion proteins. A PSI-BLAST search for sequences significantly similar to the Myo2p tail identified 16 plant, 7 animal, 1 Dictyostelium , and 3 fungal myosin Vs (not including Myo2p). All have a conserved NH 2 -terminal motor domain, six IQ repeats, a predicted coiled–coil region (COILS2) , and the conserved tail domain ( Table ), except for the mouse myosin Vb sequence, which is incomplete. Searching for sequences similar to the Myo4p tail failed to find additional myosins. Some authors have grouped the plant and Dictyostelium myosins as a separate class, myosin XI. For the sake of clarity we call all of these myosins “myosin V,” since they clearly form a distinct group with a common origin and since all of the experimentally characterized myosins of this group are called myosin V in the literature. We used a parsimony method to determine the relationship among Myo2p, Myo4p, and Schizosaccharomyces pombe myosin V, using Dictyostelium myosin II as an outgroup. The three myosin V motor domains were independently aligned with myosin II, identifying 29 informative positions (where one residue occurs in two myosins and a different residue occurs in both of the other two myosins) in unambiguously aligned regions. These positions are widely dispersed in the motor domain structure and are spaced at least five residues apart. CLUSTAL W provided an initial multiple alignment of amino acid sequences that was manually corrected by comparing with the outputs of BLAST and MEME to find obvious alignment errors. Western blotting and chemiluminescent protein detection was performed using a 1:2,000 dilution of affinity-purified anti-Myo2p antibody overnight at 6°C. Invertase accumulation was measured and cells tested for elevated specific gravity by mixing early log-phase cells with 92% Percoll (colloidal silica; Sigma Chemical Co.), then centrifuging at 12,000 g for 5 min. Wild-type cells float, whereas sec6-4 and other sec mutants sink after incubation at the restrictive temperature. Much of the current knowledge about MYO2 function comes from studies of the original myo2 allele, myo2-66 , a mutation in the NH 2 -terminal motor domain . To investigate the COOH-terminal domain's function, we introduced random mutations into the chromosomal MYO2 gene and screened for heat- and cold-sensitive mutants. We found seven new heat-sensitive alleles of myo2 ( Table ) but no cold-sensitives. Temperature sensitivity is recessive in all myo2 ts tail mutants and none are complemented by myo2-66 . All seven tail mutants have doubling times within ±15% of wild-type (95% confidence interval for each allele) in synthetic minimal medium (SD) at 24°C, showing that all are highly functional at the permissive temperature. However, the growth rate of myo2-12 yeast at 24°C is slowed if wild-type MYO2 is introduced on a low-copy number plasmid, indicating a partial gain-of-function effect of this allele at permissive temperatures. The myo2-12 mutation appears to make more drastic amino acid changes in Myo2p than any other myo2 ts tail mutation, replacing a polar residue and a charged residue, both conserved in nearly all myosin Vs, with hydrophobic residues ( Table ). A phenotype common to myo2-66 and most of the new myo2 ts alleles is sensitivity to rich media: rich media cause a 1–3°C reduction of the restrictive temperature ( Table ) in all alleles except myo2-14 . In addition, myo2-12 and myo2-16 mutants have slowed growth in rich media even at 24°C. The reason for this rich medium sensitivity is unclear; addition of filter-sterilized peptone (2%) strongly inhibits growth of myo2-12 yeast (the most YPD-sensitive mutant) in SD, accounting for most of the inhibitory effect of rich media, whereas reduction of the glucose concentration partially restores growth to myo2-12 yeast in YPD. Addition of 1 M sorbitol to provide osmotic support has no effect on myo2-12 mutant growth in YPD. To identify components that interact with the Myo2p tail, we screened for genes whose overexpression suppress myo2-17 , myo2-18 , or myo2-20 temperature sensitivity. Our screen identified one clone of SMY1 , a gene recovered previously as a multicopy suppressor of myo2-66 . Multicopy SMY1 (YEpSMY1) dramatically suppresses myo2-12 , myo2-13 , myo2-16 , myo2-17 , and myo2-18 temperature sensitivity and moderately suppresses myo2-20 . Intriguingly, suppression of myo2-66 in an isogenic strain is weaker than for the alleles mentioned above, and multicopy SMY1 does not suppress myo2-14 at all ( Table and data not shown). The original myo2-66 mutation is lethal in combination (synthetically lethal) with mutations in a broad spectrum of other genes, including a subset of genes affecting post-Golgi steps in secretion . Since we are interested in the role of Myo2p in the polarized delivery of secretory vesicles, we wished to examine whether a myo2 ts tail mutation shows synthetic lethality with these mutations as well. We chose myo2-13 because it is neither one of the most nor one of the least severe temperature-sensitive alleles we isolated. myo2-13 is not synthetically lethal at 30°C on SD or YPD with sec1-1 , sec2-56 , sec5-24 , sec6-4 , or sec9-4 . In contrast, sec2-41 , sec5-24 , and sec9-4 have been reported to be synthetically lethal with myo2-66 at 25°C . We also checked for synthetic lethality of all the new myo2 ts alleles with sec4-8 . Like myo2-66 , myo2-14 is synthetically lethal with sec4-8 , whereas myo2-12 , myo2-13 , myo2-16 , myo2-17 , and myo2-18 have only mild synthetic growth defects with sec4-8 ( Table ). Interestingly, myo2-20 shows no synthetic growth defect at all with sec4-8 . Like myo2-66 , the new myo2 ts tail mutations block bud initiation and polarized growth at the restrictive temperature, with most of the cells arresting as very large, unbudded spheres ( Table ), indicating a block in polarized secretion even though secreted proteins still reach the cell surface. Furthermore, like myo2-66 yeast, the new temperature-sensitive tail mutants deposit chitin diffusely over the cell surface at 37°C in SD ( Table ). In myo2-66 yeast, this diffuse chitin distribution is due to a failure to target a membrane protein, the main chitin synthetase Chs3p, to the correct locations . To investigate whether loss of polarized secretion is a direct result of loss of Myo2p function, we looked at how rapidly the polarized distribution of Sec4p, a Rab GTPase component of late secretory vesicles , disappears in myo2 ts mutants after shift to the restrictive temperature. In wild-type yeast, shifting to temperature above 35°C depolarizes actin, Myo2p, and Sec4p, which then return more than an hour later . The polarized Sec4p distribution fails to return in myo2-66 and myo2 tail mutants ( Table ). To see the immediate effects of disrupting Myo2p function, we chose to look at myo2-16 and myo2-66 because their restrictive temperatures are low enough that shifting to these temperatures leaves the distribution of Myo2p, Sec4p, and the actin cytoskeleton relatively unaffected in wild-type yeast . In myo2-16 yeast, the Sec4p distribution begins to depolarize within 1 min of temperature shift and is completely depolarized within 5 min . In earlier studies, Walch-Solimena et al. 1997 showed that myo2-66 blocks accumulation of Sec4p at growth sites after 1 h at 37°C, and Govindan et al. 1995 showed that myo2-66 blocks the accumulation of secretory vesicles in small buds. Here we show that myo2-66 yeast has a partially polarized distribution of Sec4p at 25°C that disappears very rapidly upon shift to a restrictive temperature . The redistribution of Sec4p is complete within a 1-min shift to 30.5°C (data not shown). We conclude that the loss of Myo2p head or tail function depolarizes Sec4p, and presumably secretory vesicle targeting in <5 min. Shifting myo2-16 yeast to 35°C for 5 min, conditions that completely depolarize Sec4p, has no effect on the Myo2-16p distribution . MYO2 wild-type yeast shows a slight depolarization of Myo2p upon shift to 35°C, such that Myo2-16p retains slightly greater polarity than wild-type Myo2p. Myo2-12p, another tail mutant, also remains polarized despite a depolarized Sec4p distribution . Furthermore, Myo2-17p, Myo2-18p, and Myo2-20p are at least as polarized as wild-type Myo2p at 37°C ( Table ). In contrast to Myo2-16p, the polarized distribution of Myo2-66p disappears very rapidly (within 5 min of shifting to 35°C) , even though Myo2-66p is still present . As is the case with Sec4p, the redistribution of Myo2-66p is nearly complete within 1 min shift to 30.5°C (data not shown), and even at the permissive temperature there is a substantial defect in Myo2-66p polarization . To test whether this polarized distribution of Myo2-16p reflects continued cable-dependent transport or simple trapping of the myosin at growth sites, the behavior of the tail mutant was examined in a background conditionally defective for cables . tpm1-2 tpm2Δ myo2-16 cells shifted to 34.5°C lose cables as well as the polarized distribution of Myo2-16p . When restored to a permissive temperature, cables reform and the mutant myosin repolarizes within 2 min, just as seen for wild-type myosin ( MYO2 + tpm1-2 tpm2Δ yeast) . Notably, Myo2-16p repolarizes despite the lack of Sec4p-associated cargo . Again, a slightly greater proportion of myo2-16 than MYO2 + cells show a polarized Myo2p distribution. myo2-16 has very little effect on the overall appearance of the actin cytoskeleton at 35°C. As seen by immunofluorescence, actin cables are as abundant as in wild-type yeast and remain aligned towards presumptive regions of growth even after prolonged incubation (1.5 h) at the restrictive temperature. Actin patches remain polarized at early timepoints and then slowly redistribute to an even distribution over the entire surface of the cell. Substantial depolarization of actin patches requires ∼15 min of temperature shift and complete depolarization requires >30 min. At their restrictive temperatures, the other myo2 ts tail mutants show a similar actin distribution to myo2-16 , except for myo2-12 after prolonged incubation at a restrictive temperature, where a small proportion (≤1%) of cells contain actin bars similar to those seen in myo2- 66 cells . The bars presumably represent aberrant aggregates of unpolymerized actin, because antiactin antibody detects the bars whereas rhodamine-phalloidin, which binds actin filaments, does not. The actin distribution in myo2-66 yeast is remarkably similar to wild-type and myo2-16 , except that even at the permissive temperature myo2-66 yeast has subtle defects in the actin patch distribution. These results are consistent with the finding that functional Myo2p is unnecessary for the rapid assembly of actin cables . Actin bars are much less abundant (≤1% of cells) than reported by Johnston et al. 1991 , a discrepancy probably accounted for by a difference in strain background and growth conditions. If Myo2p moves along actin cables, it might partially colocalize with them. Indeed, in myo2 tail mutants shifted for a long time to 37°C such that actin patches no longer obscure cables at polarized sites, the mutant Myo2p is concentrated at the point where cables converge . Furthermore, Myo2p staining in wild-type yeast sometimes reveals faint cable-like structures and rows of Myo2p patches along actin cables . This colocalization is not due to bleed-through between fluorescence channels, because the bright actin patches are not generally visible in the Myo2p images and the bright patch of Myo2p at the bud tip is not seen in the F-actin images . In addition to its role in polarized secretion, MYO2 is also needed for polarized transport of the vacuole into the bud , and even at a permissive temperature, myo2-66 blocks transfer of any part of the vacuole from the mother cell to the growing bud ( Table ). In addition, a screen for mutants defective in vacuole inheritance identified myo2-2 , a Myo2p tail point mutation that does not dramatically affect polarized secretion . In contrast, none of the new myo2 ts mutants have any vacuole inheritance defect at 24°C ( Table ). Even at 34°C, a semipermissive temperature, 51% of myo2-17 cells, 75% of myo2-18 cells, and 64% of myo2-20 cells show transfer of vacuole membrane to the bud, demonstrating only a partial defect (95% of wild-type cells transfer vacuoles to the bud). A conditional mutant of actin shows a partial defect in invertase secretion , but neither myo2-66 nor the new myo2 tail mutations shows a detectable defect in invertase secretion at the restrictive temperature (data not shown), showing that the myo2 mutations disrupt polarized secretion without affecting fusion of invertase-containing secretory vesicles with the plasma membrane. The new myo2 tail mutations also fail to show the increase in specific gravity at the restrictive temperature seen in mutants blocking the secretory pathway . Endocytosis requires many of the known components of the actin cytoskeleton , but does not require a functional Myo2p motor domain . After 20 min at 37°C, none of the new myo2 ts alleles affect endocytosis, assayed by uptake of Lucifer yellow, indicating that the Myo2p tail, like the motor, is dispensable for endocytosis. We expressed fusion proteins containing the Myo2p COOH-terminal domain to see whether this domain would interfere with polarized delivery of secretory vesicles by competing with endogenous Myo2p. Expression of GST fused at the COOH terminus with residues 1131–1574 of Myo2p from a galactose-inducible promoter on either a multicopy plasmid (pEGM) or low-copy number plasmid (pCGM) completely blocks growth. Galactose concentrations of 0.02 and 0.08% are sufficient to block growth of cells bearing pEGM and pCGM, respectively, but expression of GST alone from either a high-copy (pEG) or low-copy (pCG) plasmid has no effect on growth at 2% galactose. At high galactose concentrations (≥1%) >50% of the cells with pEGM or pCGM are lysed by 6 h of induction, but at lower concentrations (≤0.1%), cells with pEGM become spherical, very large, and mostly unbudded, like myo2 ts mutants. Also, like myo2-66 yeast that has been shifted to the restrictive temperature for a long time , a population of very tiny cells is also present, and presumably represent buds that have separated from the mother cell without growing to a normal size. Cells with pEGM and induced with 2% galactose show a normal polarized Sec4p distribution except at late timepoints, after 8 h in galactose, when most of the cells become inviable. 8 h is also the amount of time needed for full induction from the GAL promoter in the strains we use. To confirm that the tail fusion protein is interfering with normal MYO2 function rather than interfering with an unnatural target, we tested whether myo2 mutants are hypersensitive to GST–Myo2p tail fusion protein by transforming isogenic MYO2 + and myo2 ts strains with pEG and pEGM and testing growth on raffinose plates in the presence or absence of leucine. pEG and pEGM contain a partially defective LEU2 allele, leu2d , which selects for two- to fourfold higher plasmid copy number in the absence of leucine than in the presence of leucine , such that in the absence of leucine there is some expression from the GAL promoter despite the lack of galactose. Under these conditions, MYO2 + yeast is unaffected by GST–Myo2p tail, but myo2-12 , myo2-14 , myo2-16 , and myo2-20 yeast grow poorly and become spherical and very large. Growth of myo2-13 and myo2-18 yeast is moderately slowed, whereas myo2-17 yeast is only slightly affected . We were unable to test myo2-66 yeast because myo2-66 , unlike the other myo2 alleles, causes very poor growth at any temperature when raffinose is the sole carbon source, and we were unable to construct myo2-66 strains capable of growing on nonfermentable media. To see the subcellular distribution of Myo2p tail fusion protein, we tagged residues 1147–1574 of Myo2p with LexAp, a bacterial protein . Expression of LexA–Myo2p tail fusion protein from a GAL promoter on a low-copy number plasmid (pCLM) is lethal . Immunofluorescence shows that 1–2 h after addition of 2% galactose, 10–40% of cells (depending on strain background) transformed with pCLM show a polarized distribution of LexAp similar to the polarized distribution of Sec4p and Myo2p . Sec4p and the endogenous Myo2p distribution (using an antibody raised to a region outside the tail) is normal in these cells. With longer induction times (>4 h in galactose) the distribution of LexA–Myo2p tail in cells bearing pCLM becomes depolarized. Some of the larger-budded cells (1–9% at 1–2 h induction, rising to 20% at 4 h) show depolarized LexA–Myo2p tail and bright lumps of staining near the bud neck. Cells expressing LexAp alone (bearing pCL) show diffuse cytoplasmic staining, with a small proportion of cells that also show bright lumps of staining randomly distributed in the cell (data not shown). The clumping seen in both these cases might represent aggregation in those cells that express high levels of protein, since LexAp multimerizes at high concentrations , but notably, only the Myo2p tail fusion protein polarizes within the cell. To test whether this polarized distribution of LexA–Myo2p tail fusion protein depends on polarized targeting of secretory vesicles, we looked at the distribution of LexA–Myo2p tail fusion protein in a myo2-16 mutant. After 5 min shift to 34.5°C, myo2-16 yeast shows a greatly reduced number of cells with polarized LexA–Myo2p tail protein compared with MYO2 + yeast . The distribution of endogenous Myo2-16p is normal in the myo2-16 cells. These results indicate that the polarized distribution of the Myo2p tail fusion protein requires an endogenous Myo2p motor protein with a functional tail domain. This could be because either the tail fusion protein binds directly to full-length Myo2p and this site is defective in myo2-16 , or the tail fusion protein binds to secretory vesicles normally transported by full-length Myo2p. To distinguish between these two possibilities, we examined whether the fusion protein becomes depolarized in a sec2-41 mutant. Walch-Solimena et al. 1997 have shown that Sec4p becomes depolarized in sec2 cells after 10-min shift to 37°C, indicating that polarized delivery of secretory vesicles requires Sec2p, the nucleotide exchange factor for Sec4p. The polarized distribution of LexA–Myo2p tail is reduced in sec2-41 yeast after 5-min shift to 35°C . The polarized distribution of Sec4p also disappears, showing that secretion is no longer polarized. However, the distribution of endogenous full-length Myo2p is normal in the sec2-41 cells, showing that the mechanism whereby sec2-41 disrupts Sec4p and LexA–Myo2p tail targeting does not involve disrupting the targeting of full-length Myo2p. In contrast, sec1-1 , which blocks vesicle fusion but not targeting, increases the frequency of polarized LexA–Myo2p tail distribution upon temperature shift . The two myosin V genes of yeast, MYO2 , required for polarized membrane transport, and MYO4 , required for polarized mRNA targeting , afford an opportunity to find protein sequences specifically involved in membrane binding. Saccharomyces has undergone a genome duplication sometime after it diverged from another Ascomycete, Kluyveromyces , and MYO2 and MYO4 lie in an unambiguously duplicated region . In support of a recent duplication, the Myo2p and Myo4p tails are more similar to each other than to any other myosin V. Furthermore, phylogenetic analysis of motor domain sequences shows that Myo2p and Myo4p diverged from each other after they diverged from a Schizosaccharomyces pombe myosin V, the most similar known protein to Myo2p and Myo4p. Despite this evidence for a relatively recent origin of two different myosin Vs in yeast, animal myosin Vs are much more similar to Myo2p than to Myo4p in the COOH-terminal half of the tail. Fig. 8 C shows the two extensive regions that can be unambiguously aligned among animal myosin V tails and both Myo2p and Myo4p (matches highlighted in blue). Chicken p190, a biochemically well-characterized myosin V , is shown as an example of an animal myosin V. Strikingly, in both regions animal myosin Vs are much more similar to Myo2p than to Myo4p . Plant myosin Vs (also known as myosin XIs) show less similarity to yeast myosin Vs, but nevertheless tend to be more similar to Myo2p than to Myo4p in this region . Since the animal myosin Vs are known to transport membranes , the simplest interpretation is that the distal half of the Myo4p tail has lost features involved in membrane transport which are conserved in Myo2p and animal myosin Vs. Notably, most of the myo2 ts tail mutations make at least one nonconservative change in this region, and changes in the more distal regions correlate with sec4-8 synthetic growth defects . Since the original description of the myo2-66 mutation's effect on polarized secretion , Myo2p's precise role has been elusive. Models for Myo2p function have proposed that the motor transports secretory vesicles tethered to the COOH-terminal globular domain down tropomyosin-containing actin cables to their site of exocytosis , or alternatively, that the COOH-terminal domain binds the plasma membrane at polarized sites, which indirectly leads to targeting of secretory vesicles to growth sites , possibly by polarizing the actin cytoskeleton . To distinguish between these models and to shed more light on the role of Myo2p, we have characterized new conditional alleles of myo2 with defects in the COOH-terminal domain. Our results provide strong support for a model in which Myo2p binds secretory vesicles through its COOH-terminal domain and transports them down actin cables to regions of cell growth. We show that polarized secretion requires a functional Myo2p COOH-terminal globular domain: the tail mutants show depolarized growth, isotropic chitin deposition and loss of bud emergence, like the original motor mutant. These results are the first to directly implicate the Myo2p tail in polarized delivery of secretory vesicles. Loss of the secretory vesicle component Sec4p from polarized sites occurs within 5 min of shifting the tail mutant myo2-16 to the restrictive temperature. By contrast, both Myo2-16p and the actin cytoskeleton remain polarized for >15 min, demonstrating that the redistribution of Sec4p in myo2-16 mutants is not due to Myo2p mistargeting or actin cytoskeleton depolarization. Since the Sec4p and Myo2p distributions are normally identical but temperature-shifting myo2-16 yeast disrupts only the Sec4p distribution, the simplest interpretation is that the tail tethers vesicles to Myo2p. Additional evidence that the tail binds secretory vesicle cargo comes from yeast expressing fusion proteins containing the Myo2p tail. Isotropic growth and loss of bud emergence, the same phenotypes as myo2 ts mutants, combined with the hypersensitivity of myo2 tail mutants to tail fusion protein, show that tail fusion proteins disrupt Myo2p's normal function in polarized secretion. These fusion proteins presumably compete with the endogenous Myo2p tail for a factor in limited supply, such as a receptor for Myo2p on secretory vesicles. Accordingly, fusion protein initially concentrates at the same polarized sites as Myo2p and Sec4p. As we describe below, this initial distribution probably represents fusion protein bound to secretory vesicles, which are carried by endogenous Myo2p to growth sites, before fusion protein expression reaches saturating levels. While this study was in the final stages of assembly, Reck-Peterson et al. 1999 reported a similar approach with an epitope-tagged Myo2p tail construct that, like our constructs, lethally disrupts polarized secretion and initially concentrates at polarized sites. Although Reck-Peterson et al. 1999 concluded that the Myo2p tail localizes independently of the rest of the molecule, we found no evidence to support that conclusion. Instead, we found that the polarized distribution of tail fusion protein depends on functional full-length Myo2p, as demonstrated in a myo2-16 mutant shifted to the restrictive temperature. More specifically, polarized secretion is essential for a polarized tail fusion protein distribution. The sec2-41 mutation disrupts the targeting of secretory vesicles to growth sites . Shifting sec2-41 yeast to the restrictive temperature for 5 min also disrupts the tail fusion protein's polarized distribution, even though endogenous Myo2p stays at growth sites, showing the tail fusion protein does not bind to endogenous Myo2p directly but probably binds to its cargo instead. Furthermore, since sec1-1 blocks vesicle fusion with the plasma membrane but not vesicle targeting, resulting in an initial hyperaccumulation of vesicles at growth sites , the increased frequency of polarized tail fusion protein in sec1-1 yeast shifted to the restrictive temperature also suggests that the tail fusion protein resides on secretory vesicles. Catlett and Weisman 1998 recently proposed direct binding of the vacuole to the Myo2p tail and describe myo2-2 , a tail mutant that blocks vacuole transport with relatively little effect on polarized growth, in contrast to the tail mutant myo2-12 described here, which has no effect on vacuole transport at a temperature where the Sec4p distribution is highly defective and growth is sensitive to rich media. For comparison, myo2-66 completely blocks vacuole inheritance at permissive temperatures. These combinations of phenotypes are difficult to reconcile with simple models that do not involve binding of the tail to specific cargo membranes. In light of Myo2p's role in secretory vesicle transport, the rich medium sensitivity of myo2 mutants might be related to the rich medium sensitivity reported for sec3 and snc1Δ snc2Δ mutants . SEC3 and the two SNC synaptobrevin genes are needed for efficient fusion of secretory vesicles with the plasma membrane , but are not absolutely essential. Since nutrient compositions that speed the growth of wild-type yeast slow the growth of most myo2 ts mutants, it is plausible that the increased production of secretory vesicles in rich media exceeds the mutant Myo2p's capacity to transport vesicles, leading to vesicle fusion at inappropriate sites in addition to the appropriate sites. Mistargeted secretion might be harmful if secretory vesicles carry a spatial signal that normally helps maintain cell polarity. Given the polarized distribution of parts of the vesicle docking and fusion machinery, including Sec3p , the same principle might apply to sec3 and snc1Δ snc2Δ mutants, where increased vesicle production in rich media could swamp the capacity of polarized fusion sites leading to fusion at inappropriate sites. The myo2-66 mutation removes a salt bridge in a conserved actin-binding loop of the Myo2p NH 2 -terminal motor domain . Lillie and Brown 1994 have shown the loss of Myo2-66p localization in a myo2-66 mutant shifted to 32°C for 75 min, whereas Walch-Solimena et al. 1997 have shown the loss of Sec4p localization in a myo2-66 mutant shifted to 37°C for 1 h. Here we show that the depolarization of Myo2-66p and Sec4p in myo2-66 yeast is extremely rapid, indicating that the motor domain is directly responsible for targeting Myo2p to the correct parts of the cell. Conversely, temperature shifting myo2-16 , a tail mutant, does not depolarize the Myo2-16p distribution, even though it completely depolarizes Sec4p. Instead, the Myo2-16p distribution is slightly more polarized than in wild-type yeast, which might be expected if Myo2-16p is released from vesicles that otherwise sterically slow down access of Myo2p to actin cables. The retention of Myo2-16p's polarized distribution reflects active cable-dependent transport rather than trapping of Myo2-16p at the growth site, as the loss of cables in myo2-16 tpm1-2 tpm2Δ yeast depolarizes Myo2-16p but the restoration of cables rapidly repolarizes Myo2-16p. How, then, do we account for the Ayscough et al. 1997 finding that some Myo2p still concentrates at polarized sites despite depolymerization of actin with latrunculin-A? Growth sites may nucleate or stabilize actin filaments so strongly that actin filaments remain there, but are too short or sparse to be detectable by fluorescence microscopy. Alternatively, parts of the vesicle docking machinery may remain at polarized sites such that Myo2p can bind there. For example, latrunculin-A has no effect on the polarized distribution of Sec3p, a protein required for efficient vesicle fusion or docking . However, this is unlikely to be the normal mechanism for Myo2p localization, as actin cable–dependent targeting of Myo2p is much more rapid and robust. Our attempts at reproducing the polarized Myo2p distribution in the presence of latrunculin-A have failed: all of the treated cells have a diffuse Myo2p distribution, even the few cells that still have cortical actin patches faintly visible by rhodamine-phalloidin staining, whereas untreated cells have a normal Myo2p distribution. We also need to account for the depolarized Myo2p distribution that has been reported for cells with disrupted Myo2p tail function, namely cells expressing the Myo2p tail construct of Reck-Peterson et al. 1999 and cells with the tail mutation myo2-2 that Catlett and Weisman 1998 isolated on the basis of a vacuole inheritance defect. The simplest explanation is that the Myo2p distribution represents a balance between rapid translocation of Myo2p along cables to growth sites, and slower release or diffusion from those sites. The tail construct and myo2-2 may disrupt interactions that would otherwise retain Myo2p at growth sites or slow Myo2p diffusion. Since the Myo2p tail construct used by Reck-Peterson et al. 1999 contains an additional 44-residue segment and the single amino acid change in myo2-2 is at residue 1248, another explanation is that the relatively unconserved proximal region of the tail might regulate Myo2p's motor activity and translocation to the growth site. A precedent for autoregulation of a myosin by its COOH-terminal tail comes from a nonmuscle myosin II and a myosin I whose tails inhibit Mg 2+ -ATPase activity . Nevertheless, cargo does not seem to regulate Myo2p motor activity very strongly if at all, given the normal intracellular distribution of Myo2p in the absence of bound secretory vesicle cargo. Note that our results show that Myo2p continually cycles into the bud whether or not secretory vesicles are present. Indeed, the Myo2p distribution is unaffected by sec12-4 , which blocks the production of secretory vesicles at the restrictive temperature (data not shown). We show that the formation and polarity of actin patches and cables do not directly require functional MYO2 . Specifically, the actin cytoskeleton is unaffected in myo2 ts mutants in the first minutes after shift to the restrictive temperature during which secretion becomes delocalized. However, cortical patches do eventually depolarize after 15–30 min of loss of either Myo2p protein function or tropomyosin-containing actin cables . In contrast, actin patches depolarize within 1 min in wild-type yeast in response to stresses such as heat shock and centrifugation , indicating that the effect of Myo2p and tropomyosin on actin patches is indirect. Among those post-Golgi secretory mutants that are synthetically lethal with myo2- 66, the small Rab GTPase mutant sec4-8 is particularly interesting, because Sec2p, its guanine nucleotide exchange factor, is the only SEC gene product known to be needed for polarized vesicle delivery , suggesting that Sec4p marks vesicles for targeting to growth sites. We have found that a sec2 mutation rapidly depolarizes secretion at the restrictive temperature without affecting the Myo2p distribution , indicating that the sec2 mutation uncouples secretory vesicles from Myo2p. Furthermore, Sec4p resides specifically on the surface of late secretory vesicles in wild-type yeast , and of the late sec mutations, sec4-8 and sec2-41 are the only ones known to be synthetically lethal with Δsmy1 , implying a relatively direct interaction with MYO2 . The new myo2 ts alleles provide additional insight into these interactions. Of the COOH-terminal myo2 ts alleles, myo2-14 is by far the most deleterious in combination with sec4-8 . The trivial explanation, that myo2-14 is the most severe allele below 30°C, is unlikely; both myo2-12 and myo2-16 have milder synthetic growth defects with sec4-8 , but have lower restrictive temperatures than myo2-14 and impair growth in rich media, even at 24°C. The region altered in myo2-14 , the COOH-terminal 123 residues , is therefore likely to be important for Sec4p- and Sec2p-dependent vesicle targeting. This strong allele specificity and the uncoupling of secretory vesicles from Myo2p in a sec2-41 mutant support a model in which a vesicle-associated Rab protein and its guanine nucleotide exchange factor direct vesicle transport either by physically tethering the myosin V COOH terminus to vesicles or by activating a latent myosin V COOH terminus–binding receptor on the vesicle surface. A screen for multicopy suppressors of myo2 ts tail mutants identified SMY1 , which has been isolated previously as a suppressor of myo2-66 . SMY1 encodes a kinesin homologue that, like Myo2p, concentrates in regions of growth and aids MYO2 function by a mechanism involving neither a kinesin-like motor activity nor microtubules . Since myo2-14 is the only myo2 ts mutant whose growth is unaltered by multicopy SMY1 , some part of the COOH-terminal 123 residues of Myo2p must affect functional interaction with SMY1 in addition to SEC4 , most likely the final 30 residues because myo2-14 is the only allele that changes this part of the extreme COOH terminus . It is unlikely that suppression of myo2-14 by SMY1 was overlooked due to a narrow temperature range where myo2-14 is only partially functional, as sec4-8 lowers myo2-14' s restrictive temperature by at least 13°C. The strong suppression of some but not all COOH-terminal globular domain mutants by multicopy SMY1 , but comparatively weak suppression of the motor domain mutant, taken together with Smy1p's and Myo2p's identical subcellular distributions , and Smy1p's apparent lack of its own motor activity , suggests that Smy1p binds to the Myo2p globular tail domain directly. Binding of a kinesin to a myosin V tail has been demonstrated in an animal system , consistent with organelle transport occurring along microtubules using kinesin motors and then switching to myosin-driven transport along actin filaments near the final delivery site. Binding of a kinesin homologue to the Myo2p tail could have originated in yeast's hyphal ancestors. If so, Smy1p would have been kept because it stabilizes a multiprotein complex with the Myo2p tail needed for docking onto organelles, but Smy1p's microtubule-binding and motor activities would have been lost in a cell too small and compact to need microtubule-based vesicle transport. Finally, comparison of the Myo2p sequence and the sequence of Myo4p, an RNA-transporting myosin V in yeast, with type V myosins in other organisms supports a model in which, after duplication of the yeast genome resulting in two myosin V genes, one yeast myosin V selectively retained and the other yeast myosin V selectively lost features needed for membrane transport. These features are concentrated in the final 183 amino acid residues of Myo2p, a region that also contains the tail sequences most conserved among fungi, plants, animals, and cellular slime molds. In particular, animal myosin Vs known to be involved in membrane transport are much more similar in tail sequence to Myo2p than to Myo4p, despite overwhelming evidence that MYO2 and MYO4 arose from a comparatively recent duplication. Most of the tail mutations we isolated that affect polarized secretion make at least one nonconservative change in this region . Nevertheless, the varying effects of tail mutations on vesicle transport compared with vacuole transport implies that the recognition of different organelles involves different features of the myosin tail. On the other hand, plant and Dictyostelium myosin V tails are more similar to Myo4p than to Myo2p in a more proximal part of the tail (data not shown), but the functions of these myosin Vs are unknown. It would be interesting to know whether these myosins transport RNA, membranes, or both. In summary, the data presented here support a model for polarized secretion in which a myosin V COOH-terminal globular domain binds vesicles, and the NH 2 -terminal motor domain drives translocation of the myosin and its attached cargo along tropomyosin-containing actin cables to the cargo's destination. This cycle of myosin V transport does not depend on the secretory cargo marked by Sec4p, and we have identified regions of Myo2p tail that might bind cargo. | Other | biomedical | en | 0.999997 |
10562282 | Horse heart cytochrome c was obtained from Sigma Chemical Co. or from Amersham. SOx was purified from rat and chicken and anti–rat SOx antibodies raised in rabbits as previously described . Ac-DEVD-CHO and zVAD-fmk were purchased from Biomol and dissolved in DMSO. Xenopus egg extracts were prepared as described . The final egg cytosolic extracts were approximately two parts cytoplasm and one part buffer A (250 mM sucrose, 20 mM Hepes/KOH, pH 7.5, 50 mM KCl, 2.5 mM MgCl 2 , 1 mM DTT, 5 μg/ml cytochalasin B, and 50 μg/ml cycloheximide). Incubations were comprised of either crude egg extract that contained mitochondria, or of isolated Xenopus egg mitochondria (1 mg/ml as protein) incubated in either buffer or Xenopus egg cytosol. All cell-free incubations, including those with buffer only, were supplemented with an ATP regenerating system (10 mM phosphocreatine, 2 mM ATP, and 150 μg/ml creatine phosphokinase). The buffer used in incubations was buffer A supplemented to 100 mM KCl (buffer B), as we found that 80 mM KCl was required for complete dissociation of cytochrome c (but not AK) from mitochondrial membranes after hypotonic lysis or exposure to either Bid or Bax . To measure caspase-3-like activation, extract aliquots (2 μl) were incubated with DEVD-pNA ( N -acetyl-Asp-Glu-Val-Asp- p -nitroanilide, 40 μM; Biomol) in 200 μl of a buffer (250 mM sucrose, 20 mM Hepes/KOH pH 7.5, 50 mM KCl, 2.5 mM MgCl 2 , and 1 mM DTT) similar to that used to make the egg extracts. Incubations were kept at 22°C and A 405 development monitored over 30 min (SPECTRAmax 250 microplate spectrophotometer). Male Sprague Dawley rats (300–500 gm body weight) were killed with carbon dioxide and their livers were removed and placed in ice-cold sucrose solution (300 mM sucrose, 10 mM Tris-HCl pH 7.4, and 1 mM EDTA) for transport. All subsequent procedures were performed at 0 or 4°C. Tissue was placed in ice-cold buffer C (60 mM sucrose, 210 mM mannitol, 10 mM KCl, 0.5 mM DTT, 10 mM succinate, 10 mM Hepes/KOH, pH 7.5, 5 mM EGTA, 1 mM PMSF, 0.1% BSA, 10 μg/ml aprotinin, and 10 μg/ml leupeptin), diced, and aliquots transferred to fresh buffer in a 10-ml Wheaton Potter-Elvehjem tissue grinder. Using a drill-driven Teflon-coated pestle, aliquots were homogenized by 3–5 strokes. Total homogenate was diluted to 100 ml with buffer C and centrifuged twice at 2,000 g for 5 min to remove unbroken cells. Mitochondria were then pelleted from the supernatant by 10 min at 8,500 g (Sorvall RC5C), and washed twice before a final wash in buffer C without PMSF or BSA, then resuspension in a minimal volume. Protein concentration was estimated by the Biuret method. Mitochondrial swelling was assessed by measuring the decrease in light scattering at 520 nm . Light scatter measurements of reconstituted extracts needed to be performed on aliquots diluted with buffer A (which already constitutes 1/3 of the extract), as A 520 of undiluted extract was in the nonlinear range (>3). Aliquots (40 μl) of reconstituted extracts were diluted with 260 μl buffer B and added to a 1-ml cuvette for measurement of A 520 in a Hitachi 2000 spectrophotometer. As a positive control, mitochondria were swollen by incubation with the membrane disrupting peptide mastoparan from wasp venom (Sigma Chemical Co.), sometimes used as an inducer of permeability transition . Samples of extract containing mitochondria (5 μl) were placed in an Eppendorf tube and fixative (5 μl of 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) layered on top, so that after a few minutes on ice the pellets formed a congealed plug. After full fixation (2 h), samples were washed in cacodylate buffer, followed by secondary fixation in 4% osmium tetroxide in 0.1 M cacodylate buffer pH 7.4. Samples were then washed in double distilled water and stained en bloc with 2% aqueous uranyl acetate overnight. Dehydration was carried out stepwise in 30, 50, 70, 90, 95, and 100% acetone/water solutions before embedment in Durcopan (EMS). Thin sections ∼100 nm thick were cut and imaged with transmission electron microscopy on either a JEOL 100CX or Philips 410 microscope. Cross-sectional area measurements were performed using NIH Image program (1.61) and presented as mean ± SD, with statistical significance determined using the paired Student's t test. High resolution scanning electron microscopy was performed using the recent technology afforded by high-brightness sources used in in-lens field emission instruments (FEISEM), which provide resolution to 1 nm . Reconstituted extracts were incubated at 22°C with BaxΔTM or alamethicin (2 μg/ml; Sigma Chemical Co.) to induce cytochrome c release (confirmed by DEVDase activation and Western blot). Aliquots (10 μl) were mixed with buffer B (400 μl) to wash the mitochondria, and then placed on 5-mm-square silicon chips in chambers constructed from Eppendorf tubes. Mitochondria were spun onto the chips (5 min, 14,000 g ), followed by removal of supernatant, and fixation in 100 μl 150 mM sucrose, 80 mM Pipes-KOH, pH 6.8, 1 mM MgCl 2 , 2% paraformaldehyde, 0.25% glutaraldehyde on ice, followed by postfixation in OsO 4 in 0.2 M cacodylate buffer, pH 7.4. After 10 min in 1% aqueous uranyl acetate, the specimens were dehydrated through an ethanol series, and critical point dried from CO 2 using Arklone (Freon112; ICI), as intermediate solvent. The chips were then sputter coated with 4 nm chromium, and examined in a Topcon ISI DS 130F field emission, in-lens SEM at 30 kV accelerating voltage, with digital image acquisition. To determine mitochondrial and cytosolic content of various proteins, incubation aliquots were centrifuged (12,000 g ) to pellet mitochondria and the supernatant carefully removed. For SDS-PAGE, supernatant samples (10 μl) were mixed with 2× loading buffer (10 μl) and mitochondrial pellets were resuspended in a volume of 1× loading buffer equivalent to the initial volume of the centrifuged aliquot. Supernatant (20 μl) and mitochondrial (10 μl) samples were heated at 95°C for 5 min, loaded onto 12% SDS–polyacrylamide gels for electrophoresis, and then transferred to nitrocellulose (BioRad) membranes. Membranes were then blocked for 1 h in TBST (25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 27 mM KCl, and 0.02% Tween 20) containing 5% nonfat dried milk. Membranes were probed with monoclonal anti–cytochrome c antibody (clone 7H8.2C12; PharMingen), rabbit antiserum to rat SOx, mouse anti-mtHsp70 antibody (kindly provided by Richard Morimoto), or rabbit anti-serum against bovine heart Rieske's iron-sulfur protein . Recognized proteins were detected using horseradish peroxidase-labeled secondary antibodies (Amersham) and enhanced chemiluminescence (Amersham). Mitochondrial AK activity was measured by a modification of the method of Schmidt et al. 1984 . Extract aliquots (50 μl) containing ∼0.05 mg mitochondrial protein were pelleted at 12,000 g for 3 min, and the pellet washed twice in 800 μl buffer D (60 mM sucrose, 210 mM mannitol, 10 mM KCl, 0.5 mM DTT, 10 mM succinate, 10 mM Hepes/KOH, pH 7.5, and 5 mM EGTA) to remove contaminating cytosolic AKs. The mitochondrial pellets were lysed with 50 μl of 1% Triton X-100 in buffer D to release remaining AK and the sample stored at –80°C. AK activity was measured in a mixture composed of 1 ml of 130 mM KCl, 6 mM MgSO 4 , 100 mM Tris-HCl, pH 7.5, 15 μl of 0.1 M NADH, and 5 μl each of 0.1 M ATP, 100 mM phosphoenol pyruvate, 1 mM rotenone, 1.5 mM oligomycin, a mixture of pyruvate kinase and lactate dehydrogenase (80 U/ml each), and 0.15 M AMP. 200 μl of buffer mix was added to 6 μl of sample in a microtiter plate. The absorbance decrease of NADH was measured at 366 nm in a microtiter plate reader (SPECTRAmax 250) for 10 min at 22°C. The rates were calculated (SOFTmax PRO) and calibrated against chicken muscle myokinase (Sigma) activity. Rates obtained in the presence of the inhibitor di-adenosine pentaphosphate (400 μM; Sigma) were minimal and subtracted as background. To examine the intactness of the outer mitochondrial membrane we measured the accessibility of cytochrome c oxidase to exogenous cytochrome c . The assay involved the addition of extract (2 μl) containing mitochondria (final protein concentration ∼9 μg/ml) to 450 μl assay solution in a 1-ml cuvette. The assay solution consisted of 100 μM horse heart cytochrome c , 60 mM KCl, 125 mM sucrose, 20 mM Tris-HCl, pH 7.4, 1 μΜ each of carbonyl cyanide m-chlorophenylhydrazone (CCCP), rotenone and antimycin, and sufficient sodium dithionite crystals to obtain near fully reduced cytochrome c (A 550 /A 565 between 6 and 10). On addition of mitochondria, samples were quickly mixed and A 550 recorded every 6 s . The rate of cytochrome c oxidation at 550 nm integrated over 30 s reflected complex IV accessibility to the solution. Maximal complex IV accessibility was obtained by premixing 2 μl extract with 2 μl Triton X-100 (2%) and used to calculate percent complex IV accessibility. An additional method used to assess permeability of the mitochondrial outer membrane was the reduction of exogenous cytochrome c by complex III . Mitochondria from treated extracts (250 μl) were washed by dilution in 1.25 ml buffer E (125 mM sucrose, 60 mM KCl, 20 mM Tris-HCl, pH 7.4), followed by pelleting for 5 min at 12,000 g . The mitochondrial pellet was resuspended in 250 μl buffer E and 25 μl (final concentration 0.15 mg/ml) added to 300 μl buffer E containing 2 mM potassium cyanide (to block oxidation by complex IV). Decyl benzoquinol (DBH 2 , 5 μl of 3.5 mM stock in ethanol) was added and the sample sealed with parafilm and mixed. Finally, 10 μl of 2.5 mM ferricytochrome c (horse heart; Sigma Chemical Co.) was added with a Hamilton syringe and the rate of cytochrome c reduction at 550 nm was integrated over 30 s. Bcl-2 and the control β-galactosidase were expressed in baculovirus-infected Sf-9 cells, and lysates were prepared as described . Human Bid (hBid) cDNA was amplified with PCR and subcloned into the BamHI and NotI sites in pGEX4T-1 (Pharmacia). BL21(DE3) cells were transformed and GST-hBid induced with 1 mM isopropyl β- d -thiogalactopyranoside (IPTG) at 37°C for 4 h. The induced protein was incubated with glutathione sepharose (Pharmacia) and eluted with 20 mM reduced glutathione. Eluted GST-hBid was digested with thrombin (Pharmacia) to cleave GST, which was subsequently removed by incubation with a new batch of glutathione sepharose. The hBid product was >90% pure with minor contamination of GST. Human caspase-8 lacking the prodomain in PET15b vector (Novagen) was expressed in BL21(DE3) cells by induction with 1 mM IPTG at 37°C for 4 h and affinity-purified with Ni ++ -NTA (Qiagen). The protein was eluted with 50 mM and 250 mM imidazole, and the 250 mM imidazole eluate further purified on a 1 ml Q-Sepharose FF HiTrap column (Pharmacia) using a 0–0.6 M KCl linear gradient. The product was fully processed and electrophoretically >90% pure. tBid was made by incubation of recombinant human Bid with active recombinant caspase-8 at 37°C for 2–4 h in 25 mM Hepes-KOH, pH 7.5, 80 mM KCl, and 10 mM DTT. (The tBid preparation was used without removing caspase-8; however, control experiments showed that caspase-8 alone did not release cytochrome c from mitochondria.) COOH-terminally deleted Bax (BaxΔTM) was produced as previously described . Bak BH3 domain (NH 2 -GQVGRQLAIIGDDINR-COOH) and mutant (NH 2 -GQVGRQAAIIGDDINR-COOH) peptides were kindly provided by S. Kataoka and Y. Tokoro (Kirin Brewery Co., Japan). Cytosol was size-fractionated using Millipore Ultrafree-MC centrifugal ultrafiltration membranes. Cytosol was dialyzed against 4 changes of buffer B (500 ml each) over 24 h at 4°C in 3500 MW cutoff dialysis tubing (Spectra/Por). Cytosolic proteins were hydrolyzed by incubation with 0.1 mg/ml proteinase K or 1.0 mg/ml trypsin (Sigma Chemical Co.) for 30 min at 22°C, followed by addition of phenylmethylsulfonyl fluoride (1 mM). Heat-denatured cytosol was prepared by heating at 50°C for 15 min or 95°C for 1 min, followed by clearing at 12,000 g for 5 min. To determine if mitochondrial swelling might participate in the efflux of mitochondrial cytochrome c during apoptosis in the Xenopus cell-free system, we assessed mitochondrial swelling by three methods. First, we measured the light scatter of mitochondrial suspensions. As mitochondria swell, their refractive index decreases, resulting in a drop in spectrophotometric absorbance at higher wavelengths . Xenopus mitochondria were induced to release cytochrome c by incubation in Xenopus cytosol for 4 h at 22°C. We found that these mitochondria did not exhibit decreased light scattering despite complete cytochrome c release. As a control to determine if mitochondrial swelling could be measured with Xenopus mitochondria using this technique, we used mastoparan, a membrane disrupting peptide that causes mitochondrial swelling associated with permeability transition and cytochrome c release . As shown in Fig. 1 , mastoparan at 300 and 400 μg/ml rapidly induced both a decrease in light scattering and loss of mitochondrial cytochrome c . We conclude that the constitutive pro-apoptotic factor present in the Xenopus cell-free system initiates cytochrome c release by a mechanism independent of mitochondrial swelling, as measured by light scatter. Second, we used transmission electron microscopy to compare the size and morphology of apoptotic and non-apoptotic mitochondria . Crude egg extracts were incubated at 22°C to induce mitochondrial cytochrome c loss. In the Xenopus system, essentially all mitochondrial cytochrome c becomes cytosolic ∼30 min before the detection of DEVDase activation . Samples fixed immediately after full DEVDase activation exhibited no obvious mitochondrial swelling or morphological changes when compared with (non-apoptotic) extracts that had been incubated on ice. Quantitative assessment of mitochondrial cross-sectional area indicated no difference ( P = 0.32, n = 38) between apoptotic (0.75 ± 0.25, arbitrary units) and non-apoptotic mitochondria (0.77 ± 0.26). The integrity of the outer membrane appeared equal in apoptotic and non-apoptotic samples. Comparison of several individual mitochondria by three-dimensional electron tomography (Renken, C., and G. Perkins, unpublished data) also failed to reveal obvious changes in outer membrane ultrastructure. Mitochondria fixed at 3 h after cytochrome c release also were unswollen (not shown), indicating that neither activated caspases nor other cytosolic factors cause gross morphological mitochondrial changes. The majority of mitochondria in Xenopus extracts was observed to be in the condensed form (dark matrix and wide cristae), rather than the orthodox configuration (light matrix and narrower cristae). A condensed matrix is thought to reflect low respiration . Thus, the condensed morphology of Xenopus extract mitochondria may indicate low respiratory rates in response to the high external energy status caused by addition of the ATP regenerating system (see Materials and Methods). Alternatively, unfertilized Xenopus eggs may contain a factor similar to that implicated in the condensed mitochondrial morphology of unfertilized sea urchin eggs . Condensed mitochondria have recently been observed in association with apoptosis . With the opening of the permeability transition pore, mitochondria in the condensed state are converted irreversibly (in the presence of sucrose) to the orthodox conformation. Thus, simply on ultrastructural grounds, as both apoptotic and non-apoptotic Xenopus mitochondria are condensed, it is unlikely that a permeability transition had taken place. To examine further the ultrastructure of apoptotic mitochondria, we used a third approach, a high-resolution form of scanning electron microscopy . As Fig. 2 shows, FEISEM revealed that mitochondria depleted of cytochrome c were of similar size and appearance to control mitochondria. Outer membrane discontinuities were not observed. This technique did reveal outer membrane pores, 10–13 nm in diameter, in mitochondria treated with the antibiotic peptide alamethicin , which also induced complete release of cytochrome c (Kluck and Newmeyer, unpublished). However, no such pores were observed in mitochondria depleted of cytochrome c by the factors present in Xenopus cytosol, or by Bid or Bax, suggesting that if these factors form pores, they are either transient or considerably smaller than those formed by alamethicin . In summary, multiple approaches demonstrated the absence of gross ultrastructural changes in mitochondria accompanying cytochrome c release in the Xenopus cell-free system. To continue investigating the mitochondrial events leading to cytochrome c release, we next determined whether other soluble proteins present in the intermembrane space were released during apoptosis. One such protein is adenylate kinase (25,200 Da). The amount of mitochondrial adenylate kinase (AK) released into the cytosol is difficult to measure because cytosol already contains ∼50% of the cell's AK activity (not shown). However, the AK activity remaining in the mitochondrial pellet can readily be assayed. We incubated mitochondria in cytosol for various times, washed them free of cytosol, and measured adenylate kinase content in the final pellet. As Fig. 3 shows, with apoptosis induced by Xenopus cytosol, almost all AK activity was lost from mitochondria before DEVDase activation, with kinetics similar to those observed for cytochrome c . Another mitochondrial protein, sulfite oxidase (SOx), is found only in the intermembrane space. As antisera raised to rat SOx failed to recognize the Xenopus protein, we determined whether rat liver mitochondria incubated in Xenopus extracts coreleased SOx with cytochrome c . Many of the apoptotic features of the Xenopus egg cell-free system were duplicated if rat liver mitochondria were incubated in Xenopus cytosol. That is, mitochondrial cytochrome c was released after a period of incubation at 22°C, but not at 0°C . (It should be noted that, for reasons unknown, some preparations of rat liver mitochondria failed to exhibit cytochrome c release in response to incubation with Bid, Bax or Xenopus egg cytosol.) Near the time of rat cytochrome c release, Xenopus cytosolic DEVDases became activated, and coincubated rat liver nuclei exhibited chromatin condensation, margination and beading, with subsequent nuclear disintegration (not shown) typical of apoptosis in the Xenopus system. Swelling of the rat liver mitochondria was not observed by cross-sectional area measurements of transmission electron micrographs ( P = 0.5, n = 21), with non-apoptotic mitochondria showing an average area of 2.45 ± 1.1 (arbitrary units), compared with 2.45 ± 1.0 for apoptotic mitochondria. Light scatter measurements (not shown) also demonstrated a lack of swelling. Despite this absence of swelling, we found that SOx (a dimer of 52 kD subunits) was coreleased from the intermembrane space with cytochrome c and with AK (not shown). Loss of these proteins from mitochondria occurred over a period of under 1 h. As SOx is normally found only in the intermembrane space, it was of interest to examine whether the presence of this protein in cytosol altered the pro-apoptotic activity of cytochrome c . Purified rat or chicken SOx added to Xenopus cytosol, either in the presence or absence of cytochrome c , did not alter DEVDase activation (not shown), indicating that SOx does not affect caspase activation. (It is unlikely that released mitochondrial AK has any effect on the activation of caspases, as much of the cellular AK is already present is cytosol.) Western blot analysis of rat SOx released from mitochondria suggested that this protein was degraded in apoptotic cytosol, but not in either buffer or an ultrafiltrate obtained by passing cytosol through a 10-kD cutoff membrane (not shown). Chicken SOx was cleaved to a 45-kD fragment after a 1-h incubation with either caspase-3 or cytochrome c– activated cytosol, and this cleavage was blocked by the caspase inhibitor Ac-DEVD-CHO (1 μM, not shown). However, while chicken SOx appeared to be a good caspase substrate, rat SOx was cleaved in a much less efficient manner to several products of different molecular mass. In summary, the corelease of cytochrome c , AK and SOx with apoptosis, argues against an outer membrane transport channel specific for cytochrome c , and is consistent with the formation of a nonspecific opening or protein channel in the outer mitochondrial membrane. To examine in more detail the apoptotic changes occurring in mitochondrial outer membrane permeability, we measured the accessibility of exogenous cytochrome c to complex IV (cytochrome c oxidase), present on the outside face of the inner mitochondrial membrane. When detergent-treated or mechanically disrupted mitochondria are suspended in a solution containing reduced cytochrome c , this exogenous cytochrome c becomes gradually oxidized by complex IV, leading to a steady change in the absorption spectrum of cytochrome c . In intact mitochondria, this change does not occur, because the permeability barrier of the outer membrane prevents the interaction of exogenous cytochrome c with the catalytic site exposed within the intermembrane space . The rate of change of absorbance at 550 nm is taken as a measure of the accessibility of complex IV, and hence the permeability of the outer membrane. The values obtained were expressed as percentages of the rate of change of A 550 observed after exposure of mitochondria to 1% Triton X-100, as this treatment was assumed to cause complex IV to be completely accessible. Carefully isolated Xenopus egg and rat liver mitochondrial preparations exhibited <1% of this value, while osmotic lysis of the outer membrane gave complex IV accessibility values close to 100%. We used this assay to examine the permeability changes occurring in mitochondria incubated in Xenopus cytosol. With the onset of apoptosis, complex IV accessibility increased at around the time of cytochrome c release , indicating that outer membrane permeability to cytochrome c in apoptotic mitochondria (in the presence of cytosol) is bidirectional. The addition of Bcl-2 to the extracts blocked both the increase in complex IV accessibility and the release of endogenous cytochrome c . Next, we examined the effects of two known pro-apoptotic human proteins, Bid and Bax. Addition of recombinant Bid or Bax to reconstituted Xenopus egg extracts (mitochondria mixed with cytosol) caused the rapid release of mitochondrial cytochrome c and the activation of DEVDases (not shown). Cytochrome c release was complete and highly synchronous. Recombinant Bax exhibited a 1,000-fold lower specific activity than recombinant Bid (not shown); this may be due to the absence of the Bax transmembrane domain or to undefined differences in the way these proteins are expressed in bacteria. Cleavage of full-length Bid by caspase-8 (see Materials and Methods) to give truncated Bid (tBid) consistently enhanced its potency (not shown), as reported by others . Transmission electron microscopy of mitochondria depleted of cytochrome c by treatment with Bax or caspase-activated Bid revealed no significant changes in mitochondrial size ( P = 0.44, n = 21 and P = 0.33, n = 21 for comparisons of control mitochondria with Bid- and Bax-treated mitochondria, respectively). The mean cross-sectional areas of control, Bid- and Bax-treated mitochondria, were 0.684 ± 0.2, 0.70 ± 0.2, and 0.71 ± 0.2, respectively (arbitrary units). Thus, Bid and Bax, like the factors present in Xenopus egg cytosol, induced cytochrome c release without gross changes in mitochondrial morphology. As cytosol-treated mitochondria coreleased AK with cytochrome c , we examined whether this was also the case for tBid- and Bax-treated mitochondria. Indeed, tBid and Bax both released AK with kinetics similar to the release of cytochrome c . This release occurred in the presence of either buffer or cytosol. The constitutive presence of large amounts of AK activity in the cytosol prevented us from measuring the AK released from mitochondria incubated in cytosol; however, for mitochondria incubated in buffer, the amount of AK released into the supernatant of tBid and Bax-treated mitochondria accounted for that lost from the mitochondrial pellet (not shown), indicating that the protein was translocated, rather than inactivated. These data support the hypothesis that similar cytochrome c– releasing mechanisms are used by various cytochrome c –releasing proteins. Cytochrome c release from Xenopus mitochondria incubated in Xenopus cytosol is associated with high accessibility of the inner membrane protein cytochrome c oxidase (complex IV) to exogenous cytochrome c . This high accessibility is also seen in Xenopus mitochondria treated with tBid and Bax in the presence of Xenopus cytosol . It was surprising to note, however, that mitochondria treated with tBid and Bax in buffer displayed an almost undetectable level of accessibility of complex IV to exogenous cytochrome c , despite their complete loss of endogenous cytochrome c . These results imply that a novel cytosolic activity, which we call Permeability Enhancing Factor, or PEF, is responsible for a striking increase in complex IV accessibility beyond that produced by factors such as tBid or Bax. PEF might function either simultaneously with factors like tBid or subsequent to their action. To address this question, we did the following experiment. Mitochondria were first depleted of cytochrome c by an initial incubation with tBid in buffer, and then washed and resuspended in cytosol for a second incubation. As Fig. 5 D shows, complex IV accessibility rapidly increased during this second incubation in cytosol. This is consistent with data showing that tBid stays strongly associated with mitochondria after washing (Kuwana, O., T. von Ahsen, and D.D. Newmeyer, unpublished results), but further implies that the effect of tBid is sustained. Some samples were incubated in fresh buffer before reincubation with cytosol, in an attempt to encourage maximum repair of the tBid-induced permeability. However, this intermediate incubation had no effect (not shown). We conclude from this experiment that PEF can function subsequent to tBid-induced cytochrome c translocation. This experiment also demonstrates that PEF activity is not dependent on downstream effects of cytochrome c release, because the cytosol used in Fig. 5 D contained neither cytochrome c (nor any other soluble proteins from the mitochondrial intermembrane space) nor active caspases. It could have been argued that the quantitative differences in accessibility of complex IV reflected some effect specific for that enzyme complex, rather than changes in the permeability of the outer membrane. To rule out this possibility, we measured the accessibility of another mitochondrial inner membrane component, complex III. This assay is similar in principle to the complex IV assay, except that cytochrome c reduction rather than oxidation is measured, and an electron donor (DBH 2 ) is added to ensure saturating substrate (see Materials and Methods). These experiments showed that cytosol-induced cytochrome c release was accompanied by an increase in the accessibility of both complexes III and IV , confirming the bidirectional permeability of the outer membrane under these conditions. Again, however, in the presence of buffer alone, tBid induced a nearly undetectable accessibility of complexes III and IV , which was greatly enhanced in the presence of cytosol . The ability of cytosol to increase outer membrane permeability was not due to a feedback effect of cytochrome c– activated caspases, as Ac-DEVD-CHO (10 μM) did not prevent the development of high complex III and IV accessibility (not shown). In summary, the enhancing effect of cytosol on accessibility of complexes III and IV is explained by the presence of a factor in Xenopus cytosol, which we have named permeability enhancing factor (PEF). PEF does not release cytochrome c by itself, but rather enhances outer membrane permeability after a pro-apoptotic factor has caused the release of endogenous cytochrome c . To address the possibility that PEF increases accessibility of complexes III and IV by grossly perturbing the integrity of mitochondria, we examined whether a soluble mitochondrial matrix protein, mtHsp70, is released after the actions of tBid and PEF. First, to show that mtHsp70 can be released if the inner mitochondrial membrane is permeabilized, we treated mitochondria with increasing concentrations of digitonin and followed the redistribution of mtHsp70 from the mitochondrial pellet fraction to the soluble supernatant. Fig. 7 A shows that cytochrome c is released at a low concentration (0.1%) of digitonin, but that mtHsp70 becomes soluble at higher digitonin concentrations (0.2%). Only at still higher concentrations (0.6%) did the membrane-associated Rieske protein become solubilized, showing that at the intermediate concentrations sufficient to release mtHsp70, digitonin did not solubilize the mitochondrial membranes, but merely permeabilized them. Next, we examined the behavior of mtHsp70 in mitochondria induced to release cytochrome c by cytosol or by tBid (in the presence or absence of cytosol). As seen in Fig. 7 B, mtHsp70 remained in the mitochondrial pellet fraction under all conditions, even in the presence of cytosol containing active PEF. We conclude that cytosolic factors and tBid can permeabilize the outer mitochondrial membrane while leaving the inner membrane intact. To begin characterizing PEF, we treated cytosol in various ways before incubating it with Xenopus mitochondria and tBid . Treatment with proteinase K , trypsin, or heat (50°C, 15 min, or 95°C, 1 min; not shown) greatly diminished or abolished cytosol's ability to increase complex IV accessibility, indicating that PEF is proteinaceous. PEF activity was not observed in the ultrafiltrate obtained by passing cytosol through a 300-kD cutoff membrane, and was not lost on dialysis, suggesting that PEF is macromolecular and possibly present in a large complex. The complex IV accessibility produced by PEF accumulates over a few hours to approach that seen in mitochondria treated with Triton X-100 (see Materials and Methods) and digitonin (not shown). PEF activity is reduced substantially on dilution of cytosol. A PEF-like activity may also be present in mammalian cells, as a lysate (40 mg/ml) of human Jurkat cells caused an increase in complex IV accessibility of isolated Xenopus mitochondria (not shown). This Jurkat cell PEF-like activity was observed in the presence or absence of tBid, suggesting that the Jurkat cell lysate contained endogenous cytochrome c– releasing factors. To address the possibility that PEF is a substrate of caspases, we examined the effect of cytochrome c– activated cytosol on fresh Xenopus mitochondria. Fresh cytosol was first incubated for 3 h with a low level (0.5 μM) of cytochrome c to activate cytosolic caspases (DEVDases). Subsequent addition of mitochondria to this activated cytosol resulted in rapid loss of mitochondrial cytochrome c and AK (by 1 h, not shown). The factor responsible for this cytochrome c efflux is likely to be a caspase-activated Bid-like factor , rather than caspases themselves, as addition of caspase inhibitors just before the addition of mitochondria did not block cytochrome c release. Strikingly, however, cytochrome c– activated cytosol did not produce high complex IV accessibility, even though it caused the complete release of cytochrome c from mitochondria . Thus, this cytochrome c– treated cytosol contained activated DEVDases, inactivated PEF, and activated Bid-like activity. Further, we found that the addition of caspase inhibitors to cytosol substantially enhanced and prolonged PEF activity. Typically, in the presence of cytosol, complex IV accessibility increased over 1–2 h after cytochrome c release and then reached a plateau . If caspase inhibitors were added, however, this plateau was surpassed and complex IV accessibility approached 100% of that seen with Triton-treated mitochondria . In summary , cytosolic PEF begins to act on mitochondria once they have lost their cytochrome c , and over 2–3 h, progressively allows a higher permeability of the outer membrane to exogenous cytochrome c . If caspases are activated by the released cytochrome c , PEF activity is inactivated after perhaps 1–2 h. Addressing whether this inactivation by caspases might be direct or indirect awaits the molecular identification of PEF. We have investigated how mitochondrial cytochrome c is released during apoptosis in a cell-free system based on Xenopus egg extracts . Four pro-apoptotic cytochrome c– releasing activities were examined: (a) the spontaneous cytochrome c– releasing activity of Xenopus cytosol, (b) the Bid-like activity of cytochrome c– activated Xenopus cytosol, (c) recombinant human Bid activated with caspase-8, and (d) recombinant Bax. These factors all caused a permeability of the outer membrane that allowed the corelease of AK (and SOx, where measurable) with cytochrome c . None of these cytochrome c– releasing factors caused detectable mitochondrial swelling . From this and other data, we conclude that neither mitochondrial permeability transition nor hyperpolarization occur in this apoptotic system. A potentially attractive mechanism for the release of multiple proteins from the intermembrane space would involve swelling of the mitochondrial matrix, leading to mechanical disruption of the outer mitochondrial membrane. A single break in the outer mitochondrial membrane is theoretically sufficient to allow cytochrome c to diffuse rapidly out of mitochondria down its concentration gradient (10 mM in intermembrane space and cristae). Ruptures in the outer membrane ensue if the matrix swells past the point that the outer membrane can expand. Such matrix swelling is proposed to occur during apoptosis in response either to the formation of a 1.5-kD permeability transition pore in the inner membrane or to hyperpolarization of the inner membrane . However, mitochondrial swelling has not been observed as a frequent accompaniment to apoptosis. In the studies reported here, light scatter measurements and transmission and scanning electron microscopy all failed to provide evidence of mitochondrial swelling in association with apoptotic cytochrome c efflux from Xenopus mitochondria. Another group has reported that, in whole cells of Xenopus embryos treated with gamma irradiation, apoptotic mitochondria display normal size and morphology . In other systems also, mitochondrial swelling has not been observed with apoptosis , and in some apoptotic cells mitochondria even shrink . Previously, Bid was reported to release cytochrome c from isolated mitochondria without swelling . Bax-mediated cytochrome c release has been associated in some reports with mitochondrial swelling or permeability transition , but in other reports with an absence of swelling . The presence of swollen mitochondria in some apoptotic systems, including whole cells, may reflect either secondary necrosis or a fundamentally distinct apoptotic cytochrome c release mechanism. Cytochrome c efflux is not a specific export process, as AK (25,200 Da) was also released by each of the four pro-apoptotic factors we studied. Furthermore, SOx (104 kD) was released from rat liver mitochondria incubated in cytosol . Several intermembrane space proteins, including AK , a cytochrome c– GFP fusion protein , AIF , and certain caspases have been reported to be coreleased with cytochrome c in other systems, often in the absence of a permeability transition. Whereas proteins such as Bid, Bax, and certain endogenous Xenopus egg cytosolic factors produced only a limited outer membrane permeability, we detected another cytosolic activity that caused a much stronger permeabilization effect. We termed this novel cytosolic activity PEF, as it enhanced, rather than initiated, outer membrane permeabilization . The initial permeability caused by cytochrome c releasing factors is limited as it allows a relatively small cytochrome c flux across the outer membrane. This flux is sufficient to allow the loss of endogenous intermembrane space proteins, but not the rapid exchange of exogenous cytochrome c measured as accessibility of complexes III and IV . However, if fresh cytosol (PEF) is present when cytochrome c– releasing factors act on mitochondria, an additional mitochondrial change occurs which strikingly enhances the permeability of the outer membrane to exogenous cytochrome c . From measurements of complex IV accessibility such as those presented in Fig. 4 , we calculate that 1 μl of apoptotic mitochondria can oxidize ∼60 nmol of cytochrome c per minute. As the same volume of Xenopus egg mitochondria contains ∼12 pmol of cytochrome c , we conclude that the rate of cytochrome c exchange across the outer membrane produced by PEF is at least 5,000-fold greater than that needed for complete efflux of endogenous cytochrome c per minute. A potential explanation for the PEF activity we observed is that PEF could be an analogue of Bax or Bid that is present in cytosol at higher concentrations than those we used with recombinant Bid and Bax. However, this notion can be ruled out for the following reasons: (a) when cytosol activated by pretreatment with cytochrome c is subsequently mixed with mitochondria, rapid release of cytochrome c is observed, likely due to the cleavage and activation of endogenous Xenopus Bid. This activated cytosol, however, has lost PEF activity rather than gained it; (b) recombinant tBid was added at five-fold higher concentrations (170 ng/ml) than those required for cytochrome c release (not shown); and (c) (most compelling) PEF-containing cytosol does not possess the ability to cause rapid release of cytochrome c , and therefore cannot contain Bid or Bax activity greater than the amounts of recombinant protein we added. Further dilution of PEF-containing cytosol results in diminution of PEF activity, but rapid cytochrome c release is never seen. Thus, PEF does not behave like Bax and Bid. The high permeability caused by PEF is consistent with a major rearrangement of the outer mitochondrial membrane, as a similar permeability (complex III or IV accessibility) was obtained with Triton X-100 , osmotic breakage of the outer membrane , and with digitonin in the absence of cytosol. The permeability increase by PEF was not, however, accompanied by observable changes in mitochondrial ultrastructure . Furthermore, evidence suggests that the inner mitochondrial membrane remains intact even after PEF has acted to permeabilize the outer membrane. We observed that the mtHsp70 protein was released from the matrix of Xenopus mitochondria treated with certain concentrations of the detergent digitonin but was not released with apoptosis , in agreement with another report that the inner membrane remains impermeable to proteins . In addition, our earlier data show that inner membrane potential (as measured by mitochondrial retention of the dye, DiOC 6 ) remains high for at least 3 h after cytochrome c release, a finding inconsistent with inner membrane permeabilization. Thus, the effects of tBid, Bax, and PEF are confined to the outer membrane, rather than reflecting a global alteration in mitochondrial integrity. The outer membrane permeability changes we observed were not due to a feedback effect of cytochrome c– activated caspases. Caspase inhibitors failed to block cytochrome c and AK efflux induced by Bid, Bax and the pro-apoptotic proteins in Xenopus cytosol. Moreover, Bid and Bax induced cytochrome c and AK release from mitochondria incubated in buffer alone . Caspase inhibitors also did not block the permeabilizing activity of PEF, but rather enhanced it . Preliminary characterization showed that PEF is proteinaceous and apparently large (>300 kD). While the slow kinetics of PEF action may suggest enzymatic behavior, PEF is not a caspase, as it was not inhibited by caspase inhibitors. Indeed, PEF is inactivated by caspases, because cytosol in which caspases had been activated by the addition of cytochrome c contained no PEF activity, and caspase inhibitors prolonged the effects of PEF in fresh cytosol. A PEF-like activity was also detected in human Jurkat cell extracts (not shown). It is possible that PEF is active in whole cells, as a similar assay monitoring oxygen consumption (complex IV activity) in permeabilized cells found that apoptosis induced high oxygen consumption in the presence of exogenous reduced cytochrome c . There are several mechanisms by which pro-apoptotic Bcl-2 family members might induce outer membrane permeability without affecting the inner membrane. These membrane-associated proteins may form channels large enough for the passage of soluble proteins or cooperate with channels already present such as VDAC . Alternate mechanisms might be similar to the manner in which 15-lipoxygenase can interact specifically with organellar membranes to cause visible changes (3–10 nm) in the lipid structure and permeability to high molecular mass proteins , or to that in which Bax causes instabilities in artificial membranes . Also unclear is the mechanism of PEF action, in particular how PEF is permitted to function only after a prior permeabilization of the outer membrane by factors such as Bid and Bax. One possibility is that PEF could interact with a target located on the lumenal side of the outer membrane. In this case, PEF would only gain access to its sites of action when the outer membrane becomes permeable to some degree. Alternatively, PEF could require the cooperation of a nonsoluble cofactor that is located within the intermembrane space . Yet another possibility is that PEF interacts directly with channels produced by tBid or Bax. It is thought that eukaryotic mitochondria originate from a symbiotic relationship between primitive host cells and primitive gram-negative bacteria, which allowed the host cells to use oxygen to produce energy . It can be argued that pressures to maintain this symbiotic relationship may have led to the utilization of death pathways involving mitochondria, i.e., apoptosis via cytochrome c or necrosis via reactive oxygen species . From this perspective, it is interesting to consider whether the Bcl-2 family might have evolved from primitive antibiotics produced by the host cell. All known effects of the Bcl-2 family members, with the exception of Bcl-2 cell cycle effects , can be linked to their regulation of cell death, often via effects on mitochondria. Indeed, the structures of Bcl–x L and Bid reveal similarities to those of the pore-forming domains of bacterial toxins. Moreover, like bacterial toxins, Bcl-2 proteins form channels in synthetic lipid membranes. Other factors, derived from the host cell, might also have evolved to destroy mitochondrial integrity. Such products include defensins , granulysin , bactericidal permeability-increasing proteins , and channel-forming peptides , such as mastoparan and alamethicin, both of which we have shown can release mitochondrial cytochrome c . PEF can now be added to this list of molecules affecting mitochondrial permeability. What are the implications of PEF activity for a cell? If PEF is present, attempts to rescue doomed cells by using caspase inhibitors would likely be futile, and may even enhance mitochondrial damage by leaving PEF fully active. Some cells given an apoptotic stimulus undergo necrotic death instead of apoptosis when caspase inhibitors are present , and in such cases PEF might be responsible for ensuring that mitochondria are irreversibly damaged. On the other hand, in Apaf-1 and caspase-9 knockout mice, where the cytochrome c– initiated caspase activation pathway is missing, many neurons survive despite likely mitochondrial cytochrome c release . Moreover, in the presence of caspase inhibitors, trophic factor-deprived primary sympathetic neurons survive for many days despite mitochondrial cytochrome c translocation . In such saved cells, neuronal mitochondria can survive the loss of cytochrome c and, upon restoration of trophic support, actually recover their content of cytochrome c after 24 h . It has not yet been determined whether the recovery of mitochondrial cytochrome c in these neurons is due to multiplication of a few intact mitochondria, or to the reimport of (apo-)cytochrome c into mitochondria that recover from their permeabilization. We hypothesize that in most nonneural tissues, PEF provides a backup mechanism for killing the cell in the event of a disabled caspase pathway. This kind of redundancy in death pathways might be desirable in lymphocytes and Xenopus eggs, but not in irreplaceable cells such as neurons in the adult organism. | Study | biomedical | en | 0.999998 |
10562283 | The EJ human bladder carcinoma cell line, 293T human embryonic kidney cell line, and MCF7 human breast carcinoma cell line were maintained in DMEM supplemented with 10% FBS (GIBCO BRL). H1299 human lung carcinoma cells were maintained in RPMI 1640 supplemented with 10% FBS. Both EJ-p53 and EJ-p73 cells were maintained in DMEM supplemented with 10% FBS, penicillin-streptomycin (50 U/ml), hygromycin (100 μg/ml), and geneticin (750 μg/ml). To repress the expression of p73α, p73β, or p53, tet was added to the medium every 3 d to a final concentration of 1 μg/ml. To induce p73 expression, cells were washed three times with PBS and seeded directly in culture medium without tet. The NH 2 -terminal hemagglutinin (HA)-tagged coding sequence of p73α (or p73β) (obtained from M. Kaghad, Sanofi Recherche, Innopole, France) was released with BamHI and StuI from pcDNA3-p73α (or p73β) and then ligated with pBluescript SK digested with BamHI and EcoRV. The resulting plasmid pBluescript-p73α (or p73β) was then digested with BamHI and SalI, and the fragment encoding p73α (or p73β) was cloned downstream of the tet-regulated promoter into pUHD10-3 (generously provided by H. Bujard, Universitat Heidelberg, Heidelberg, Germany), resulting in plasmid pTet-p73α (or p73β). EJ-tTA cells, generated as described previously , were transfected with pTet-p73α (or p73β) using the standard calcium phosphate method. Transfectants were doubly selected in the presence of hygromycin (100 μg/ml) and geneticin (750 μg/ml). Individual clones of stable transfectants, designated EJ-p73α or EJ-p73β, were selected for further analysis. Cells cultured in the presence or absence of tet were washed twice with ice-cold PBS with 2 mM sodium vanadate and lysed in EBC lysis buffer as described previously . Lysates were cleared by centrifugation at 14,000 rpm for 20 min at 4°C. Protein concentrations were determined using the BCA protein assay kit (Pierce). About 40 μg of cellular protein per sample were subjected to 12 (for p73) or 7.5% (for hMDM2) SDS-PAGE and transferred to Immobilon (Millipore) polyvinylidene difluoride filter. p73 was detected using an HA polyclonal antibody (Santa Cruz Biotechnology) or Ab-2 mAb (clone ER-15; NeoMarkers), and wild-type as well as mutant hMDM2 were detected using Ab-1 mAb (Oncogene Science), followed by ECL detection system (Amersham Pharmacia Biotech). 293T cells were transiently cotransfected with p73α or p73β and mdm2 using Fugene 6 (Boehringer Mannheim). Cell lysates were made using EBC lysis buffer and 200 μg of cellular protein was incubated with HA or MDM2 antibody at 4°C for 1 h, followed by another hour incubation with protein A beads. Immunoprecipitation complexes were washed three times with NET-N buffer , and subjected to SDS-PAGE followed by immunoblot with the reciprocal antibody. Cells were cultured in the presence or absence of tet for the indicated times, washed in PBS, and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min at room temperature. The method for senescence-associated β-galactosidase (SA-β-gal) (pH 6.0) staining was performed as described . Subconfluent cultures were pulse labeled for 30 min with 10 μM 5-bromo-2′-deoxyuridine (BrdU) (Sigma). Both adherent and floating cells were harvested, fixed in 70% ethanol, and then double stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody (Becton Dickinson) and 5 μg/ml propidium iodide (Sigma Chemical Co.). Cell cycle analysis was performed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson). Data were analyzed using Elite software (Becton Dickinson). EJ-p53, EJ-p73α, and EJ-p73β cells were seeded in the presence of 2 ng/ml tet to induce submaximal levels of either p53 or p73. Cells were then treated with 2 or 5 μg/ml mitomycin C, 0.02 or 0.1 μg/ml doxorubicin, or 5 or 10 ng/ml actinomycin D for 24 h. MCF7 cells were treated under the same condition. Cell lysates were prepared and aliquots containing 40 μg of cell protein were subjected to SDS-PAGE followed by immunoblot analysis with 1801 mAb for p53, HA polyclonal antibody, or ER-15 mAb for p73. Plasmid DNA was transiently transfected into H1299 cells using Fugene 6 (Boehringer Mannheim). Approximately 2 × 10 6 cells were cotransfected with plasmids as indicated. Cells were harvested 48 h after transfection, and luciferase activity was measured using a luciferase assay kit (Promega). The assay was normalized by cotransfection of a pCMV–β-gal plasmid and measurement of β-galactosidase activities. It has been shown that p53 can induce growth arrest, apoptosis, or senescence depending on the cell context . Since p73 is a homologue of p53, we attempted to examine the biological consequences of induced p73 overexpression in EJ cells, which lack functional p53 due to a mutation in exon 5 . To obtain tightly regulated p73α or p73β expression, the tet-regulatable expression system was used. EJ-tTA cells, which contain the transactivator with a hygromycin-resistant marker, were transfected with either pTet-p73α or pTet-p73β containing HA-tag and a neomycin-resistant marker. Stable clones were isolated by double selection. More than 10 tet-regulatable clones for each gene were selected and two independent clones were analyzed in detail. The phenotypes in each case (designated EJ-p73α or EJ-p73β) were similar. As shown in Fig. 1 A, there was no detectable amount of HA-p73α in the presence of 1 μg/ml tet as determined by immunoblot analysis . Within 24 h of tet removal, p73α was induced and became readily detectable , with p73α levels further increasing to a steady-state level by 48 h . To test whether p73α induction was reversible, tet was added back to the medium after induction for 24 h, and p73α levels examined 24 h later. It was apparent that p73α returned to an undetectable level under these conditions, indicating that p73α expression was fully reversible. Similar results were obtained with EJ-p73β as shown in Fig. 1 B. It can also been observed that two p53 transcriptional target genes, p21 and mdm2, were induced by both p73α and p73β . The kinetics of the induction paralleled that of p73, and was also reversible following readdition of tet . In response to p73 induction in EJ-p73α or EJ-p73β cells, we observed profound alterations in both cell proliferative capacity and morphology. Whereas EJ-p73 cells grew as small, rounded, refractile cells in the presence of tet and reached confluence, similar to parental EJ cells, the induction of p73 expression caused cells to stop growth and exhibit increased size and flattened morphology as well as enlarged nuclei . Of note, there were no characteristics of apoptosis detected in these cells as determined by 4′,6-diamidino-2′-phenylindole dihydrochloride nuclear staining (data not shown). To examine the reversibility of p73-induced growth arrest in EJ cells, we performed a colony formation assay. EJ-p73α or EJ-p73β cells were seeded at about 100 cells per 60-mm plate and maintained in the absence of tet for varying time periods followed by tet readdition. Cultures were subsequently maintained in the presence of tet for another 2 wk, followed by fixation and Giemsa staining. The number of colonies were counted and plotted as shown in Fig. 3 . Maintenance of the cells in the absence of tet for three or more days resulted in a marked reduction of the ability to form colonies. Indeed, the kinetics of permanent inhibition of colony formation by p73α or p73β was comparable to that observed with p53 . These experiments demonstrated that induced expression of either p73α or p73β in EJ cells causes irreversible growth arrest. To investigate in which specific cell cycle stage(s) p73 arrested EJ cells, we performed fluorescence-activated cell sorting analysis using EJ-p73α or EJ-p73β cell. EJ-p73 cells were maintained in the presence or absence of tet for varying time periods, followed by analysis using simultaneous flow cytometry for both DNA content and DNA synthesis, with propidium iodide staining and BrdU labeling, respectively. After tet removal, EJ-p73 cells exhibited a dramatic reduction in BrdU incorporation within 3 d, with the population of S phase cells declining from 45.2 and 51.7% in (+) tet to 5.9 and 12.4% in (−) tet for EJ-p73α or EJ-p73β, respectively . Conversely, the percentage of cells in both G1 and G2/M phases increased from 34.1 and 20.2% in (+) tet to 60.5 and 33.6% in (−) tet for EJ-p73α, and from 31.5 and 16.0% in (+) tet to 56.3 and 26.5% in (−) tet for EJ-p73β by 3 d, respectively. Thus, induced expression of p73α or p73β arrested EJ cells in both G1 and G2/M phases. Induction of both G1 and G2/M arrest has also been observed with p53 overexpression in EJ-p53 cells . Of note, there was no evidence of a sub-G1 population as usually seen in apoptosis in either EJ-p73α or β cells. It has been shown that senescent but not presenescent, quiescent, or terminally differentiated cells express a SA-β-gal, which can be detected by incubating cells at pH 6.0 with 5-bromo-4-chloro-3-indolyl β- d -galactoside (X-gal) . Since a striking feature of induced p73 expression was a morphological change characteristic of senescent cells, we examined whether EJ-p73 cells expressed this senescent-specific marker after p73 induction. As shown in Fig. 5 , >90% of EJ-p73 cells became positive for SA-β-gal staining within 7 d after induction of p73α or p73β, whereas EJ-p73 cells grown in the presence of tet over the entire time course of the experiment showed no staining (only [+] tet 7 d of EJ-p73α is shown). These results indicated that expression of p73 can promote a senescence-like program in EJ cells. It has been reported that the product of mdm2, a p53 transcriptional response gene, can interact with p53 and target it for degradation . Since p73 shares high homology with p53, we sought to investigate whether mdm2 also interacted with p73. 293T cells were cotransfected with p73α or p73β, together with wild-type or a mutant human MDM2 with the first 58 aa deleted (ΔN-hMDM2). This deletion is known to abolish MDM2's ability to interact with p53 . Reciprocal coimmunoprecipitation was performed using anti-mdm2 or HA antibody, followed by Western blot analysis. As shown in Fig. 6 B, p73α or p73β was detected in the immunocomplexes precipitated by the anti-mdm2 antibody; similarly, hMDM2 was also detected in the immunocomplexes precipitated by the anti-HA antibody. However, there was no detectable p73α or p73β associated with the mutant hMDM2; similarly, mutant hMDM2 was not detected in the immunocomplexes associated with p73α or p73β. These experiments demonstrated that p73α and p73β interact with wild-type but not NH 2 -terminal deleted hMDM2, despite the comparable expression level of these proteins . Next, we attempted to investigate whether the interaction between hMDM2 and p73 had any effects on p73's transcriptional activity. To do so, p73 or vector was cotransfected into H1299 cells along with a luciferase reporter plasmid that contains the genomic sequence from the p21 promoter. As shown in Fig. 7 , p53, p73α, and p73β increased the luciferase activity by 15-, 18-, and 38-fold compared with vector, respectively. Neither hMDM2 nor ΔN-hMDM2 alone had any effect on the luciferase activity. When wild-type mdm2 was cotransfected with p53, p73α, or p73β, the luciferase activity decreased three-, four-, and sixfold, respectively. However, when ΔN-hMDM2 was cotransfected, there was no significant change in the p21 promoter response. These experiments demonstrated that wild-type hMDM2 interacts with p73 and specifically inhibits its transcriptional activation of the p21 promoter, consistent with recent reports . It has been shown that p53 is induced by various stresses such as DNA damage, hypoxia, or nucleotide pool depletion , and this induction is mainly regulated at the level of p53 protein stability . To investigate whether p73 could also be stabilized by DNA damaging agents, we titrated the amount of tet required to induce moderate increases in tet-regulatable p53 or HA-p73 levels, and treated the cells with different concentrations of mitomycin C (MMC), doxorubicin (Dox.), or actinomycin D (Act.D) to activate DNA damage checkpoints, followed by immunoblot analysis with p53 or HA antibodies. As shown in Fig. 8 A, p53 levels increased in response to each DNA damaging agent, consistent with previous reports . In striking contrast, the levels of both p73α and p73β did not increase after exposure to any of these DNA damaging agents. Since the HA-tagged p73 was transcriptionally active, it is unlikely that it would respond differently from endogenous p73 to DNA damage, although we cannot exclude this possibility. Thus, we next tested whether endogenous p73 behaved similarly. MCF7 cells were treated with different concentrations of DNA damaging agents, followed by immunoblot analysis with p53 or p73 antibodies. As shown in Fig. 8 B, p53 levels increased after each treatment. However, the levels of both p73α and p73β did not increase in response to any of the DNA damaging agents tested. These results suggested that unlike p53, p73 protein stability was not increased in response to several different genotoxic agents. These studies demonstrate that in tumor cells lacking functional p53, the induced overexpression of either p73α or p73β, an alternative product of the p73 gene, promoted a cellular response leading to irreversible growth arrest with markers of replicative senescence. This conclusion is supported by the following observations: induction of a flattened, enlarged cell morphology, commonly observed with senescent fibroblasts; and SA-β-gal staining (pH 6.0), a specific biochemical marker of senescent cells . The commitment to senescence became irreversible within 3 d and no longer required p73 expression. Similar results have been observed with overexpression of p53 or p21, an effector of both p53 and p73, in these same cells . p73 has been reported to induce apoptosis when overexpressed in some tumor cells, independent of p53 status . In our studies, there were no findings consistent with apoptosis in response to p73, p53, or p21 overexpression in any of the assays used , suggesting that p73, like p53, induces apoptosis in a cell context–dependent manner. We also found that p73 can induce mdm2 and p21, two known transcriptional targets of p53, consistent with previous studies . It has been shown that p53 interacts with the NH 2 -terminal 58 aa of hMDM2, since removal of this segment abolishes this interaction . Similarly, we showed that hMDM2 coimmunoprecipitates with both p73α and p73β, and this interaction was also disrupted by deletion of the NH 2 -terminal 58 aa residues of hMDM2, indicating that p73 interacts through the same NH 2 -terminal 58 residues. We further observed that hMDM2 inhibited the transcriptional response from the p21 promoter in response to p73, as has been reported for p53 . All of these findings indicate striking similarities in several aspects of p53 and p73 biology. Unlike the case with p53, hMDM2 interaction did not target p73 for degradation, since p73 protein levels did not decrease . These results indicate that although hMDM2 can interact with both p53 and p73, its inhibition of p73 transcriptional activity is not mediated by a mechanism involving p73 protein degradation. Similar findings have been reported recently by Zeng et al. 1999 . We observed another major difference in p53 and p73 biology. In EJ tumor cells in which p53 function had been inactivated, exogenously expressed p53 but not p73 showed increased protein level in response to several different DNA damaging agents. Since transcription of each gene was under the control of the same tet-regulatable promoter, these findings likely reflect p53 protein stabilization in response to genotoxic stress by mechanisms that remained intact in these tumor cells. The lack of response of p73 to the same agents further implies differential regulation of these genes at the level of protein stabilization in these tumor cells. These findings could help to explain a selective pressure for inactivation of p53 but not p73 function in the evolution of this tumor despite their comparable ability of inducing permanent growth arrest in these cells. We also observed that in MCF7 breast cancer cells with intact p53, neither endogenous p73α nor p73β was induced by DNA damaging agents under conditions in which p53 overexpression was readily observed, consistent with a previous report . Recent studies have indicated that in mouse embryo fibroblasts, certain other DNA damaging agents such as cisplatin were able to induce protein stabilization through a mechanism involving c-Abl . We have also observed variation in responsiveness among different tumor cell lines to p73 induction by DNA damaging agents (our unpublished observations). Thus, cell context or the specific agent may be critical determinants of p73 induction in response to DNA damage. Our present findings that p73 can induce permanent growth arrest, in combination with previous studies that p73 can induce apoptosis in other cells imply that p73 can mimic two major p53 effector functions used in its role of guardian of the genome. Thus, the paucity of p73 mutations in human tumors may reflect its lack of responsiveness to genotoxic stresses which commonly induce p53, or to a more restricted tissue expression pattern . | Study | biomedical | en | 0.999997 |
10562284 | Human microvascular endothelial cells (HMEC-1) cells were maintained and passaged in endothelial growth medium (Clonetics) supplemented with an additional 8% FBS (Gemini) for a final concentration of 10%. For some experiments, cells were transferred to endothelial basal medium supplemented with 0.2% FBS (Clonetics). Cells were maintained at 37°C in a humidified incubator containing 7% carbon dioxide. cDNA expression plasmids containing full-length PAK1 and its various mutants in pCMV6M (CMV promoter, NH 2 -terminal myc tag) have been described elsewhere . Anti-PAK antiserum was raised as described . Cells were extracted with buffer containing 1% NP-40 plus protease and phosphatase inhibitors, and PAK was immunoprecipitated using a polyclonal antiserum as described . Kinase activity was determined using an in-gel kinase assay with myelin basic protein, and PAK protein levels were determined by Western blotting using the same antibody . Cos-1 cells grown to 75% confluence on 10-cm tissue culture dishes were transiently transfected using the lipofectamine transfection protocol (GIBCO BRL) with a total of 7.5 μg of pCMV6M expression vectors containing various myc-tagged constructs. The cells were allowed to express the protein for 40 h after transfection and were then washed in PBS and scraped into 250 μl of lysis buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 5 mM MgCl 2 , 1 mM DTT, 5% glycerol, 1% NP-40) at 4°C. Lysates were passed four times through a 21 gauge needle and then clarified by centrifuging for 5 min at 7,000 rpm in a benchtop Eppendorf microfuge to remove unbroken cells and large cellular debris. Supernatants were decanted and 30 μl of each lysate was run on SDS-PAGE gels and transferred to nitrocellulose before probing for the expression of myc-tagged protein using an anti-myc antibody (9E10); the remainder was immediately frozen at −80°C until required. To assay migration, HMEC-1 cells were plated on coverslips coated with 2 μg/ml of fibronectin (FN; Sigma Chemical Co.). Dishes were prepared by cutting a hole in the bottom of the dish and attaching a coverslip to the outside of the dish with silicone grease. These were placed in an open chamber with atmospheric and temperature control and viewed with a Nikon DiaPhot microscope equipped with a SenSys cooled CCD video camera linked to a Silicon Graphics workstation running the Inovision ISEE software program. HMEC-1 ECs were injected with cDNAs at 0.2 mg/ml as described . Protein expression was detectable by immunofluorescence ∼30 min after injection and cell migration was assessed by time-lapse imaging beginning 60 min after injection. At the end of the experiment, images of cells were outlined and the centroid (cell center) calculated. Displacement of the centroid was then used to determine movement over time . NIH 3T3 cells were plated on 50 μg/ml of FN in medium with 10% calf serum to match the conditions used by Sells et al. 1999 . They were allowed to spread for 2 h before injection and migration was measured as described. Cells were fixed for 10 min in 3% paraformaldehyde in PBS, permeabilized for 10 min with 0.01% Triton X-100/PBS, and rinsed twice with PBS. Coverslips were blocked by incubating for 40 min in 10% goat serum at room temperature, then stained for 60 min in a 1:10 dilution of rhodamine-phalloidin (ICN Immunobiologicals). Coverslips were then washed twice in PBS before being incubated with a 1:100 dilution of antivinculin (Sigma Chemical Co.) antibody , followed by washing with PBS. Coverslips were incubated in a 1:100 dilution of CY5-conjugated goat anti–mouse F(ab′)2 fragment (Sigma Chemical Co.) or CY5 goat anti–rabbit (H & L; Sigma Chemical Co.) for 60 min, washed twice in PBS, and mounted in Immunofluore mounting medium (ICN Immunobiologicals). Slides were viewed using a BioRad 1024 MRC scanning confocal microscope. ECs, which were injected with DN, active CAAX tagged (AC), or wild-type (WT) PAK were fixed and stained for actin and P-MLC as described above. Images were then processed using a Silicon Graphics workstation running the Inovision ISEE software program. Cells were outlined and total fluorescence staining intensity determined by determining the sum of the pixel intensities per cell. Background staining in cell-free areas was negligible. At least 20 cells were analyzed from four experiments for each value. HMEC-1 ECs were coinjected with cDNAs at 0.2 mg/ml of various PAK constructs, together with 0.2 mg/ml of cDNA coding for green fluorescent protein (GFP)–α-actinin fusion protein. Starting at ∼6 h after injection, at which time the fluorescence signal was sufficiently intense, cells were monitored for up to 16 h after injection under fluorescence optics on the inverted Nikon microscope using a 20× oil immersion lens. Images of cells were collated and viewed by time-lapse using a Silicon Graphics workstation running the Inovision ISEE software program. To visualize cell contractility, flexible rubber substrates were generated by a protocol described in Chrzanowska-Wodnicka and Burridge 1996 . In brief, silicone rubber (dimethyl polysiloxane), viscosity 10,000 centistokes (Dow Corning Co.), was coated onto the coverslip at the bottom of 35-mm tissue culture dishes and allowed to spread for 2 h. The silicone was then coated with a thin layer of gold-palladium using a Hummer VI sputter coater. The UV glow discharge during the coating process polymerized the silicone rubber and the gold-palladium coating decreased the hydrophobic nature of the rubber surface; the surface was additionally coated with 2 μg/ml of FN. ECs were plated on the rubber substrate and allowed to adhere for ∼30 min, then, only cells that had wrinkled the substrate beneath were coinjected with the various PAK constructs and GFP, and incubated for 4 h. Expressing cells were recognized by GFP fluorescence and the ability of the cells to create wrinkles in the substratum was scored by phase-contrast microscopy. Statistical analysis was performed using Microsoft Excel statistics software. The displacement of the centroid over time and thus the rate of cell migration was obtained with the Inovision ISEE software program from images obtained using a 10× objective. Approximately 25 cells were injected per experiment, with at least three independent experiments for each PAK construct. Results were expressed as mean ± SEM. The t test was used to determine whether the observed differences were statistically significant ( P = 0.05). Previous work in fibroblasts has shown that PAK1 kinase activity is stimulated by both integrin-mediated adhesion to extracellular matrix proteins and by serum or soluble growth factors . Therefore, we tested whether PAK1 in HMEC cells behaves similarly. Serum starved cells were detached and either replated on FN or kept in suspension. Additionally, adherent starved cells were stimulated with growth medium containing serum and basic fibroblast growth factor. PAK1 was immunoprecipitated from cell lysates and its activity toward myelin basic protein assayed. Fig. 2 (right) shows that PAK activity in suspended cells was nearly undetectable, and was stimulated by about tenfold upon reattachment to FN. PAK activity was maximal by ten minutes after replating and was sustained, as it was similar to the level in serum starved stably adherent cells. A separate experiment shows that the level of PAK activity in stably adherent cells in basal medium was further increased by addition of growth medium by ∼1.7-fold. These data show that both adhesion and growth factors contribute to PAK activity in HMECs, confirming that PAK in ECs behaves similarly to PAK in fibroblastic cells. Initial measurements of HMEC-1 migration showed that cells plated the day before the experiment in basal medium migrated at a very low rate . Indeed, much of the apparent migration was due to changes in the location of the centroid due to extension and retraction of processes, rather than true displacement of the cell body. Cells that were plated on coverslips coated with 2 μg/ml FN spread within 15 min and, after one hour, migrated at a slightly but consistently faster rate. This rate persisted for at least four hours and additional experiments showed no change for up to eight hours (not shown). These results suggest that the increase was due to migration on the FN coating as opposed to the matrix synthesized by the cells, and did not represent transient stimulation due to replating, per se. The rate of migration of cells plated on FN in growth medium was ∼2.5-fold higher than in basal medium. This rate was also maintained for at least four hours after plating , and additional experiments showed no change for up to eight hours (not shown). Examples of cell displacement for each condition are shown to illustrate typical patterns of cell movement . To analyze the effects of PAK mutants, we chose to express these constructs by microinjection, since this method gives efficient expression even in ECs that are difficult to transfect. This approach also avoids potential longer term effects of PAK mutants on gene expression. To confirm the size of the expressed proteins, all of the constructs used in these and the following experiments were overexpressed in COS-1 cells and analyzed by Western blotting. The constructs were expressed at similar levels and yielded proteins of the expected sizes . Microinjection of cells with a control GFP construct slowed the rate of movement of the cells in growth medium by ∼30%, though it had little effect on cells in basal medium . This modest inhibition presumably indicates the effect of microinjection alone and is consistent with results obtained in other studies . Coexpression of WT PAK with the GFP did not significantly alter cell migration compared with GFP alone . Injected cells that expressed the heterologous proteins could be identified either by staining with anti-myc to detect the tagged PAK or by fluorescence of the GFP. Since these two always correlated (not shown), we routinely relied upon GFP fluorescence to detect expressing cells in subsequent experiments. All of the PAK constructs gave similar staining intensity with anti-myc antibody (not shown), indicating similar expression levels in HMEC-1 cells. HMEC-1 cells were coinjected with GFP plus a DN PAK containing mutations in both the kinase domain (R299) and the GTPase binding domain (H83L, H86L). This construct lacks kinase activity and also fails to bind Rac or Cdc42, therefore, it does not sequester the endogenous GTPases to inhibit their interactions with other effectors. Previous studies showed that this DN PAK interferes with function of endogenous PAK , presumably by associating with substrates or other PAK-interacting proteins. Expression of this construct decreased the rate of migration in growth factor–stimulated cells to the level in unstimulated cells . A slight decrease was also noted in unstimulated cells, but this effect was not statistically significant ( P = 0.45). This result suggests that PAK may be involved in growth factor–stimulated EC migration. To further investigate the role of PAK in migration, four constitutively active mutants of PAK were also examined. T423E PAK and H83L,H86L PAK are activated variants; the T423E mutant shows significantly higher kinase activity in vitro , whereas the H83L,H86L mutant is also deficient in binding to Rac and Cdc42, and therefore should not inhibit other pathways downstream of the GTPases. Additionally, we tested a combined mutant, T423E,H83L,H86L PAK, which is strongly activated and deficient in binding the GTPases, and a membrane-targeted active variant of this latter mutant, H83L,H86L,AC PAK, which is activated, deficient in GTPase binding, and membrane targeted due to the presence of a COOH-terminal isoprenylation sequence . Cell migration was assayed both in basal medium and growth medium. While the weakly activated 83,86 mutant had only a small effect that did not reach statistical significance ( P > 0.05), the strongly activated T423E mutant and the membrane targeted 83,86 mutant both significantly inhibited cell movement ( P < 0.001) to an extent that was similar to DN PAK . The combined mutant T423E,H83L,H86L also significantly inhibited migration, indicating that effects of T423E were not due to sequestering the GTPases. Notably, some inhibition by the membrane targeted construct also occurred in cells without growth factors ( P < 0.05); the 83,86 mutant that did not target to membranes decreased migration of starved cells slightly, but the effect did not reach statistical significance ( P = 0.22). One might have expected that expression of active PAK constructs would stimulate migration in the starved cells, however, the effects were, if anything, weakly inhibitory. These results argue that proper regulation of PAK is required for cell migration. Thus, either unregulated increases or decreases in PAK activity inhibit motility. To further investigate interactions of PAK that were important for inhibiting HMEC-1 cell migration, we tested additional constructs designed to probe smaller regions of the molecule . These constructs allowed us to investigate the relative contributions of different domains. As shown in Fig. 6 , a full-length construct in which the third proline-rich domain, which is known to bind the Rac/Cdc42 nucleotide exchange factor PIX , was mutated to block binding to SH3 domains (A193,A194) and had no effect on migration. Mutation of the ED (glutamic and aspartic acid-rich) domain also had no effect in the context of full-length PAK. Expression of the NH 2 -terminal regulatory domain of PAK (1-205) inhibited migration, as well as the full-length DN construct, indicating that important inhibitory sequences resided in the NH 2 -terminal half of PAK. A 1-205 (H83L,H86L) construct that lacked GTPase binding inhibited migration equally well, indicating that this effect did not require blocking Rac or Cdc42 function. The extreme NH 2 terminus contains 2 putative SH3 domains . Expression of the first 74 residues that encompass these sites decreased migration as efficiently as full-length DN PAK, indicating that this region was sufficient to inhibit migration. Mutation of the first proline rich sequence (P13A) prevented the decrease in migration rate, whereas mutation of the second proline-rich sequence (P42A) had no effect. These results indicate that the first proline rich sequence of PAK is critical to its dominant effects on EC migration. Since HMEC-1 cells were studied by time-lapse microscopy, information about cell morphology was also readily available. Similar to HMEC-1 cells injected with GFP alone or WT PAK plus GFP, those injected with DN PAK continued to extend lamellipodia and ruffle. They tended to spread more extensively than control cells, especially in basal medium, despite their low rate of translocation . Cells with DN PAK also typically had circular or oval shapes with fewer extensions than either WT or AC PAK . Interestingly, cells injected with active, membrane-targeted AC PAK showed lamellipodia and ruffles similar to control cells, but translocated very little in comparison to control cells . The T423E mutant behaved similarly to the AC PAK (not shown). Because the activated constructs all gave similar results, the AC PAK construct was chosen for further studies, as it has strong effects, but avoids possible complications due to binding Rac or Cdc42. To test effects of PAK on actin organization and focal contacts, injected cells were stained for actin and vinculin. As shown in Fig. 8 , HMEC-1 cells expressing GFP alone or with WT PAK generally had modest numbers of stress fibers and focal adhesions. These cells were indistinguishable from uninjected cells (not shown). Cells expressing DN PAK showed increased stress fibers and larger focal adhesions. Similarly, expression of AC PAK also increased stress fibers and focal adhesions. The results from the previous experiments are ostensibly paradoxical, since either inhibiting or activating PAK gave superficially similar effects (though differences in shape were noted). To gain further insight into the effects of PAK on the cytoskeleton, cells were injected with an expression plasmid coding for GFP-conjugated α-actinin, which labels both stress fibers and focal adhesions, and therefore permits their visualization in living cells . The GFP–α-actinin was expressed alone, with WT PAK, or with other PAK constructs, and cells viewed using time-lapse fluorescence microscopy. Similar to results from the static images described above, both DN and AC PAK induced an increase in stress fibers and focal adhesions. Interestingly, in cells expressing GFP only or GFP and WT PAK, most stress fibers and focal adhesions were relatively stable, appearing or disappearing relatively slowly. However, once formed, these structures showed surprising flexibility. They changed positions extensively, with stress fibers lengthening, shortening, bending, and moving relative to the substrate and each other as the cells changed shape . By contrast, the α-actinin–containing structures in cells expressing DN PAK were almost completely static . Whereas extensive lamellipodia and ruffling was observed around the periphery of these cells, the inner network of actin cables and adhesions appeared virtually fixed in place. In addition, lamellipodia failed to show any polarity and were observed around the entire cell periphery. HMEC-1 cells injected with AC PAK appeared intermediate between the DN and WT constructs. These cells were less dynamic than control cells, but more dynamic than those expressing DN PAK. Formation of new adhesions, retraction of areas of the cell edge and bending, shortening, and lengthening of actin cables still occurred, although less rapidly than in control cells. Substantial lamellipodia and ruffling were also observed as in cells expressing GFP alone and WT PAK. These results raise the possibility that DN and active PAK may in fact not have identical effects on the cytoskeleton. Cell migration requires that new adhesions form at the leading edge and that old adhesions detach at the rear of the cell. Detachment may involve both contraction and diminished adhesion strength, depending on the cell type . One hypothesis that could explain the apparently paradoxical effect of PAK constructs on migration and focal adhesion formation is that it plays a role in coordinating adhesion formation at the leading edge with contraction and detachment at the trailing edge (these ideas are explained more fully in the Discussion). To test the involvement of PAK in contraction, cells were plated on silicone rubber membranes that can be deformed by cell-generated tension . HMEC-1 cells plated on this deformable surface create wrinkles that are easily observed by phase-contrast microscopy. Expression of GFP alone or GFP plus WT PAK had little effect on these wrinkles. Cells injected with the AC PAK construct maintained or increased wrinkling of the substrate. This increase was difficult to quantitate, however, an increase in wrinkle depth or intensity was consistently observed . The DN PAK, by contrast, caused a substantial decrease in wrinkling of the rubber substrate . Quantification of these results showed that after injection with GFP, 79% of cells still wrinkled the rubber substrate. 80 and 81% of cells expressing WT PAK and AC PAK, respectively, also produced wrinkles. However, only 30% of cells expressing DN PAK induced wrinkles . Addition of cytochalasin D at 1 μg/ml completely abolished all wrinkles (not shown), demonstrating that wrinkling required an intact actin cytoskeleton. These results show that PAK is required for contractility in HMEC-1 cells. Effects of PAK mutants on contractility and stress fiber formation suggest that myosin phosphorylation might be involved. Recent reports in different systems have been contradictory, however, since an active PAK mutant decreased phosphorylation of MLC in baby hampster kidney and HeLa cells , but increased MLC phosphorylation in 3T3 cells . The microinjection strategy employed in our studies precludes biochemical assays of myosin phosphorylation, hence we resorted to staining with an antibody developed by Matsumura and colleagues that specifically recognizes phosphorylated MLC . Staining intensity was then quantified. These experiments revealed an increase in staining with anti–P-MLC in cells expressing the active PAK mutant, indicating an increase in MLC phosphorylation . By contrast, the DN PAK construct caused only a slight and statistically insignificant change in staining with anti–P-MLC, even though it induced an increase in actin stress fibers. Other groups have reported that expression of the H83L,H86L–activated PAK in fibroblasts decreased actin stress fibers and focal adhesions . Additionally, stable expression of that construct in NIH 3T3 cells increased migration . To determine whether these discrepancies were due to distinct cell types or might be explained by different methods or cell lines used by different laboratories, we tested effects of the same construct in 3T3 cells by microinjection. We observed that microinjection of NIH 3T3 cells with cDNA for the 83,86 active PAK mutant triggered formation of lamellipodia and loss of actin stress fibers as described in the above reports . When cell migration was assayed under the conditions employed by Sells et al. 1999 , cells microinjected with vector coding for the active PAK construct moved at a rate of 15.7 ± 1.4 μm/h compared with 9.4 ± 1.3 for GFP vector only (a 70% increase, statistically significant P < 0.05). This result is consistent with the data obtained by Sells et al. 1999 and suggests that the observed differences are most likely due to cell-type specificity rather than different methods of expression. In light of these results on myosin phosphorylation, the data indicate that the cell-type–specific effects of PAK cannot be explained by differences in MLCK activity, suggesting the existence of a second pathway by which PAK modulates cytoskeletal dynamics. Our results show that expression of either inhibitory or activated PAK in HMEC-1 cells decreases growth factor–stimulated migration to the level in unstimulated cells. The effect of the DN construct was mapped to a single short proline-rich sequence that is known to bind the SH3 domain of the adapter protein Nck . Whether Nck itself or another SH3-containing protein is involved in this effect on migration is currently unknown. The DN PAK construct also induced an increase in focal adhesions and stress fibers and a decrease in contractility and mobility of these structures. Remarkably, cells expressing the DN PAK still showed extensive ruffling and lamellipodia, though these structures no longer appeared in a polarized manner. Expression of constitutively activated PAK variants also inhibited cell migration. Active PAK also increased stress fibers and focal adhesions, however, these structures retained some degree of dynamic behavior, consistent with increased cell contractility and phosphorylation of MLC. Cellular responses to the overexpressed PAK constructs show striking cell-type specificity. In HeLa, CHO, and 3T3 cells, active PAK mutants disrupt stress fibers and stimulate lamellipodia formation . Active PAK mutants can also stimulate migration in 3T3 cells . These effects may be mediated via phosphorylation of MLC kinase by PAK and/or an interaction with PIX . However, a distinct set of effects on cytoskeletal organization occurred in ECs. As we obtained the expected effects on 3T3 cells, it appears that these apparent discrepancies are most likely due to the distinct cell types used in these studies. Most likely, substrates and/or adapter proteins that modulate PAK function differ between cell types. The effects of PAK constructs on phosphorylation of MLC in HMEC-1 cells were similar to those of Sells et al. 1999 in 3T3 cells, but differ from those of Sanders et al. 1999 in HeLa and BHK cells. The increase in MLC phosphorylation in cells with active PAK is consistent with the measurements of contractility . By contrast, DN PAK caused, if anything, a slight (though statistically insignificant) increase in MLC phosphorylation, but a large decrease in contractility. It also triggered a large increase in actin stress fibers. Thus, DN PAK must induce or stabilize stress fibers by a pathway unrelated to MLC phosphorylation. This idea suggests that PAK may have additional substrates that regulate formation or stability of stress fibers and focal adhesions. That DN PAK increased stress fibers and focal adhesions, but decreased MLC phosphorylation and contractility may seem to be at odds with the dogma that myosin-generated tension is required for stress fiber formation . There is, however, some precedent for this distinction, since caldesmon decreases actomyosin-dependent tension, yet stabilizes actin stress fibers . Conversely, active PAK would be predicted to induce a global, nonpolarized increase in contractility. Such an increase could give rise to enhanced actin stress fibers, due to the increase in tension . However, the nonpolarized nature of the contraction could prevent efficient cell movement. Past studies of migration suggested a link between extension at the leading edge and retraction at the trailing edge . Productive movement requires that cells extend processes, make new adhesions at the front, and then detach old adhesions at the rear of the cell. Detachment may occur by ripping of the cell membrane to leave integrins and actin bound to the substratum, indicating that substantial tension is required . In constitutively adhesive cells, such as fibroblasts and ECs, these events appear to occur in cycles. Extension of a lamellipodium at the leading edge leads to formation of new adhesions at the front of the cell . New adhesions then initiate signals that stimulate contraction and detachment at the rear of the cell . Detachment then allows new protrusive activity at the leading edge. In ECs injected with α-actinin, we also observed cycles where lamellipodial extension and focal adhesion formation preceded the initiation of tail detachment, which was followed by new lamellipodia formation (our unpublished observations). These considerations suggest a model for the role of PAK in EC migration. It may be highly relevant that PAK is activated by formation of new adhesions and is inactivated by detachment . Thus, formation of new adhesions should trigger an increase in PAK activity. We hypothesize that this stimulation leads to contraction and detachment of the cell's tail. Thus, inhibition of detachment is the most likely target of DN PAK. This idea fits the observation that in ECs, DN PAK decreases, whereas active PAK appears to increase, contractility. There are several aspects of this model that remain to be tested. In particular, localization of PAK activity (as opposed to total protein) and its role in localized contraction or focal adhesion stability need to be investigated. In conclusion, our results indicate that PAK function is required for migration of microvascular ECs. These effects are distinct from those observed in fibroblasts and other cell lines. The available data are consistent with a model in which PAK is not required for formation of lamellipodia or filopodia by Rac or Cdc42, but instead plays a role in coordinating leading edge adhesion formation and trailing edge detachment to produce polarized cell movement. The data also raise the possibility that PAK may be a suitable target for pharmacological inhibitors of angiogenesis for development of therapies against cancer or other diseases involving pathological angiogenesis. | Study | biomedical | en | 0.999998 |
10562285 | The following procedure was used to construct an integrating plasmid encoding a tagged form of Cla4 that would be degraded rapidly by the N-end rule pathway following a shift to 37°C. The open reading frame (ORF) of the temperature-sensitive cla4-75 allele was amplified with flanking HindIII sites by using Pfu polymerase, and inserted into the HindIII site of the integrating plasmid pPW66R , producing p cla4-td (temperature-sensitive degron). This procedure produced an in-frame fusion between a hemagglutinin (HA)-tagged, thermolabile form of dihydrofolate reductase (DHFR) and the NH 2 terminus of the temperature-sensitive Cla4 protein; expression of the fusion protein is driven from the inducible CUP1 promoter. Cleavage of p cla4-td with NcoI was used to direct integration of the promoter fusion protein cassette at the URA3 locus. All yeast strains used were isogenic derivatives of W303 ( Table ), except the cdc42-1 mutant. Standard genetic techniques were performed according to standard methods ; disruptions were confirmed by PCR. CLA4 was disrupted in W303 by one-step gene deletion, resulting in KBY209. A ste20 Δ cla4 Δ double mutant (KBY211) expressing the temperature-sensitive cla4-75 allele on YCp TRP1-cla4-75 was obtained as a segregant from a cross between KBY209 carrying YCp TRP1-cla4-75 and a MAT α ste20 Δ mutant (KBY210) carrying a GAL-STE20 plasmid (pRD56) . The pRD56 plasmid was eliminated from KBY211 by selection on 5-fluoroorotic acid (5-FOA). The cla4-td allele was integrated at the URA3 locus of wild-type cells (SWY518, a W303 derivative), producing KBY213, and the URA3 locus of a cla4 Δ ste20 Δ mutant (KBY211) carrying YC pTRP1-cla4-75 , producing KBY212. Integration was confirmed by the ability of cells to lose YCp TRP1-cla4-75 , by a temperature-sensitive growth phenotype, and by detection of the DHFR-HA-Cla4 protein. A green fluorescent protein (GFP)-Tub1 fusion (encoded by plasmid pAFS91) was integrated at the HIS3 locus of a ste20 Δ cla4-td mutant (KBY212) and an isogenic wild-type strain (KBY213), producing KBY216 and KBY215, respectively; integration was confirmed by observing fluorescently labeled microtubules. Cells were grown overnight in synthetic media lacking uracil with or without 500 μM Cu-acetate, diluted into 15 ml of the same media, and grown 4 h at 23°C to an OD 600 = 0.5. For temperature-shift experiments, cells were diluted into media lacking uracil and copper at 37 or 23°C for various time periods. Cells were harvested, washed in media lacking copper, and lysed according to a NaOH extraction method . Total protein was quantified by the method of Lowry. Proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes, blocked, probed with the 12CA5 (anti-HA) mAb and a monoclonal goat anti–mouse HRP-coupled secondary antibody (Cappell Laboratories), and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech). Protein expression was quantified by analyzing immunoblots with NIH Image. G1 cells were isolated using a modification of published methods . Cells were plated as a lawn on synthetic media containing dextrose and lacking tryptophan for 3 d at room temperature, suspended in 10 ml of media lacking glucose, and sedimented through 1 M sorbitol in selective synthetic media lacking glucose. Unbudded cells remained in the supernatant fraction. Yields of unbudded cells were 85–95% of total cells. Cells in the supernatant fraction were collected by filtration and suspended for 1–2 h at 37°C in synthetic media lacking tryptophan and glucose and then transferred to rich media containing yeast extract, bactopeptone, and dextrose (YPD) at 37°C for the duration of the experiment. At various time points, cells were viewed by Nomarski microscopy or fixed in 3.7% formaldehyde and stained with 0.66 μM rhodamine-phalloidin . 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) was included in mounting media for fixed cells. For mitotic arrest assays, cells were grown in YPD containing 1% DMSO to facilitate solubilization of nocodazole. After addition of nocodazole (15 μg/ml final concentration), cells were incubated with shaking for at least 3 h at 23°C, resulting in 70–90% of cells arrested with large buds and unsegregated nuclei. Cells were pelleted, washed once in selective synthetic media lacking glucose at 37°C, which inhibited recovery from cell cycle arrest, and suspended in the same media at 37°C for 1–2 h. Cells were collected by filtration and washed at 37°C with at least 3 ml of selective synthetic media lacking glucose to remove residual nocodazole. Cells were suspended in 3 ml of 37°C YPD to resume growth. Cells were fixed with 3.7% formaldehyde at 20-min intervals. Fixed cells were subjected to Zymolyase (ICN) treatment to determine if cell separation had occurred. Cells in phosphate buffer were added to 1 M sorbitol containing 0.2 mg/ml Zymolyase and incubated at 37°C for 15 min. Septin localization was determined by staining cells with anti-Cdc11 antibodies according to published immunofluorescence protocols . In brief, cells were collected at various times after shift to the nonpermissive temperature, fixed, processed, and stained with anti-Cdc11 polyclonal antibody (1:3 dilution; gift of M. Longtine, Oklahoma State University, Stillwater, OK) and FITC-conjugated goat anti–rabbit secondary antibodies (1:500 dilution; Cappell Laboratories). Fusions encoding Cla4-GFP and Abp1-GFP were constructed by using PCR and integrative transformation to append the GFP-S65T coding region to the 3′ end of the genomic CLA4 and ABP1 ORFs. The GFP reading frame was preceded by a Gly 8 linker. The fusions, verified by PCR, were fully functional as judged by phenotypic analysis. Images of cells expressing various GFP fusion proteins were captured by using a DAGE cooled CCD camera mounted on an Olympus BX-60 or Olympus IX 70 inverted microscope equipped with a UPlanApo 100× objective. All other immunofluorescence and Nomarski images were acquired on a DAGE cooled CCD camera mounted on an Olympus BH-2 microscope equipped with a DPlanApo100UV 100× objective. Movies of actin patches labeled with Abp1-GFP were obtained according to published methods . For each time point, 9–10 z-plane images covering the entire cell were superimposed to yield a single two-dimensional image. Images were collected with a LG-3 framegrabber (Scion Corporation) and a DAGE ISIT camera mounted on an Olympus BX-60 microscope with a UPlanApo 100× objective. Nomarski movies were made by capturing images at 5-min intervals on an Olympus BH-2 microscope. Thermostatic controls were used to maintain the stage at desired temperatures. Previous studies have revealed terminal phenotypes resulting from inactivation of Ste20 and Cla4 , but they have not established when these PAK homologues execute their functions during the cell cycle. This point required further study because the terminal phenotype of a mutant can be caused by activation of a checkpoint early in the cell cycle that arrests cells at a point later in the cell cycle . Therefore, our goal was to determine when in the cell cycle Ste20 and Cla4 execute their functions. This would test the hypothesis that these PAK-family kinases function as effectors of Cdc42 to polarize cells during G1 or other phases of the yeast cell cycle. To define when during the cell cycle the activity of Ste20 and Cla4 is required, we needed a conditional means of inactivating Cla4 rapidly and completely in a cell that lacks Ste20. Inactivation of both kinases was necessary, because either one is sufficient to allow cell growth and division. To inactivate Cla4 in a cell that lacks Ste20, we constructed a temperature-sensitive degron allele by fusing a thermolabile form of DHFR to the NH 2 terminus of Cla4 encoded by the existing cla4-75 temperature-sensitive allele . Temperature-induced degradation of the DHFR-Cla4 fusion should result in rapid loss of Cla4 function. To facilitate protein detection, the DHFR-Cla4 fusion was tagged with the HA epitope and expressed from the inducible CUP1 promoter. The DHFR-Cla4 expression cassette was integrated at the ura3 locus in a cla4 Δ ste20 Δ mutant that carried a plasmid-borne copy of the cla4-75 allele. The DHFR-Cla4 fusion functioned in the expected manner. At permissive temperature and basal expression levels (absence of added copper), the DHFR-Cla4 fusion expressed from the chromosome was functional because cells could readily lose the plasmid carrying the cla4-75 allele. Cells that had lost the cla4-75 plasmid (referred to subsequently as the ste20 Δ cla4-td mutant, for temperature-sensitive degron) had a normal morphology and growth rate at the permissive temperature (23°C) (data not shown). As anticipated, the ste20 Δ cla4-td mutant did not grow at nonpermissive temperature (37°C) under basal or inducing expression conditions (data not shown; see below), indicating that DHFR-Cla4 had been inactivated. Consistent with these phenotypic effects, the cla4-td allele encoded a protein that was degraded upon shift to nonpermissive temperature . After inducing protein expression with copper and shifting cells to 37°C in the absence of copper, the steady-state levels of the DHFR-Cla4 fusion declined rapidly. In contrast, steady state levels of DHFR-Cla4 remained constant at permissive temperature (23°C) under otherwise identical conditions. All subsequent experiments were performed under basal expression conditions, which appeared to provide normal levels of Cla4 function. To determine whether Ste20 and Cla4 are effectors of Cdc42 in G1, we asked whether loss of Ste20 and Cla4 recapitulates phenotypes of cdc42-1 mutants: arrest as unbudded cells with depolarized actin patches and the continuation of the nuclear division cycle . Because various CLA4 alleles could have different effects, we compared wild-type cells with ste20 Δ mutants that expressed either the cla4-75 temperature-sensitive allele or the novel cla4-td allele. Furthermore, Ste20 and Cla4 may function at several points in the cell cycle. Therefore, a cell synchronization protocol was used to determine whether Cla4 executes an essential function in G1 when Ste20 is absent. Cells were arrested early in G1 by nutrient deprivation at permissive temperature (23°C) , shifted to the nonpermissive temperature (37°C) for 1 or 2 h ( ste20 Δ cla4-td or ste20 Δ cla4-75 mutants, respectively), released from the nutritional block at the nonpermissive temperature, and monitored for the kinetics of bud emergence . Wild-type cells treated in this way recovered from nutritional arrest and resumed budding efficiently. In contrast, the ste20 Δ cla4-td double mutant failed to undergo bud emergence even 22 h after release from the nutritional block. Similar to cdc42-1 mutants, the ste20 Δ cla4-td mutant cells enlarged over time (data not shown), indicating that isotropic growth occurred. The ste20 Δ cla4-td mutant did not lose viability at nonpermissive temperature because cells did not stain with methylene blue, and they resumed budding when returned to permissive temperature (data not shown). These results indicated that Ste20 and Cla4 are required for bud emergence, consistent with the hypothesis that they function as effectors of Cdc42 in G1. In contrast to the unbudded phenotype of the ste20 Δ cla4-td mutant, an isogenic ste20 Δ cla4-75 double mutant was able to bud at nonpermissive temperature with kinetics and efficiency similar to wild-type cells . However, the ste20 Δ cla4-75 mutant formed cells with wide necks, and cell division did not occur (data not shown). This phenotype was identical to the reported terminal phenotype of ste20 Δ cla4-75 double mutants . Because these phenotypes are less severe than those of the ste20 Δ cla4-td mutant, the cla4-td allele appeared to inactivate Cla4 function more completely. To rule out that the requirement of Ste20 and Cla4 for bud emergence was due to the synchronization process, we analyzed the phenotypes of asynchronous cultures shifted to the nonpermissive temperature ( Table ). Cultures of the ste20 Δ cla4-td mutant displayed an increase in the proportion of unbudded cells, consistent with the hypothesis that Ste20 and Cla4 are required for bud emergence. Those of the ste20 Δ cla4-75 mutant accumulated budded cells with a wide-neck morphology. If Ste20 and Cla4 are important effectors of Cdc42 in G1, loss of these PAK homologues should result in other phenotypes characteristic of cdc42-1 mutants: depolarization of the actin cytoskeleton while allowing the nuclear division cycle to proceed. To examine actin polarization, we used cells that were synchronized in G1 by nutritional deprivation, shifted to the nonpermissive temperature, released from the nutritional block at the nonpermissive temperature, and stained at various times with rhodamine-phalloidin . Wild-type cells and the ste20 Δ cla4-75 mutant formed buds and polarized actin patches and cables to the bud. In contrast, the ste20 Δ cla4-td mutant did not bud and never exhibited fully polarized actin patches or cables. However, cortical actin patches and cables were present, indicating that loss of PAK function did not result in wholesale depolymerization of F-actin structures. Similarly, when an asynchronous culture of the ste20 Δ cla4-td mutant was analyzed, the cortical actin cytoskeleton of unbudded as well as budded cells was depolarized ( Table ). To further determine whether actin polarization was defective in the ste20 Δ cla4-td mutant, we asked whether the cortical actin cytoskeleton polarized transiently. Movies were made by acquiring images at 2-min intervals of cells expressing Abp1-GFP, a component of cortical actin patches. During the course of the experiment (80–90 min) there was no evidence of transient polarization of actin patches in the ste20 Δ cla4-td mutant at nonpermissive temperature (data not shown). Furthermore, actin patch motility was unperturbed as indicated by making real time movies of cells expressing Abp1-GFP (data not shown), ruling out a role for Ste20 and Cla4 in this process. Therefore, loss of Ste20 and Cla4 results in a severe loss of actin polarity in G1. To examine the nuclear division cycle, we expressed GFP-tagged tubulin (GFP-Tub1) in wild-type cells and the ste20 Δ cla4-td mutant. Cells were incubated at permissive or nonpermissive temperature, and microtubule morphology in only unbudded cells was scored periodically over 3 h. As expected, unbudded wild-type cells always displayed astral microtubules attached to a single spindle pole body, indicating that the nuclear division cycle had not progressed beyond G1 . In contrast, most (70%) of the unbudded ste20 Δ cla4-td cells analyzed at the nonpermissive temperature had microtubule phenotypes characteristic of nuclear cell cycle progression . Most of these cells had duplicated spindle pole bodies separated by a short spindle, whereas some cells (10%) possessed longer mitotic spindles and separated spindle pole bodies typical of cells in anaphase or telophase. As expected from these results, we also found that the kinetics of nuclear division was delayed in the ste20 Δ cla4-td mutant, as indicated by the rate of appearance of binucleate cells . The delay in nuclear division could be due to a delay in DNA replication or to the activation of a checkpoint, because results that will be presented in a later section indicate that Ste20 and Cla4 do not regulate nuclear division per se. Indeed, a checkpoint activated by morphogenesis or actin cytoskeletal defects early in the cell cycle has been described by Lew, Snyder, and colleagues . Therefore, we conclude that Ste20 and Cla4 are required for bud emergence and actin polarization early in the cell cycle, indicating that PAK homologues are effectors of Cdc42. Because shifting an asynchronous culture of the ste20 Δ cla4-td mutant to the nonpermissive temperature did not arrest cells exclusively with an unbudded phenotype ( Table ), we hypothesized that Ste20 and Cla4 are required for progression through later phases of the cell cycle. To test this hypothesis, we used time-lapse video microscopy of single cells to determine whether ste20 Δ cla4-td cells that have budded (S/G2 phase) can complete bud growth and divide at the nonpermissive temperature . Wild-type and mutant cells were shifted to 37°C for 2 h, mounted on media-containing agar pads, and maintained at nonpermissive temperature. Cells with small or medium-sized buds were identified, and images were acquired at 5-min intervals for 2 h. In all wild-type cells examined (6/6), growth was polarized or asymmetric, because buds grew whereas mothers did not, resulting in a decrease in the ratio of mother to bud volume over time . Subsequently, wild-type cells divided, completing a round of cell division over the course of the experiment. In striking contrast, the ste20 Δ cla4-td mutant displayed a profound defect in polarized growth of the bud (7/7 cells examined). Bud growth was much slower than in wild-type cells because both the mother and bud increased in volume, resulting in a constant ratio of mother to bud volume , as expected for completely isotropic or depolarized growth. Consistent with a depolarized growth phenotype, cortical actin patches in small budded cells were depolarized (distributed equally between mother and bud) in the ste20 Δ cla4-td mutant ( Table ). Therefore, Cla4 and Ste20 are required for polarized growth of the bud. Polarization of the actin cytoskeleton to the mother-bud neck and the formation and contraction of an actin ring accompany cell division. Therefore, we determined whether Ste20 and Cla4 are required for these processes. Wild-type or ste20 Δ cla4-td mutant cells were grown at permissive temperature to allow bud emergence to occur. Cells were arrested at permissive temperature early in mitosis by depolymerizing microtubules with nocodazole. Arrested cells were shifted to the nonpermissive temperature for 1 h to inactivate Cla4, released from the nocodazole block at the nonpermissive temperature, and scored for the ability to divide as indicated by decreases in the proportion of cells exhibiting a large budded morphology . Whereas wild-type cells divided when released from the nocodazole block, the ste20 Δ cla4-td mutant showed a strong block or delay in cell division. To confirm that cell division had not occurred, we used cell wall digestion to distinguish cells that had failed to divide from cells that had divided but failed to separate. This treatment resulted in only a slight decrease in the number of large budded cells (data not shown), indicating that the ste20 Δ cla4-td mutant was defective in the completion of mitosis or cytokinesis rather than cell separation. The cell division defect was accompanied by the failure to polarize actin patches to the mother-bud neck . However, we found that nuclear division was unaffected, as revealed by DAPI staining (in wild-type and ste20 Δ cla4-td mutants, binucleate cells appeared within 30 min after release from the nocodazole block; data not shown). Because this result indicated that Ste20 and Cla4 are not required for nuclear division, the delay in nuclear division in unbudded ste20 Δ cla4-td cells described in a previous section is probably due to the activation of a checkpoint. Therefore, we conclude that Cla4 is required late in the cell cycle to promote actin patch polarization to the neck and the completion of cytokinesis. This is consistent with the observation that Cla4 kinase activity peaks near mitosis . Previous studies have shown that ste20 Δ cla4-75 mutants arrest growth as large budded cells with wide necks separating the mother and bud, resulting in a cytokinesis defect. However, it had not been determined whether this defect is a direct consequence of a failure to execute events at the time of mitosis or cytokinesis, or an indirect consequence of the failure to form a normal neck early in the cell cycle that results in arrest in mitosis. An indirect block could occur due to a structural defect in the neck that prevents cytokinesis, or to the activation of a checkpoint. We tested these hypotheses by subjecting the ste20 Δ cla4-75 mutant to the same nocodazole block and release protocol used above to analyze the ste20 Δ cla4-td mutant. After release of the mitotic block at nonpermissive temperature, the ste20 Δ cla4-75 mutant underwent cell division at a wild-type rate . Therefore, the ste20 Δ cla4-75 mutant was able to execute functions required at the time of cell division, provided that a proper bud neck had formed. This provided a further indication that the cla4-75 mutation partially inactivates Cla4 function. Therefore, we suggest that the previously reported cell division defect of the ste20 Δ cla4-75 mutant results from a structural defect in the mother-bud neck or from the activation of a checkpoint in response to the abnormal neck formed early in the cell cycle. Septins ( CDC3 , CDC10 , CDC11 , and CDC12 gene products) are assembled in G1 into a filament network that forms a double ring structure or collar at the mother-bud neck . Previous studies have indicated that septins are severely mislocalized in ste20 Δ cla4-75 double mutants, possibly explaining the wide-neck morphology and cytokinesis defect of these cells . To address this question further, we compared septin localization in wild-type cells, and in ste20 Δ mutants that carried a cla4 - 75 or cla4-td allele. In contrast to previous reports, we found that septins were localized to the mother-bud neck in the ste20 Δ cla4-75 mutant at the nonpermissive temperature . Only when cells were grown in rich media at nonpermissive temperature was a septin localization defect observed. However, even under these conditions septins localized to the mother-bud neck in 93% of the ste20 Δ cla4-75 cells, although the staining pattern was not as sharp as in wild-type cells, suggesting a mild defect in septin organization. The remaining 7% of the cells mislocalized septins to the bud tip, similar to previous reports. Regardless of the septin localization phenotype, the ste20 Δ cla4-75 mutant always displayed a wide-neck morphology, indicating that severe mislocalization of septins is not required to observe the wide-neck phenotype. Furthermore, septin localization was relatively normal in the ste20 Δ cla4-td mutant at the nonpermissive temperature, suggesting that Ste20 and Cla4 are not essential for septin stability. Other evidence also suggests that the wide-neck morphology and cytokinesis defect of ste20 Δ cla4-75 mutants are not due entirely to loss of septin function or assembly. First, septin mutants have narrower necks than ste20 Δ cla4-75 mutants . Second, unlike ste20 Δ cla4-75 double mutants, septin-null mutants can form microcolonies containing 80 or more cells, suggesting that several rounds of cell division occur in the absence of septins (M. Longtine, personal communication). Third, we never observed a septin localization defect as severe as that in mutants lacking Nim1-homologous kinases (Gin4, Hsl1, and Kcc4) , which localize to the septin ring and control its stability. Although our results indicated that Ste20 and Cla4 are relatively unimportant to maintain the stability of septins, these kinases could facilitate septin assembly in G1. To address this possibility, we used nutritional arrest to enrich for G1 wild-type and ste20 Δ cla4-td mutant cells at permissive temperature (23°C). Cells were shifted to the nonpermissive temperature (37°C), immediately released from the nutritional block, and analyzed for septin localization over time. The ste20 Δ cla4-td mutant displayed a significant defect in septin assembly . Therefore, it appears that Cla4 facilitates septin assembly rather than stability. The preceding results suggest that in the absence of Ste20, Cla4 is required for cell and actin polarization throughout most or all of the cell cycle, and that Cla4 facilitates septin assembly. If Cla4 directly regulates cell and actin polarization, it should be localized to sites of polarized growth independently of an intact actin cytoskeleton. Similarly, if Cla4 regulates septin localization, it may associate with the neck or neck filaments, as do Nim1-related kinases that regulate septin stability . To test these predictions, we tagged the chromosomal CLA4 locus at the 3′ end of its ORF with the GFP coding region, resulting in the production of a functional Cla4-GFP fusion protein. Cla4-GFP localization was examined under conditions where the actin cytoskeleton was intact or depolymerized . In cells with small buds, Cla4-GFP was concentrated in a crescent at the tips and sides of the bud. In cells with medium-sized buds, Cla4-GFP was detected as dots or patches distributed over the cortex of the bud. At wild-type expression levels, Cla4-GFP was undetectable in unbudded cells or in the bud at later points in the cell cycle, and it was never observed in the mother. Cla4-GFP was not observed to localize to neck filaments or the mother-bud neck, which is consistent with the ability of Cla4 to facilitate the assembly rather than the stability of septins. Cla4-GFP localization to the tips of small buds or the cortex of medium buds was not disrupted when latrunculin was used to depolymerize the actin cytoskeleton. This result is in contrast to the actin-dependent polarized localization of proteins such as Myo2 and Sec4 that are thought to function downstream of actin by delivering secretory vesicles to sites of polarized growth . Therefore, throughout much of the cell cycle Cla4-GFP localizes to sites of polarized cell growth. Interestingly, Cla4-GFP also localized to the tips of polarized cell surface projections formed when cells were stimulated with mating pheromone . Whether this suggests a previously unappreciated role for Cla4 in mating is unknown, but it reinforces the conclusion that Cla4-GFP becomes concentrated to sites of polarized cell growth. Taken together, these results and those of previous studies indicate that Cdc42, Ste20, and Cla4 function in a pathway that transmits signals that polarize the actin cytoskeleton to promote bud emergence and polarized cell surface growth. Throughout much of the cell cycle the actin cytoskeleton of budding yeast is highly polarized, mediating bud emergence, polarized bud growth, and efficient cytokinesis. Here we have shown that cell and actin cytoskeletal polarization requires the PAK homologues Ste20 and Cla4. In cells lacking Ste20, inactivation of Cla4 at various points in the cell cycle reveals that these PAK homologues are required for polarization of the actin cytoskeleton and cell growth during bud emergence, polarized growth of the bud with respect to the mother, and completion of cytokinesis. To our knowledge, Ste20 and Cla4 are the first signaling molecules that have been shown to be required for cell and actin polarization throughout much or all of the yeast cell cycle. Thus, PAK-family kinases appear to be primary regulators of yeast cell polarity. We have found that Ste20 or Cla4 is required to polarize the actin cytoskeleton and initiate bud emergence. Whereas mutants lacking either kinase can carry out these processes, loss of Ste20 and Cla4 blocks these events, displaying phenotypes like those of cdc42-1 mutants . Because results presented here and elsewhere indicate that Cla4 and Ste20 interact and colocalize with Cdc42 at sites of polarized growth , these PAK homologues function as direct signaling effectors of Cdc42 in pathways that promote bud emergence and actin polarization in G1. In contrast, Ste20 and Cla4 are not required for isotropic growth or progression of the nuclear division cycle, indicating that they have primary roles in cell and actin polarization. Several observations indicate that Ste20 and Cla4 promote bud emergence by executing functions that are at least partially distinct from those carried out by the Cdc42 binding proteins Gic1 and Gic2. Whereas ste20 Δ cla4 Δ double mutants are inviable, gic1 Δ gic2 Δ double mutants are viable, except at elevated temperature where they display bud emergence and growth defects . Furthermore, gic1 Δ gic2 Δ double mutants are nonconditionally inviable when CLA4 is also deleted . Finally, overexpression of STE20 or CLA4 suppresses the conditional growth defect of gic1 Δ gic2 Δ double mutants . Although Ste20 and Cla4 clearly do not require Gic1 or Gic2 to promote bud emergence, there could be functional interactions between these two classes of Cdc42 effectors. For example, PAKs and Gic1/2 could regulate each other's localization, stability, activity, interaction with Cdc42, or association with downstream targets. However, Ste20 and Cla4 may have unique targets, possibly including Bem1. Bem1 is involved in bud emergence, localizes to sites of polarized growth, associates with Ste20 immunoprecipitates, and is phosphorylated in vivo . By what mechanisms do Ste20 and Cla4 promote bud emergence? Evidence suggests that these PAK homologues may trigger bud emergence at least in part by stimulating actin filament assembly at the site of bud emergence. In unpolarized cells early in G1, F-actin assembly occurs independently of Cdc42 and its effectors because mutations that inactivate Cdc42 or its effectors do not lead to wholesale depolymerization of F-actin structures . Recruitment of Cdc42 and its effectors, including Ste20 and Cla4, to the presumptive bud site may stimulate the rate of actin filament assembly locally, because activated forms of purified Cdc42 or Ste20 stimulate actin filament assembly in an in vitro assay using permeabilized cdc42-1 cells . Furthermore, permeabilized cla4 Δ or ste20 Δ mutants show defects in this in vitro assay . Stimulation of actin assembly at the bud site could bias the accumulation of new patches and cables to this location at the expense of distal sites on the cell cortex. Several observations are consistent with this hypothesis. Actin monomers dissociate rapidly from patches and cables . The number of patches or total filamentous actin does not increase in polarized cells . A relatively small, invariant pool of actin monomer is present throughout the cell cycle , and inactivation of Ste20 and Cla4 does not affect the relative levels of F- and G-actin (our unpublished results). Cdc42 and its effectors may stimulate localized actin assembly and cell polarization by targeting the Arp2/3 complex. Indeed, an arp3 temperature-sensitive mutant loses actin polarity at the nonpermissive temperature . In vertebrates, the Arp2/3 complex is required for Cdc42 to stimulate actin assembly in cell free extracts . Moreover, Cdc42 and PIP 2 synergistically activate N-WASP, a mammalian protein that stimulates actin filament assembly by the Arp2/3 complex . However, the yeast WASP homologue (Bee1/Las17) may not be a direct target of Cdc42. Bee1 lacks a consensus Cdc42 binding domain and a bee1 Δ mutant does not display a defect in bud emergence . Instead, bee1 Δ mutants are partially defective in bud growth and cytokinesis, accumulate secretory vesicles, and have a disorganized actin cytoskeleton . Accordingly, Cdc42 may use Bee1-dependent and -independent mechanisms to stimulate actin assembly or polarization. The polarization complex organized by Cdc42 in yeast could also function by capturing actin cables and patches, tethering them near the incipient bud site. However, tethering of the actin cytoskeleton to the polarization complex appears to be flexible and of relatively low affinity. This hypothesis is based on observations that polarized actin patches move at rates similar to unpolarized actin patches , and that polarized patches can move away from or escape the presumptive bud site . Tethering molecules that link actin cables and patches to the polarization complex remain to be identified. Cdc42 and its effectors may promote polarization and bud emergence by executing functions in addition to polarizing the actin cytoskeleton. We suggest this because bud emergence can occur in mutants (such as tor2 ) that apparently have lost actin polarization in G1. In tor2 mutants, Cdc42 and it effectors may promote bud emergence by facilitating the docking or fusion of secretory vesicles delivered by actin patches and cables that localize by chance to the incipient bud site. This would be consistent with the essential role of F-actin in bud emergence . In this regard, two myosin I homologues (Myo3 and Myo5) have been proposed to be targets of Ste20 and Cla4. Mutants lacking Myo3 and Myo5 have a disorganized actin cytoskeleton and accumulate intracellular vesicles . Furthermore, sites in Myo3 and Myo5 that are phosphorylated by Ste20 in vitro are required for function in vivo . However, although myo3 Δ myo5 Δ double mutants have severe growth defects, they can carry out bud emergence , indicating that there are other targets of Ste20 and Cla4 involved in this process. The level of Cla4 activity appears to be critical for cells to coordinate the processes of bud emergence and neck morphogenesis. We suggest this because incubation of ste20 Δ cla4-75 double mutants at restrictive temperature allows actin polarization to the incipient bud site and subsequent bud emergence to occur, but results in the formation of cells with abnormally wide necks. In contrast, more complete inactivation of Cla4, as occurs in the ste20 Δ cla4-td double mutant, completely blocks bud emergence. Cla4 may coordinate neck morphogenesis and bud emergence by regulating septin and actin organization. Whereas our results indicate that Cla4 facilitates septin assembly during G1, it is less clear whether Cla4 also stabilizes septins once they are assembled. On the one hand, Cla4 may be involved in septin stabilization because it activates Gin4, one of three Nim1-homologous kinases that have been suggested to maintain neck filament organization after bud emergence . However, we find that inactivation of Cla4 in a ste20 Δ mutant does not lead to wholesale disassembly of septins. Perhaps in the absence of Cla4, other kinases activate Nim1 kinases and promote septin stability. Neck morphogenesis may also be regulated by the ability of Cla4 to polarize the actin cytoskeleton. We suggest this because incomplete loss of Cla4 function (in ste20 Δ cla4-75 mutants) causes a partial defect in actin polarization (less tightly clustered actin patches) (Holly, S.P., unpublished data). This in turn could result in the delivery of vesicles over a larger region of the cell cortex and the formation of a wide neck. Previous studies have suggested that Cla4 is required to inhibit apical growth of the bud, resulting in a switch to isotropic bud growth during mitosis. This hypothesis is based on the finding that cla4 Δ mutants are hyperpolarized and have long buds, suggesting that the switch to isotropic bud growth is delayed. Cla4 has been proposed to promote the apical to isotropic switch by activating Gin4 and possibly other Nim1 homologues, which in turn repress apical bud growth . Our results indicate that Cla4 is also required for isotropic growth of the bud. In the absence of Ste20, inactivation of Cla4 in G2/M leads to a complete loss of polarization, allowing both the mother and bud to grow. Cla4 may promote isotropic growth of the bud by directing the localization or assembly of actin patches over the bud cortex. Indeed, we find that Cla4-GFP localizes in a punctate pattern over the entire cortex of the bud in G2/M, while being undetectable in the mother. Furthermore, loss of Ste20 and Cla4 in budded cells results in the distribution of actin patches over the cortex of the bud and the mother. By analyzing synchronized cells lacking Ste20, we have found that loss of Cla4 early in mitosis blocks cell division. Because nuclear division is unaffected, Ste20 and Cla4 are likely to promote cell division directly. Indeed, we find that inactivation of Ste20 and Cla4 is accompanied by a failure to polarize actin patches to the neck before cytokinesis. Potentially, Ste20 and Cla4 could also regulate the formation or contraction of the actin ring at the mother-bud neck, which involves myosin II ( MYO1 gene product) and an IQ-GAP homologue ( IQG1/CYK1 gene product) . In conclusion, because PAK-family kinases in yeast are required for cell polarization throughout much or all of the cell cycle, members of this family of protein kinases are likely to be critical regulators of cell polarization and actin organization in other eukaryotes. Although many functions of mammalian PAKs remain to be established, there is a variety of evidence linking them to signaling pathways that regulate the organization of the actin cytoskeleton . Defining the mechanisms whereby yeast and mammalian PAKs regulate cell polarization and actin organization will require the identification of specific substrates. Based on our studies of Ste20 and Cla4, PAKs are likely to have several targets that regulate cell polarization during various steps in the cell cycle and in response to extracellular signals. | Study | biomedical | en | 0.999995 |
10562286 | Preparations of sheep and pig brain microtubule proteins obtained by two cycles of assembly and disassembly were used to isolate γ-tubulin complexes by immunoaffinity chromatography . γ-Tubulin complexes obtained from pig brain were used in all experiments involving mammalian complexes. Proteins from pig brain γ-tubulin complexes were separated by one dimensional gel electrophoresis under denaturing conditions, blotted onto PVDF membrane (Problott, Perkin-Elmer), and visualized by Ponceau S (Sigma Chemical Co.). The sequence analysis of the 76-kD band, carried out using a gas–liquid sequencer (model 467A; Applied Biosystems), allowed the determination of the first 20 amino acids of a single protein. In addition, the 76-kD band was excised from Coomassie blue–stained gels, digested by porcine trypsin, and the resulting peptides were purified and sequenced as described . The first 20–amino-terminal amino acids of the 75-kD band from pig γ-tubulin complexes were encoded by a human expressed sequence tag (EST) (AA115 396). The 5′ and 3′ sequences of this clone were used to obtain a full-length cDNA from a human neuroblastoma bank using a PCR approach. This cDNA was sequenced (big dye terminator on 373 DNA sequencer with internal nucleotides). The deduced 2-kb open reading frame (ORF) showed perfect matches with the peptide sequences obtained from purified 75- kD band. The Drosophila EST that showed a significant similarity to h76p, allowed by genome walking the successive identification of one genomic and three additional EST clones coding for the Drosophila orthologue (d75p) of h76p. Sequences obtained from the previously identified EST and genomic clones, together with additional sequences experimentally obtained from the AA540317 (full-length) and AA694820 cDNA clones, allowed us to generate a full-length coding sequence for d75p. The gene coding for the d75p contains seven introns and eight exons . The full-length cDNA clone from Medicago was isolated by library screening using a radioactive probe corresponding to the 850-bp EcoRI insert fragment of the EST 660560 that encoded a polypeptide showing high homology with the h76p and d75p proteins. The probe was hybridized against 8 × 10 5 plaque-forming units of a Medicago truncatula root tip cDNA library (Dr. A. Niebl and Dr. P. Gamas, LBRPM, UMR CNRS 215, Castanet-Tolosan, France). Phage plaques were plated onto nitrocellulose membranes (Optitran BA-S-85; Schleicher & Schuell, Inc.) according to the manufacturer's instructions. Prehybridization, hybridization, and washings were performed according to Sambrook et al. 1989 . 30 positive clones were detected after initial screening. Four clones containing a presumably full-length 2.8-kb cDNA insert were retained for further characterization and one of them was sequenced. The 2-kb ORF of the h76p was amplified by PCR using the 5′ primer, 5′ GCGCGCGAGCTCATCCACGAACTGCTCTTGGCT 3′, and the 3′ primer, 5′ GCGCGCAAGCTTTCACATCCCGAAACTGCCCAG 3′. The PCR fragment was digested with the restriction enzymes SacI and HindIII and inserted into the plasmid pQE30 (Qiagen) resulting in a plasmid (pQE30-p76) coding for a h76p tagged with 6-His at the amino terminus. The bacterial vector pQE30-p76 was introduced in the M15(pREP4) Escherichia coli host strain and protein expression was induced by 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 4 h at 37°C. The insoluble fusion protein was purified either as inclusion bodies and used for the purification of antibodies, or solubilized in 8 M urea, purified on a nickel agarose column (QIAexpressionist; Qiagen) and used for immunizations and quantifications. Alternatively, after induction by 0.1 mM IPTG for 3 h at 25°C, and centrifugation of the bacterial extract at 100,000 g for 45 min, soluble h76p was recovered in the supernatant where it represented ≈10% of the proteins. Buffer exchange was performed through a prepacked Sephadex G-25M column (Pharmacia). The 2-kb h76p-ORF was amplified by PCR using the 5′ primer, 5′ GCGCGCAAGCTTATCCACGAACTGCTCTTGGCT 3′, and the 3′ primer, 5′ GCGCGCAAGCTTTCACATCCCGAAACTGCCCAG 3′. The PCR product was digested with the restriction enzyme HindIII and inserted into the plasmid pEGFP-C3 (CLONTECH Laboratories). The resulting plasmid (pEGFP-C3-p76) expressed the h76p with the enhanced green fluorescent protein coding sequence fused at the amino terminus (GFP-h76p). Monkey kidney COS cells, cultured in DME (GIBCO BRL) with 7% FBS (Sigma Chemical Co.) in plastic flaskets (9 cm 2 ) were transfected (50% efficiency) with 1 μg of plasmid pEGFP-C3-p76 using the DEAE dextran/chloroquine procedure . COS cells overexpressing the GFP-h76p (103 kD) exhibited a single polypeptide of apparent molecular mass of ≈100 kD, recognized by anti–h76p and anti–GFP antibodies . Comparison of the immunolabeling with a standard curve raised with recombinant h76p showed that the GFP-h76p represented ≈0.4% of total proteins , whereas the endogenous 76p remained below the detection limit, i.e., <0.003% of total proteins in control COS cells. Since the percentage of transfection was ≈50%, transfection resulted in an average expression ≈200-fold above the basal level. Alternatively, HeLa cells were transfected using the calcium phosphate procedure and observed 65 h after transfection. Rabbit antibodies recognizing h76p were raised against h76p overexpressed in E . coli and against three synthetic peptides linked to thyroglobulin (Sigma Chemical Co.) as described . The antibodies R72 were obtained against the peptide KQLRELQSLRLIEEEN corresponding to the region 233–248 of h76p, the antibodies R801 against the peptide RQEDTFAAELHRGGC that contains 10 amino acids corresponding to the region 304–313 of h76p, and the antibodies R190 against the peptide YNKYYTQAGGTLGSFG corresponding to the carboxy-terminal region 651–666 of h76p. Antibodies specific to h76p were purified on recombinant h76p as described . Specificity was checked by preincubation with the immunizing peptide (50 μg per ml). Antibodies against γ-tubulin were raised, either in rabbits (R74, R75) or in guinea pigs (C3), against a 19–amino acid peptide corresponding to the carboxy-terminal region of human γ-tubulin and purified on recombinant γ-tubulin . The antibodies R82, raised in rabbits against the carboxy-terminal region of Arabidopsis γ-tubulin (CVDEYKASESPDYIKWG), do not cross-react with vertebrate γ-tubulin. A rat mAb (YL1/2) directed against α-tubulin was used to reveal the α/β-tubulin heterodimer. mAbs against neurofilament proteins were obtained from Dr. D. Paulin (Institut Pasteur, France). Anti–GFP antibodies were purchased from CLONTECH Laboratories. Affinity-purified antibodies were used in all experiments involving immunoblotting or immunofluorescence labeling. The immunolocalization of h76p in PtK2 cells was performed after a 6-min fixation in cold (−20°C) methanol. Double immunofluorescence staining of h76p and γ-tubulin was performed with rabbit antibodies (R801) and guinea pig antibodies (C3), respectively. Staining of γ-tubulin (R75) or microtubules (YL1/2) in COS cells overexpressing fluorescent GFP-h76p was performed after a 1-min permeabilization in a microtubule stabilizing medium and fixation in 3.7% formaldehyde . The staining was revealed with FITC and TRITC goat antibodies (Immunotech), whereas nuclei and chromosomes were stained with 4′,6-diamidino-2-phenylindole (DAPI: 0.2 μg per ml). Preparations were dried, mounted in Mowiol , sealed, and observed by epifluorescence with a Zeiss Axiophot microscope equipped with 40× (NA 1.30) plan-neofluar objectives, a 2× Optovar, a 4× TV camera, and a stabilized excitation beam. Images, recorded in the linear dynamic range by a Nocticon camera , were digitized (100 frames averaging) with an image processing system (Sapphire from Quantel) and recalculated using a linear function (Stretch program). The specific maximal fluorescence raised by h76p antibodies over the centrosome was determined (Luminance program) as the difference between the maximal fluorescence of the centrosome and the background fluorescence measured at the immediate proximity of the centrosome. Extracts were prepared using Xenopus oocytes arrested in the metaphase of the second meiotic division . Beads of protein A–Sepharose 4B (Pharmacia Biotech Sverige) carrying h76p (R801) and γ-tubulin (R74) rabbit antibodies were used for depletion experiments. Schematically, 50 μl of protein A–Sepharose beads were saturated for 2 h at 4°C with 2 ml of a solution of casein at the concentration of 5 mg/ml. They were incubated overnight at 4°C with 150 μl of rabbit serum (R801, R74) diluted fourfold in PBS, and washed successively in PBS, PBS with 0.1% Triton X-100, PBS, and acetate buffer (100 mM potassium acetate, 2.5 mM magnesium acetate, pH 7.2). The oocyte extract (50 μl, 60–80 mg/ml), diluted with 1 vol of acetate buffer containing an ATP-regenerating system and protease inhibitors, was centrifuged for 5 min at 10,000 g , mixed with the protein A–Sepharose beads, and incubated for 1 h at 4°C. After bead removal by centrifugation, the supernatant was used for microtubule assembly in the presence of permeabilized Xenopus spermatozoa . Probing the supernatant of oocyte extracts treated with beads carrying 76p (R801) antibodies, showed that these beads effectively removed x76p from extracts diluted to 12 mg/ml, but only partially depleted extracts diluted to 25 mg/ml. The nucleation experiment was performed at 22°C by incubating 15,000 sperm nuclei (1 μl) in 20 μl of oocyte extract (25 mg/ml) and 0.5 μl of rhodamine-labeled tubulin . At 5-min intervals, 2-μl samples mixed with 6 μl of Hoechst 33258 (1 μg/ml) were observed by fluorescence microscopy, and the percentage of sperm heads with an aster was determined. A sperm aster was defined by the nucleation of 15–60 microtubules at the extremity of the sperm nucleus . Alternatively, 75,000 permeabilized spermatozoa (5 μl) were incubated with 5 μM nocodazole for 20 min in 50 μl of oocyte extract, and then fixed with 200 μl of 3.7% paraformaldehyde in 80 mM Pipes pH 6.8, 1 mM EGTA, 1 mM MgCl 2 . Immediately, 50-μl samples were centrifuged at 200 g for 5 min onto a coverslip (12-mm-diam) pretreated with a solution of 0.01% poly- l -lysine (Sigma Chemical Co.), postfixed for 6 min with cold methanol (−20°C), and processed for immunolabeling. The recruitment of 76p on permeabilized spermatozoa was quantified in the same conditions as before in the presence of 5 μM nocodazole. The spermatozoa were centrifuged through a cushion of 0.6 ml 25% glycerol in Pipes buffer for 5 min at 5,700 g before processing for SDS-PAGE. γ-Tubulin complexes were purified from mammalian brain by affinity chromatography with γ-tubulin antibodies . Besides γ-tubulin and the α/β-tubulin heterodimer, the γ-tubulin protein complexes obtained from pig and sheep brain contain four polypeptide bands . The band migrating at 76 kD in pig brain γ-tubulin complexes was isolated by SDS-PAGE, blotted and submitted to microsequencing. The terminal amino acid sequence xIHELLLALSGYPGSIFTxN was identified in a putative ORF at the 5′ end of a human EST . The corresponding cDNA was obtained by PCR from a human neuroblastoma cDNA library and entirely sequenced. It contains a single ORF (h76p = hGCP4) coding for a polypeptide of 667 amino acids with a calculated mass of 76,090 and a pI of 6.14 . This ORF also contains the two sequences, xxEDTFAAExHR and xEILPTY, previously obtained from the tryptic digests of the 76-kD polypeptides from pig (sequence 1) and sheep (sequences 1 and 2). Searches in databases also identified ESTs coding for apparented proteins in evolutionary distant organisms such as Drosophila , the moss Physcomytrella , and the higher plant Medicago . A full-length cDNA sequence was first obtained for the Drosophila orthologue. The corresponding Drosophila gene contains eight exons coding for a protein of 650 amino acids (d75p). This protein exhibits ≈33% sequence identity with h76p, and shows comparable calculated mass (74,900) and pI (5.12). The presence of an orthologue of h76p in Medicago (m85p) was confirmed by the isolation and sequencing of the corresponding full length cDNA. It codes for a protein of 739 amino acids, with a calculated mass of 85,300 (m85p) and a pI of 6.23. m85p exhibits ≈34 and 31% sequence identity with its human and Drosophila counterparts, respectively. Several common amino acid motifs were identified between the three protein orthologues, particularly the conserved amino-terminal sequence . The identity of the h76p with the major protein of the 76-kD band of pig brain γ-tubulin complexes was further assessed by antibodies raised against recombinant h76p and several peptide motifs of h76p: 233–248 (R72), 303–313 (R801), and the carboxy-terminal amino acids 651–666 (R190) . The labeling specificity was checked by competition with the immunizing peptides. Moreover, h76p antibodies (R801) specifically immunoprecipitated γ-tubulin from pig brain γ-tubulin complexes in agreement with the presence of both proteins in the same complexes . The 76p was specifically recognized by R190 antibodies in mammalian cell extracts (PtK2 cells) in which it represents, similarly to γ-tubulin, ≈0.02% wt/wt of the cellular proteins . Antibodies R801 and R190 specifically labeled a single band at 76 kD in oocyte extracts (x76p), suggesting that the sequence of this protein was conserved in vertebrates. Moreover, x76p and γ-tubulin were present in similar amounts and represented ≈0.02% wt/wt of the proteins of the oocyte extract . Since h76p was associated to γ-tubulin in mammalian protein complexes, it could be assumed that it was present in the fairly homogeneous γ-TuRCs from Xenopus oocytes . An oocyte extract was sedimented on a sucrose gradient and both 76p and γ-tubulin were revealed by immunoblotting. The distribution of x76p (R801 antibodies) was coincidental with the peak of γ-tubulin (C3 antibodies). Both proteins were exclusively recovered in complexes larger than 19 S corresponding to the γ-TuRCs as defined by Zheng et al. 1995 . Immunoprecipitation of the fractions of the sucrose gradient corresponding to the γ-TuRCs by h76p antibodies (R190) and immunoblotting with γ-tubulin antibodies (C3) further demonstrated that both proteins were present in the same complexes . However, this experiment did not exclude the possibility that a fraction of x76p assembled in complexes devoid of γ-tubulin and cosedimented with the γ-TuRCs. To check this possibility, γ-tubulin from an oocyte extract (7 mg/ml) was immunoprecipitated with rabbit antibodies (R74). Despite the inability to analyze the immunoprecipitate with rabbit 76p antibodies, the experiment showed the disappearance of both γ-tubulin (C3 antibodies) and x76p (R190 antibodies) from the supernatant (not shown). Hence, in oocyte extracts, most γ-tubulin and x76p are expected to be present in γ-TuRCs in an apparent ratio of ≈1 . In contrast, stoichiometry determinations based on Coomassie staining suggested that the 76-kD polypeptide band corresponded to a minor protein both in Xenopus and mammalian brain purified γ-tubulin complexes . Using defined amounts of γ-tubulin complexes and recombinant h76p as an internal standard, we confirmed that 76p was approximately fivefold less abundant than γ-tubulin in preparations of pig brain γ-tubulin complexes . Loss of 76p in some γ-tubulin complexes and/or selective loss of some γ-tubulin complexes during purification is a likely possibility. To assess whether all purified γ-tubulin complexes contained h76p, mammalian γ-tubulin complexes were sedimented and the resuspended pellet was repeatedly subjected to immunoprecipitation either with R801 or with R190 antibodies. In each case, the first cycle of 76p depletion removed some γ-tubulin from the preparation, but further cycles failed to strongly deplete the supernatant from γ-tubulin . However, a similar result was obtained with the native γ-TuRCs directly immunoprecipitated from a crude Xenopus oocyte extract . Hence, both purified and native γ-tubulin complexes are likely to be heterogeneous. The inaccessibility of the 76p in some γ-tubulin complexes could account for these observations although two antibodies raised against distinct regions of 76p were used. Alternatively, the average stoichiometry of 76p in γ-TuRCs could be different from the actual stoichiometry in individual γ-TuRC. Most of the purified γ-tubulin complexes bind to microtubule minus extremities . Since 76p is present in a fraction of γ-tubulin complexes, we investigated whether 76p could bind to microtubules. After incubation of pig brain γ-tubulin complexes (≈100 ng of γ-tubulin) with taxol-stabilized microtubules (450 μg), free and bound γ-tubulin complexes were readily separated by differential sedimentation (at 62,000 g for 5 min) and analyzed by SDS-PAGE and immunoblotting with γ-tubulin (C3) and 76p (R801) antibodies . At 0 and 37°C in the absence of tubulin, the γ-tubulin complexes failed to sediment and both 76p and γ-tubulin remained in the supernatant. The same result was observed when γ-tubulin complexes were mixed with pure tubulin unable to assemble both at 0 and 37°C. In contrast, a definite amount of 76p and γ-tubulin sedimented in the presence of microtubules assembled in the presence of taxol (not shown). These observations suggested the following: (1) 76p could bind directly or indirectly to microtubules; and (2) 76p neither acts as an inhibitor of the fixation of γ-tubulin to microtubules nor is released from the complexes during their binding to microtubule. The centrosomal localization of 76p was assessed by immunofluorescence staining with three polyclonal antibodies (R801, R190, and R629/30) using cold methanol-fixed PtK2 cells. In all cases, 76p colocalized with γ-tubulin to the centrosome, which appeared as a diplosome during interphase , and to the spindle poles at the different stages of mitosis . Both in interphase and mitosis, the labeling raised by h76p antibodies was specific as shown by the absence of staining when the antibodies were preincubated with the immunizing peptide . Observation of the immunofluorescent figures showed that the amount of 76p to the centrosome transiently increased during mitosis. The difficulty to exactly determine centrosome limits prevented the accurate determination of the overall fluorescence (average fluorescence × area), and led us to choose the maximal centrosomal fluorescence as a quantitative parameter . The maximal fluorescence, which did not vary significantly in interphase, increased ≈6-fold from prophase to prometaphase/metaphase and decreased thereafter to its interphase level in late telophase. A similar variation has been previously reported for γ-tubulin . As was the case for γ-tubulin , h103p and h104p , treatment with colcemid (2 μM for 2 h) did not modify the presence of 76p in the interphase centrosome and its recruitment during mitosis . As for γ-tubulin , 76p was not only a centrosomal protein, but also relocalized during mitosis. Antibodies R190 directed against the carboxy-terminal region of h76p showed that some 76p was present in the metaphase spindle (not shown), although this localization was not observed with antibodies R801 , in the midzone between the two separating chromosomal masses in anaphase and in telophase as previously observed with γ-tubulin . In contrast to γ-tubulin and h104p , which are transiently present in the two regions corresponding to the minus ends of the microtubules constituting the midbody , in no case the various antibodies against h76p revealed the presence of this protein . The subcellular localization of 76p was observed in methanol-fixed cells, but no specific immunostaining was obtained using other fixation procedures. Neither formaldehyde- and glutaraldehyde-fixed PtK2 cells nor permeabilized cells with and without formaldehyde fixation showed a centrosomal staining when probed with the different h76p antibodies, whereas in all cases γ-tubulin antibodies decorated the centrosome. To confirm the centrosomal localization of h76p, a fusion protein between GFP and h76p (GFP-h76p) was transiently overexpressed in COS cells. The 103-kD fusion protein was specifically detected in protein extracts from transfected cells by immunoblotting with both h76p (R801) and GFP antibodies . Since 50% of the cells were transfected, the average overexpression was ≈200-fold as judged with a range of recombinant h76p. In interphase cells, the GFP-h76p was present at the centrosome where it colocalized with γ-tubulin , whereas in mitotic cells the GFP-h76p localized at the spindle poles and was absent from the mitotic spindle . Hence, the presence of the GFP moiety at the amino terminus of h76p did not modify its localization to the interphase and mitotic centrosomes. Since the transfection method used in PtK2 cells induced multipolar mitoses , we repeated these experiments applying another method to HeLa cells. Overexpression of h76p in HeLa cells (not shown) confirmed its immunolocalization and also failed to induce evident modifications of the microtubule cytoskeleton morphology. However, 72 h after transfection, 80% of HeLa cells expressing GFP-h76p exhibited a nuclear fragmentation characteristic of apoptosis , whereas only 12% were observed in cells expressing GFP. This suggests that h76p overexpression could be deleterious to cells by analogy with toxic effects due to Spc97p and Spc98p overexpression in yeast . It has been previously demonstrated that the γ-TuRCs present in Xenopus oocyte extracts are recruited by sperm basal bodies, where they are necessary for the nucleation of microtubule asters . In agreement with its centrosomal localization, immunofluorescence staining demonstrated that 76p was present in the basal bodies of permeabilized Xenopus spermatozoa incubated in an oocyte extract . The 76p was present at the extremities of the elongated sperm nuclei where it colocalized with γ-tubulin. The function of 76p was compared with γ-tubulin using permeabilized spermatozoa incubated at 22°C in an oocyte extract and challenged for their ability to nucleate microtubule asters. About 85–90% of spermatozoa nucleated a microtubule aster when incubated in a competent oocyte extract . When the extract was partially depleted with antibodies against either γ-tubulin (R74) or h76p (R801), only 17–19% and 0–10% of the basal bodies assembled a microtubule aster, respectively . These inhibitions were specific since they did not occur when the immunodepletion was conducted in the presence of the respective immunizing peptides , preimmune antibodies or antibodies that are unrelated to Xenopus centrosomal proteins . Moreover, addition of mammalian γ-tubulin complexes to γ-tubulin– or 76p-depleted oocyte extracts restored their capacity to induce asters on sperm basal bodies . It was likely that 76p and γ-tubulin were recruited by the sperm basal bodies. The recruitment kinetics of these two proteins, followed in the presence of 5 μM nocodazole during a 20-min incubation period , paralleled the assembly of microtubule asters . The specificity of the recruitment of γ-tubulin and 76p was further studied after a 20-min incubation period . Barely detectable in permeabilized spermatozoa (Sp) , both γ-tubulin and 76p were revealed when the spermatozoa were incubated in a crude oocyte extract . The same amounts of γ-tubulin and 76p were observed in spermatozoa incubated in an oocyte extract treated either with h76p antibodies (R801) incubated in the presence of the immunizing peptide or with R801 preimmune antibodies . Partial immunodepletion of the oocyte extract with h76p antibodies (R801) appeared highly efficient, and resulted in a severe drop in the accumulation of 76p and to a less extent of γ-tubulin in incubated spermatozoa . These observations demonstrated that 76p, like γ-tubulin, was recruited to the sperm basal bodies and that the recruitment was independent of the presence of microtubules as observed during mitosis. The quantity of 76p in permeabilized spermatozoa and spermatozoa incubated in an oocyte extract was quantified by immunoblotting by comparison with a range of recombinant h76p ≈0.45 ng (± 0.01, n = 4) and ≈4 ng (±0.5, n = 4) of 76p were observed per 10 6 spermatozoa before and after incubation in an oocyte extract, respectively. This ≈9-fold increase of 76p in nucleation-competent spermatozoa strongly suggests that this protein participates to the maturation of the basal bodies as previously suggested for γ-tubulin and the Xenopus orthologue (Xgrip109) of h104p . Since the stoichiometry of 76p in native γ-TuRCs could be variable, 76p could act as a limiting factor in the nucleation of asters especially after its immunodepletion. This hypothesis was strengthened by addition of recombinant h76p to a partially x76p-depleted extract . Although the 76p-depleted extract promoted the assembly of ≈0–10% asters, addition of recombinant h76p partially restored their formation to ≈50%. In contrast, addition of recombinant h76p to a partially γ-tubulin–depleted extract failed to restore the capacity of the basal bodies to nucleate asters: ≈19 and 21% without and with recombinant h76p, respectively. These observations suggested that recombinant h76p could complement a 76p-depleted extract, but not a γ-tubulin–depleted extract. Therefore, it is likely that 76p does not act directly in aster nucleation, but participates in the assembly of active centrosomes. It is possible that recombinant h76p could bind to the γ-TuRCs still present in the partially x76p-depleted Xenopus extract and, thus, restore the nucleating activity. But the capacity of recombinant h76p to restore aster nucleation in a partially x76p-depleted extract (≈50%) was lower than the capacity of mammalian γ-tubulin complexes (≈78%) although the amount of recombinant h76p was ≈4,000-fold higher than the amount of 76p added when mammalian γ-tubulin complexes were used. The presence of inactive recombinant h76p in the preparation could account for this difference. Alternatively, some other limiting factors from the 76p-depleted extract could be necessary to recover complete nucleation. Soluble γ-tubulin complexes are expected to constitute the functional unit of the MTOCs . The characterization of their constituents is an essential step towards the elucidation of the molecular mechanisms of microtubule nucleation. We report the identification of the h76p protein, which is a new evolutionary conserved centrosomal constituent associated with γ-tubulin. The h76p was initially characterized by microsequencing from mammalian brain γ-tubulin complexes. The amino-terminal and internal amino acid sequences as well as the mass distribution of the peptides resulting from tryptic digestion (not shown) revealed only the 76p in the electrophoretic band of the pig brain γ-tubulin complexes migrating at 76 kD. The multiple polypeptide bands observed in the 76-kD range in Xenopus γ-tubulin complexes could result from posttranslational modifications of a highly predominant constituent. Alternatively, it is not possible to exclude the presence of other proteins since we detected the light neurofilament protein in the 76-kD band of sheep brain γ-tubulin complexes both by microsequencing and immunoblot (not shown). Identification of numerous human and murine ESTs homologous to 76p reveals that 76p mRNA is present in various tissues (skin, mammary glands, liver, heart, retina, colon, testis, and placenta) and could be ubiquitously expressed in animal cells, as are γ-tubulin and the two other γ-tubulin–associated proteins, h103p (hGCP2) and h104p (hGCP3). In somatic cells, 76p, like γ-tubulin , represents ≈0.02% of the total cellular proteins. It is not only present in soluble Xenopus γ-TuRCs and heterogeneous mammalian γ-tubulin complexes, but like γ-tubulin, h103p and h104p, is a bona fide centrosomal protein. This is demonstrated by immunofluorescence staining of 76p in PtK2 cells and mature Xenopus sperm basal bodies, and the localization of GFP-h76p fusion protein in COS cells. Moreover, like γ-tubulin , h103p and h104p , 76p is associated with the centrosome independently of the presence of microtubules. An average of ≈4 × 10 4 molecules of 76p per centrosome was present in sperm basal bodies incubated in an oocyte extract, a value similar to the number of molecules of γ-tubulin per Xenopus centrosome . Moreover, 76p is not only an integral centrosomal protein, but relocalized in the mitotic apparatus like γ-tubulin and h104p , although the localization of these proteins can differ as observed in the midbody. Besides its centrosomal location, 76p associates in vitro with a microtubule similar to γ-tubulin, h103p, and h104p . Hence, 76p does not act as an inhibitor of the interaction between γ-tubulin and the α/β-tubulin heterodimers and neither dissociates before the binding of the γ-tubulin complexes to the centrosome nor inhibits their binding. The amounts of 76p (this report), γ-tubulin , h103p, h104p , and pericentrin on the centrosome increase concomitantly in early mitosis. The involvement of 76p in microtubule nucleation processes is further demonstrated by the failure of Xenopus sperm basal bodies to assemble asters when incubated in 76p-depleted oocyte extracts and the recovery of the capacity to assemble asters after addition of mammalian γ-tubulin complexes or recombinant h76p. This observation confirmed that, in addition to γ-tubulin , other proteins of the γ-TuRC, 76p (this report) and the Xenopus orthologue (Xgrip109) of h104p are necessary for the nucleation process. In purified γ-tubulin complexes, 76p is at least fivefold less abundant than γ-tubulin, in agreement with previous quantifications of the 76-kD band by Coomassie staining . The loss of 76p and/or the loss of some γ-tubulin complexes during purification could account for this observation since equivalent quantities of γ-tubulin and 76p are observed in the native γ-TuRCs present in Xenopus oocyte extracts. But the number of 76p in γ-tubulin complexes could be also heterogeneous as suggested by several observations. First, using two different antibodies, we failed to immunoprecipitate all γ-tubulin in preparations of mammalian γ-tubulin complexes and in Xenopus oocyte extracts. Second, addition of recombinant h76p to an x76p-depleted Xenopus oocyte extract partially restored the capacity of basal bodies to nucleate asters. Orthologues to human 76p were cloned from insects (d75p) and angiosperms (m85p) , and are likely present in mosses . The identity between the three sequenced orthologue proteins varies from ≈31 to 34%. Therefore, the 76p proteins seem to generally occur in eukaryotic cells. The identification of a 76p orthologue in higher plants is particularly of note. The exact nature of the MTOCs remains poorly understood in plants. However, γ-tubulin has been identified in a variety of plants and found to localize at numerous microtubule nucleation sites . The isolation of the m85p gives additional support to an evolutionary conservation of the constituents of the MTOCs. Sequence alignments based on the entire sequence of h76p, d75p, and m85p, or on highly conserved sequences between these three proteins failed to identify a 76p orthologue in Caenorhabditis and Saccharomyces genomes. Although it could be argued that the yeast spindle pole body could differ from the centrosome and that the 76p has been lost during the course of evolution, this view would not apply to the typical centrosome of nematodes. Rather, the inability to detect a orthologue in Caenorhabditis could result from a rapid divergence of 76p as previously observed for γ-tubulin . Alignments based on sequence homologies and on hydrophobic cluster analysis showed that the protein 76p exhibits significant sequence similarities with the two related centrosomal human h103p and h104p and their Drosophila (Dgrip84 and Dgrip91) and yeast orthologues (Spc97p and Spc98p) that are associated with γ-tubulin in common protein complexes . The amino-terminal regions of h103p (hGCP2) and h104p (hGCP3) are absent from 76p orthologues , and the homologies are distributed throughout the complete sequence of 76p . In the common region between these proteins, the identities between h76p and h103p (≈23%) or h104p (≈27%) were comparable with the identity observed between h103p and h104p (≈33%), suggesting that this region corresponds to the core of these three γ-tubulin–associated proteins . Hence, it is likely that these proteins originate from a unique gene family that diverged early in the evolution of eukaryotic cells. Identities between h76p, h103p, and h104p could possibly imply some common functional properties such as the positioning of γ-tubulin at the MTOCs . It is tempting to speculate that h76p, h97p, and h98p could orient the binding of γ-tubulin through specific interactions with different docking proteins and possibly specify the nucleation of different microtubule arrays as observed for Spc98p at the inner plaque of the yeast spindle pole body . The mode of interaction between h76p and the other components of the human γ-tubulin complexes together with the analysis of Drosophila d75p mutants could shed some light on the function of h76p, and are currently under investigation. | Study | biomedical | en | 0.999996 |
10562287 | Experiments were performed on MLC3F-nlacZ, Myf-5-nlacZ, c-Met double tyrosine mutant, and Splotch mouse lines . Embryos were dissected and embryonic structures were isolated as described . The tissues were then either separated into a single cell suspension by gentle pipetting or cultured as explants for periods ranging from 3–7 d. At the end of the culture period, tissues were either stained for β-galactosidase activity, immunofluorescence, or used for reverse transcriptase (RT) 1 -PCR. Cultures were grown as described . Northern Blot analysis and RT-PCR were performed as described . Oligos for Pax3 amplification were 5′-TGT GGA ATA GAC GTG GGC TGG TA-3′ and 5′-AGG AGG CGG ATC TAG AAA GGA AG-3′; for MyoD : 5′- CAC TAC AGT GGC GAC TCA GAC GCG-3′, nt 730–753, 5′-CCT GGA CTC GCG CAC CGC CTC ACT-3′, nt 873–850. The antibodies used against MyoD, myosin heavy chains, and c-Met have been described . The antibodies against M-cadherin and MNF were donated by A. Starzyski-Powitz (Humangenetik fur Biologen, Goethe-Universitat, Frankfurt, Germany) and R. Bussel-Duby (University of Texas SW Medical Center, Dallas, TX), respectively . The antibodies against vascular-endothelial (VE) -cadherin, CD34, and PECAM were donated by E. Dejana and C. Gherlanda (Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy). Antibodies against VEGR2, αM-integrin (MAC-1), β3-integrin, and von Willebrand (Santa Cruz and Pharmingen) were donated by A. Stoppacciaro (Dipartimento di Medicinal Sperimentale e Patologia, Universita ‘La Sapienza,’ Rome, Italy). Immunocytochemistry on tissue sections and cultured cells was carried out as described . Genetically labeled cells were injected into the regenerating Tibialis anterior (TA) of SCID/bg mice as described . Fetal limbs were isolated from E16–17 wild-type (wt) embryos and, after removal of the skin, were transplanted subcutaneously into newborn (P1–2) MLC3F-nlacZ mice . Alternatively, freshly dissected dorsal aortas from E9 MLC3F-nlacZ embryos were transplanted into the TA of newborn (P4–5) SCID/bg mice. At different periods after transplantation, the mice were killed, the transplanted and the contralateral TA muscles or the transplanted fetal limb were recovered, and stained for β-galactosidase activity or cryostat-sectioned and processed for immunofluorescence. Mouse satellite cells grown in culture under clonal conditions appear as round-shaped cells expressing myogenic markers, such as MyoD , desmin , and c-Met , and differentiate into multinucleated myotubes upon mitogen deprivation. Satellite cells can be cloned from skeletal muscle of postnatal mice and of fetuses older than E15, but not from earlier stages . We found that when limbs from E13 or E11, or even forelimbs from E9.5 (20–24 somites) embryos were precultured as explants for three, five, or seven days, respectively, and then successively dissociated to a single cell suspension and cultured at low density, myogenic clones with the phenotype of satellite cells appeared . Thus, cells competent to generate satellite cell-like clones must enter the limb field as early as the first migratory population of somite-derived myoblasts, but seem not to acquire this competence until later developmental stages and upon maintenance of interaction with other limb cells. We set out to investigate, by the explant preculture method described above, the possible presence of satellite cell-like cells in different tissue anlagen of the mouse embryo. We cultured newly formed somites or segmental plates, neural tube, dorsal ectoderm, lateral mesoderm, dorsal aorta (from the corresponding body level), and heart. Careful digestion with pancreatin-trypsin allowed clean dissection of the epithelial somites from both the neural tube/notochord complex axial and lateral mesoderm . Possible contamination not detected by microscopy was ruled out by RT-PCR analysis, which did not reveal expression of the medial markers Pax3 and MyoD in the dorsal aorta or in other lateral structures . After one week, half of the explants were stained for the expression of myosin heavy chains and, as expected, only cultures from somites, limb buds, and heart contained hundreds of positive cells. Virtually no myosin positive cells were present in cultures from other tissues (not shown). The rest of these explants were dissociated to single cell suspensions and cloned by limited dilution under conditions that favor clonal growth of satellite cells. Fig. 2 A shows the typical morphology of a satellite cell-like clone derived from precultured E9.5 forelimb bud explants, indistinguishable from a clone directly derived from older limbs (not shown). Unexpectedly, the vast majority of clones with this morphology came from explants of dorsal aorta . In contrast, most clones derived from precultured somites had a fibroblast-like morphology . After shifting the clonal cultures to differentiation medium, all round-shaped satellite cell-like, but not the fibroblast-like, clones differentiated into myosin positive cells . Quantitative analysis showed that a high proportion of satellite cell-like clones (defined by morphology and myosin expression) were derived from explants of dorsal aorta . These precultured explants gave rise to an average of five times more clones than the somites, whereas the latter contained ten times more cells for the same segment length. Thus, each segment of dorsal aorta can give rise to 50 satellite cell-like clones on a per cell basis, versus one clone originating from somites. Very few clones of satellite cell-like cells originated from lateral mesoderm and neural tube, and none from ectoderm and heart. As reported above, many satellite cell-like clones were obtained from cultures of forelimb bud from 20–24 somite embryos. At these stages the limb bud already contains both myoblasts and vascular endothelial cells. Thus, all the tissues that gave rise to satellite cell-like clones contain endothelial cells. The only tissue that, despite the presence of abundant endothelium (endocardium), never gave rise to satellite cell-like clones was heart. This may reflect unique tissue interactions that occur during endocardium specification and may result in a more restricted developmental potential, as compared with noncardiac endothelium. We next investigated whether the aorta would maintain the potential of generating clones of satellite cell-like cells during later developmental stages. Fig. 3 B shows that this potential declines during development, but at E17 is still approximately one third of the maximal value obtained at E9.5. Postnatal or adult aorta, as well as smaller vessels dissected from limb muscle, did not give rise to satellite cell-like clones, either directly or after explant culture (not shown). Forelimbs from the same stage were used as positive controls. These novel results led us to compare the phenotype of these clonable satellite cell-like cells with that of adult satellite cells and fetal myoblasts. Table and Fig. 4 show that most known myogenic markers ( MyoD , Myf-5 , desmin , MNF , c-Met , and M-cadherin ) were expressed by clones of dorsal aorta, as well as by postnatal muscle satellite cells and fetal myoblasts. Unexpectedly, vascular-endothelial markers, such as VE-cadherin , VEGF-R2 , αM-integrin , β3 integrin , P-selectin , smooth α-actin , and PECAM were also expressed by the first two cell types, while fetal myoblasts did not express VE-cadherin , P-selectin and β3 integrin (all clones were negative for von Willebrand factor). Fig. 5 shows clones of aorta-derived myogenic cells and of adult satellite cells that all coexpress VE-cadherin on the surface and MyoD in the nucleus. Clones from dorsal aorta also expressed CD34 , but only during the first two days in vitro. Muscle fibroblasts (used as controls) were negative for all of these markers. Thus, clones from the dorsal aorta were indistinguishable from clones of satellite cells in terms of differential expression of any of 15 different markers analyzed. Expression of VE-cadherin was confirmed by Northern blot analysis in satellite cells . It was unexpected, and is notable that satellite cells derived from adult muscle express a number of endothelial markers. In embryos null for c-Met , or expressing a Met receptor unable to transduce the HGF signal (Met D ), myoblasts fail to emigrate from the somite and to colonize the limb bud . Although devoid of myogenic cells, the developing limbs of these mutants have normal blood vessels, as shown in Fig. 7A and Fig. B . Forelimb buds from Met D homozygous embryos were cultured and then dissociated into single cell suspensions and grown as clones. Satellite cell-like clones emerged from forelimbs of homozygous Met D embryos (∼50% of those obtained from heterozygous or wt siblings). This demonstrates the presence of clonogenic satellite cell-like cells that are not derived from somitic myoblasts. The clones obtained from mutant embryos were notably smaller (averaging 4 to 6 cells) than those derived from the heterozygous siblings (averaging 20 to 60 cells), and expressed high levels of MyoD . With time in culture, some of these clones disappeared and occasionally fragmented chromatin was observed, but the surviving clones differentiated into myosin-positive mono- or binucleate muscle cells. We thus conclude that at least a fraction of satellite cell-like myogenic progenitors are present in the limbs of Met D mutant embryos, which are colonized by endothelial cells, but not by somite-derived myoblasts. Their growth is impaired, likely because they cannot respond to SF/HGF, and eventually apoptosis may occur. Still, they represent a considerable fraction (∼50%) of those obtained from wt embryos. Similar results were also obtained using limb buds of Splotch embryos as a source of myogenic cells (not shown). Splotch mice are defective in the Pax-3 gene and their phenotype overlaps with the Met null in terms of lack of migration of somite-derived precursors in the limbs . We tested the potential of transplanted aorta-derived satellite cell-like cells to participate in in vivo perinatal growth and regeneration of skeletal muscle, in three different sets of experiments. In the first experiment, a cell suspension from cultured explants of E9.5 embryonic aorta of MLC3F-nLacZ transgenic mice, in which transgene expression is restricted to heart and skeletal muscle , was mixed with a tenfold excess of satellite cells from wt P10 mice. Part of this suspension was grown in culture. Fig. 8 A shows a myotube containing one β-galactosidase positive (β-gal+) nucleus which, because of the genetic label, must be derived from the dorsal aorta explant. The rest of the mixed cell suspension was injected into a regenerating TA muscle of SCID/bg mice, where they gave rise to small clusters of several β-gal+ nuclei within regenerating fibers, surrounded by a laminin-positive basal membrane . Thus, cells derived from the dorsal aorta can fuse with satellite cells in vitro and participate in skeletal muscle regeneration in vivo. All these results show that myogenic cells derived from the aorta cannot be distinguished from bona fide satellite cells. For the second experiment, to test whether myogenic cells derived from the vasculature may also contribute to normal development of skeletal muscle, we transplanted E16 fetal limbs from wt embryos under the skin of a newborn (P2) MLC3F-nLacZ transgenic mouse . After one week, the transplanted limbs had grown and were vascularized by the host. Whole-mount staining revealed the presence of many β-gal+ nuclei clustered in the area where the vessels had penetrated the transplant . Sections of the same samples revealed β-gal+ nuclei within myosin-positive fibers adjacent to VE-cadherin–positive vessels . In the third experiment, isolated dorsal aortas from E9 MLC3F-nLacZ transgenic embryos were transplanted into the TA on one side of newborn (P 4–5) SCID/bg mice. After two weeks, the TA anterior contained a cluster of fibers with many β-gal+ nuclei in the area of the transplant . However, the untreated contralateral TA was also found to contain many β-gal+ nuclei dispersed throughout the whole muscle , indicating that these cells had reached the developing fibers through the circulation. In contrast, when somites from the same embryos were similarly transplanted into the developing TA muscle of newborn immune-deficient mice, many β-gal+ nuclei were observed surrounding a core of cartilage (presumably from the sclerotome), but no β-gal+ nuclei were found in other muscles (data not shown). Our data show that cells derived from embryonic vessels can give rise to skeletal myogenic cells. There is no skeletal muscle surrounding the developing vessels in the mouse embryo. In vertebrates, somitic muscles derive from dorsal mesoderm, whereas the intraembryonic vasculature mainly derives from ventral splancnic mesoderm , although the dorsal portion of the aorta is derived from the paraxial mesoderm and is not endowed with hemopoietic capacity . In explant cultures, we found that dorsal aorta did not contain differentiated muscle cells, which typically appear in similar cultures of somitic or limb tissues. However, when dorsal aortas were first explant-cultured for a week, and then dissociated to single cell suspensions and cloned under conditions that favor expansion of adult satellite cells, many myogenic clones appeared, undistinguishable from bona fide satellite cells. The fact that such clones could not be obtained directly from the aorta suggests a requirement for growth factors provided by neighboring cells during the incubation as organ culture, and absent in our culture medium. The myogenic clones express all the early myogenic markers considered, including M-cadherin , MNF , and desmin . Virtually 100% of these cells express MyoD , while only a minority of them express Myf-5 . Although the majority of postnatal satellite cells, upon clonal expansion in vitro or activation in vivo, express both MyoD and Myf5 , in the MyoD knockout mouse regeneration is impaired via an effect on the satellite cell population . The unexpected coexpression of a number of endothelial and myogenic markers in cells derived from the dorsal aorta suggests that these myogenic cells may be derived from true endothelial cells or from a common precursor. It is interesting to note that endothelial cells appear to be the only cell type capable of generating immortal clones in early postimplantation mouse embryos and that circulating endothelial progenitor cells can be isolated from adult human blood . These endothelial progenitors typically express CD34 . Aorta-derived myogenic clones only express CD34 at the beginning of clonal expansion, suggesting early loss of this antigen upon entry in the myogenic pathway. Other endothelial markers, such as VE-cadherin , are lost upon terminal differentiation, but are coexpressed with MyoD throughout clonal expansion. To our surprise, endothelial markers were also coexpressed with MyoD in bona fide satellite cells, a puzzling observation for cells presumed to be of somitic origin. The problem of the embryonic origin of satellite cells has never been directly approached, except in a study that was inconclusive due to technical difficulties in identifying quail nuclei in chick–quail chimeras at the ultrastructural level . It is interesting to note that, unlike postnatal satellite cells, fetal myoblasts were found not to express VE-cadherin , β3-integrin , or P-selectin , in keeping with the possibility that several phenotypically distinct populations of myogenic cells may appear sequentially during muscle histogenesis . Our data suggest that, differently from embryonic and fetal myoblasts derived from somites, satellite cells may derive, at least in part, from progenitors in the dorsal aorta. Coexpression of endothelial and myogenic markers supports this hypothesis. In normal development, endothelial cells first migrate into the limb bud from the lateral edge of newly formed somites and are soon followed by myogenic cells . In contrast, only endothelial cells (but not myogenic precursors) enter the limb bud of embryos mutant for c-Met or Pax3 . The embryos do not survive until the stage when satellite cells appear in vivo. However, explant cultures from limb buds of these embryos yielded a significant number of satellite cell-like clones (∼50% of wt), supporting the idea that they are the progeny of endothelial cells, rather than myoblasts. Interestingly, all the cells of these clones express MyoD , do not grow to more than four to eight cells, and, for the most part, eventually die, but the few surviving undergo terminal muscle differentiation. Met signaling in response to SF/HGF thus appears to be required for the clonal growth of these newly identified myogenic cells. Expression of myogenic markers in vitro suggests a potential for in vivo myogenic differentiation. Our results indicate that these myogenic cells do participate in both postnatal muscle growth and regeneration. When directly injected into the regenerating TA of an immune-deficient mouse, genetically labeled nuclei of cells from the dorsal aorta are incorporated into newly formed muscle fibers, much as bona fide satellite cells. Indeed, skin fibroblasts also are incorporated into regenerating fibers when similarly injected, although at an extremely low frequency . Thus, while regenerating muscle must be a rich source of signals recruiting competent cells to myogenesis, it is also possible that within a population of skin fibroblasts, there may be a small fraction of still multipotent progenitors and it is only this fraction that is capable of differentiating in vivo. When a fetal limb is transplanted under the skin of a transgenic MLC3F-nlacZ newborn mouse, it is vascularized by the host. These limbs contain β-gal+ nuclei, usually clustered in the area of the vessel in-growth, suggesting that they were associated with it, rather than deriving from neighboring host muscle. Finally, when an embryonic dorsal aorta is transplanted into the growing TA of a newborn immune-deficient mouse, it gives rise to many β-gal+ nuclei clustered in the area of the transplant. This does not happen if the aorta is grown in vitro, indicating that signals from the surrounding developing skeletal muscle recruit some of the aorta cells (endothelium and/or pericytes) to myogenesis. Remarkably, several dispersed β-gal+ nuclei are also present in the contralateral untreated TA, indicating that these myogenic cells must have reached this site through the circulation, much as happens for the adult progenitors in bone marrow . Possible contamination from adjacent somitic tissue is ruled out because when somites are similarly transplanted, they give rise to differentiated muscle cells only in the area of transplant. Our data may simply represent one example of transdifferentiation leading to the formation of skeletal muscle cells, as reported for the esophagus , the neural tube , the kidney , or mesenchymal cells from bone marrow and dermis . We propose a different explanation: that, at least in this case and in the case of mesenchymal cells, multipotent progenitors may be present in the endothelium (or closely associated cells). When invading developing muscle anlagen, these progenitors will be subject to a muscle field, and thus will adopt a satellite cell fate. When the vessels develop inside a different tissue, these cells may adopt the specific fate of that tissue, and contribute to its histogenesis. The only tissue in which these progenitors remain demonstrable may be the bone marrow, and this would explain our recent observation that cells from the bone marrow can contribute new myogenic cells to regenerating skeletal muscle . Multipotent mesenchymal cells, capable of producing osteoblasts, chondroblasts, adipocytes, and even skeletal muscle, have long been known to be present in the bone marrow . We do not know whether the cells we describe in embryonic vessels represent the progenitors of multipotent mesenchymal cells or a separate lineage with at least part of the same developmental potential. Preliminary observations suggest that clones of dorsal aorta can give rise to osteoblast-like cells in the presence of BMP-2. Indeed, multipotentiality is preserved, even in adult muscle satellite cells, as shown by the fact that BMP2 can switch them to an osteogenic fate . In vivo work will clarify whether the contribution of aorta-derived myogenic cells is quantitatively relevant during fetal and/or postnatal growth and regeneration of skeletal muscle. | Study | biomedical | en | 0.999997 |
10562288 | The following wild-type peptides were synthesized at the UNC Peptide Facility: WW domain of human YAP65 (VPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRK; YAP WW), the COOH-terminal 10 amino acids of YAP65 (YAPwt), and the COOH-terminal 10 amino acids of human CFTR . Mutant peptides (YAPmut and CFTRmut), where the final four amino acids of YAP65 or CFTR were changed to glycine, were also synthesized. All peptides contained an amino-terminal biotin and a 4–amino acid spacer (SGSG) and were purified by high pressure liquid chromatography. Rabbit antibodies directed against amino acids 142–432 of chicken YAP65 were described previously . Mouse anti-Src antisera was provided by Dr. Patricia Maness (University of North Carolina). Commercial antisera were obtained from the following sources: mouse anti-Yes and mouse anti-ezrin from Transduction Laboratories; rabbit anti-Yes from Upstate Biotechnology, mouse anti-Src from Santa Cruz Biotechnology, and rat anti-ZO-1 and mouse anti-GFP from Chemicon International, Inc. EBP50 antisera were generated in rabbits using hexahistidine-tagged full-length EBP50 as an immunogen. The bacterial expression plasmid pET.EBP50 was generated by subcloning the full-length EBP50 cDNA from pGEX.EBP50 to pET28c (Novagen, Inc.). Hexahistidine-tagged EBP50 protein was purified on a Ni 2+ affinity resin, and the purified protein was used to immunize two New Zealand white rabbits (Covance Laboratories). EBP50 antisera were analyzed by Western blot analysis using CalU3 cell lysates known to contain high levels of EBP50. Before immunolocalization studies, complement proteins were removed from the whole serum by incubation with DEAE-blue dextran (Pierce Chemical Co.). In addition, some experiments were performed with EBP50 antisera provided by Dr. Anthony Bretscher (Cornell University), and similar results were obtained. pGEX plasmids encoding GST fusions of EBP50, PDZ1, and PDZ2 were described . For in vitro translation experiments, pCDNA3.CFTR-CT encoding the COOH-terminal 330 amino acids of human CFTR (amino acids 1,151–1,480) was generated by PCR using pBQ.CFTR as template. PCR products were digested with the appropriate enzymes and ligated into the polylinker of pET.28c digested with the same enzymes (Novagen, Inc.). For in vitro translation and mammalian expression, full-length human YAP65 and a mutant human YAP65 (YAP65/−4) were generated by PCR. The mutant YAP65/−4 construct has an engineered premature stop codon after amino acid 450, resulting in expression of a YAP65 protein lacking the final four amino acids. PCR products were digested with the appropriate enzymes and ligated into the polylinker of pCDNA3.1 (Invitrogen Corp.) and pEGFP.C2 (CLONTECH Laboratories). All plasmids generated by PCR were sequenced at the UNC sequencing facility. Type II MDCK cells, human bronchial epithelial 16HBE14o− , and human colonic epithelial T84 cells were maintained in DME-F12 (Life Technologies, Gaithersburg, MD) + 10% fetal clone serum (Hyclone) at 37°C and 95% humidity. Primary cultures of human nasal epithelium were prepared as described previously . To generate stable 16HBE14o− cell lines expressing GFP.YAP65 or GFP.YAP65/−4, cells were transfected as described and grown in media containing 400 μg/ml G418. Drug-resistant colonies were expanded, and clonal lines were selected by fluorescence microscopy and Western blot analysis using rabbit anti-GFP antisera (Chemicon International, Inc.). Confluent monolayers of cultured cells were washed with PBS (50 mM NaH 2 PO 4 ·H 2 O, 150 mM NaCl, pH 7.4) and lysed in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholic acid, and 5 mM EDTA (RIPA) containing 250 μM sodium orthovanadate and a cocktail of protease inhibitors. Lysates were cleared by centrifugation at 40,000 g for 20 min at 4°C. Protein concentrations were determined using the BCA assay kit (Pierce Chemical Co.), and 30 μg of cell lysate was electrophoresed on 10% SDS-PAGE gels and transferred to Immobilon-P (Millipore). Western blot analysis was performed using rabbit anti-YAP65 (1:1,000), mouse anti-Yes (1:1,000), mouse anti-Src (1:1,000), or rabbit EBP50 antisera as described . 16HBE14o− cell monolayers were fixed in 4% paraformaldehyde for 5–20 min at room temperature and blocked in 4% nonfat dry milk, 2 mg/ml BSA, 0.1% Triton X-100 for 3 h (for 16HBE14o−), or 10–20% normal goat serum (primary cultures). Cultures were incubated in primary antisera for 4–12 h in PBS containing 2 mg/ml BSA. After washing in PBS + 0.1% Triton X-100, the filters were incubated in Alexa 488– (1:200; Molecular Probes) or Texas red–conjugated (1:500; Jackson ImmunoResearch Laboratories, Inc.) secondary antiserum diluted in PBS + 2 mg/ml BSA. The samples were analyzed using a Leica TCS-NT confocal microscope as described . Biotinylated peptides or GST fusion proteins (10 μg) were immobilized on streptavidin agarose or glutathione agarose as described . To assess binding between immobilized peptides or GST fusion proteins and in vitro translated proteins, plasmids containing the appropriate insert were used as templates for coupled in vitro transcription/translation (Promega TNT), in the presence of 2 μCi [ 35 S]methionine (NEN). 5 μl of radiolabeled protein was incubated with the affinity resins in binding buffer + 0.1% Triton X-100 for 3 h at 4°C. The samples were centrifuged to collect the beads, and the beads were washed three times in TEE containing 1 M NaCl + 1% Triton X-100 followed by three washes in binding buffer + 1% Triton X-100. The bound fractions were eluted from the beads and the supernatants were precipitated with ice-cold acetone. Bound and unbound fractions were analyzed by SDS-PAGE and phosphorimage analysis. For competition experiments, GST-EBP50 was immobilized on glutathione agarose beads, and the beads were incubated for 2 h in the presence of competing peptide, before the addition of in vitro translated protein or cell lysate. In some experiments, affinity resins were incubated with whole cell lysates prepared from 16HBE14o− cells. The cell lysates were prepared in RIPA buffer containing orthovanadate and protease inhibitors as described above, and ∼200 μg of cell lysate diluted in binding buffer + 0.1% Triton X-100 was incubated with the affinity resin for 3 h at 4°C. The beads were washed as described above, proteins were eluted from the beads, and fractionated by SDS-PAGE. Bound fractions were analyzed by Western blotting using the appropriate antisera. All binding assays were performed at least three times with similar results. Cells were rinsed in PBS and lysates were prepared in RIPA buffer. Rabbit anti-EBP50, preimmune sera, or purified normal rabbit IgG (2 μg) was prebound to protein A beads in binding buffer + 0.1% Triton X-100. Beads were washed in the same buffer + 1% Triton X-100. Cell lysates were added and the samples were incubated for 4–12 h at 4°C in RIPA buffer (500 μl final volume). The samples were centrifuged to collect the beads, and washed three times with RIPA buffer. Bound proteins were analyzed by blotting with the appropriate antisera or subjected to in vitro kinase assays. Kinase activity was measured after addition of a Src family substrate peptide derived from p34 cdc2 (KVEKIGEGTYGVVYK; Upstate Biotechnology) according to the manufacturer's instructions. While NHE3 is known to associate with EBP50 via a nontraditional PDZ interaction , there are no known proteins that associate with PDZ2 via a COOH-terminal interaction. We identified YAP65 as a candidate protein to associate with PDZ2 of EBP50. In addition to the potential PDZ interaction motif at the extreme COOH terminus, YAP65 contains a proline-rich sequence that mediates association with SH3 domains and a WW domain . Computer-based structural analyses of the human YAP65 protein sequence using the Coils algorithm with a window size of 28, predicted a single coiled coil from residues 259–292 with the probability of 1. We first asked whether EBP50 and YAP65 were able to associate in vitro. We immobilized GST or GST-EBP50 on glutathione agarose beads, incubated the affinity resins with whole cell lysates prepared from 16HBE14o− cells, and examined the bound and unbound fractions by Western blot using YAP65 antisera . Endogenous YAP65 bound to the GST-EBP50 beads, but not to GST beads, indicating that EBP50 and YAP65 are capable of interacting . To determine whether the COOH terminus of YAP65 was involved in the interaction, we immobilized biotinylated peptides corresponding to the COOH-terminal 10 amino acids of YAP65 on streptavidin beads, and incubated the immobilized peptide with whole cell lysates prepared from 16HBE14o− cells. We performed parallel incubations using CFTRwt peptide affinity resins since we previously showed that CFTR and EBP50 associate in vitro . We found that EBP50 bound to both CFTR and YAP65 COOH-terminal peptides and was depleted from the cell lysate . Affinity resins containing the YAP65 WW domain or mutant YAP65 peptide bound no EBP50. Therefore, the interaction between YAP65 and EBP50 is specific, and requires the COOH terminus of YAP65. To determine whether the association between YAP65 and EBP50 is direct, we tested the ability of YAP65 peptides to bind radiolabeled, in vitro translated EBP50. YAPwt peptide, but not YAPmut or WW domain peptides, bound labeled EBP50 . The YAPwt peptide also bound GST-EBP50, but not GST, in blot overlay assays (not shown), further supporting our conclusion that EBP50 and YAP65 directly associate. EBP50 is expressed in many cells, and especially high levels are found in epithelial tissues . We analyzed the expression of YAP65 protein in MDCK cells, human intestinal T84, and two cultured human airway epithelial cell lines, 16HBE14o− and CalU3 cells. YAP65 protein is expressed in whole cell lysates prepared from each of the cell lines tested . Furthermore, similar to ezrin and EBP50, a significant amount of YAP65 protein was found in the particulate fraction of these cells . We showed previously that ezrin and EBP50 are preferentially accumulated at the apical surface of human airway epithelia ; however, the subcellular localization of YAP65 has not been previously studied. Therefore, we compared the distributions of ezrin, EBP50, and YAP65 in 16HBE14o− cells, a clonal cell culture model system derived from human bronchus. In our culture system, 16HBE14o− cells form monolayers with transepithelial resistances of >400 ohm·cm 2 and exhibit vectorial ion transport properties when grown on permeable filter supports. These cells also exhibit an asymmetric distribution of proteins including ZO-1 (tight junctions) and ezrin . Thus, 16HBE14o− cell monolayers provide a well polarized airway epithelial cell culture system to examine the distribution of proteins in EBP50 complexes. Cells that were grown on transwell filters for 7–10 d after confluence were stained with rabbit antisera directed against EBP50 or YAP65. Both proteins were preferentially accumulated at the apical membrane . The amount of each protein present in the apical compartment increased when cells were maintained on transwell filters for increasing lengths of time (not shown), suggesting that the recruitment of EBP50 and YAP65 to the membrane is partially dependent upon cell polarization. We also examined the distribution of YAP65 in well differentiated primary human nasal epithelial cells grown on transwell supports at the air–liquid interface for 40–50 d. These cultures recapitulate the morphology of the well differentiated pseudostratified epithelia lining the normal airway surface, and ciliated columnar epithelial cells and mucin-producing goblet cells are present . As observed in 16HBE14o− cells, ezrin and EBP50 were preferentially accumulated at the apical membrane and their distributions overlapped . The overlap between ezrin and EBP50 was observed in ciliated and nonciliated cells, and did not significantly vary from culture to culture. YAP65 was also accumulated at the lumenal surface of cultured primary nasal epithelial cells, in a pattern that largely overlapped the distribution of apical actin . In all cultures examined the distributions of YAP65 and ezrin partially overlapped , but regions where the distributions of the two proteins were juxtaposed, but not overlapping, were also observed. In addition, we also observed that in some cultures, YAP65 was more broadly distributed in the subapical space . Nonetheless, these localization studies clearly establish that ezrin, EBP50, and YAP65 are codistributed in the apical compartment of human nasal and bronchial epithelial cells. In vitro data indicate that the COOH terminus of CFTR binds with high affinity to the first PDZ domain of EBP50 . To determine whether YAP65 bound to the same or different sites on EBP50, we immobilized GST-EBP50 on affinity resins and incubated the resins with radiolabeled YAP65 or the COOH terminus of CFTR (CFTR-CT; residues 1,150–1,480 of human CFTR). As expected, both proteins bound to the GST-EBP50 affinity resins . The interaction between CFTR-CT and EBP50 was blocked by the addition of 400-nM CFTRwt peptide, but not by the CFTRmut peptide lacking the terminal DTRL . Likewise, we found that the YAPwt peptide competed for binding between GST-EBP50 and radiolabeled YAP65, whereas the YAPmut peptide lacking LTWL failed to compete . Binding between GST-EBP50 and CFTR-CT was not blocked by 400-nM YAP65 peptide , nor was binding between GST-EBP50 and radiolabeled YAP65 blocked by addition of CFTR COOH-terminal peptide . Thus, CFTR and YAP65 do not compete for binding to EBP50 in vitro, suggesting that the two proteins associate with EBP50 via distinct sites. However, both YAP65 and CFTR appear to bind EBP50 with high affinity. When increasing amounts of YAP65 COOH-terminal peptide ranging from 1–500 nM were added to YAP65-EBP50 binding assays, association was inhibited ∼50% by 100-nM competing peptide and completely abolished by 500-nM peptide . Our competition experiments indicate that YAP65 and CFTR associate with EBP50 via distinct binding sites. To directly determine which PDZ domain of EBP50 was involved in binding YAP65, we tested the association between in vitro translated full-length YAP65 and equal amounts of GST-EBP50 fusion proteins. We found that YAP65 associated with GST-EBP50 and GST-PDZ2 of EBP50, but not with GST or GST-PDZ1 . Taken together, these data indicate that YAP65 preferentially associates with the second PDZ domain of EBP50. Our in vitro binding assays demonstrate that YAP65 and EBP50 are capable of interaction, and our localization studies place the two proteins within the apical compartment of airway epithelial cells. Therefore, we sought to determine whether EBP50 and YAP65 associate in cells. We incubated whole cell lysates from 16HBE14o− cells with normal rabbit IgG or with rabbit antisera directed against EBP50, collected immune complexes on protein A agarose, and analyzed the immunoprecipitates using ezrin or YAP65 antisera. As previously reported , ezrin was found in EBP50 immunoprecipitates but not in control immunoprecipitates . Moreover, YAP65 was not associated with normal rabbit IgG, but a significant fraction of the YAP65 expressed in the 16HBE14o− cells was associated with EBP50 . By comparing the amount of YAP65 present in the EBP50 immunoprecipitates to the amount present in the whole cell lysate, we estimate that ∼20% of the total YAP65 expressed in these cells was associated with EBP50. To determine whether the COOH terminus of YAP65 mediated its association with EBP50 in cells, we generated stable 16HBE14o− cell lines expressing GFP fused in-frame with YAP65 (GFP-YAP65) or YAP65 lacking the last four amino acids shown to be involved in association with EBP50 (GFP-YAP65/−4). Cell lines expressing similar levels of GFP-YAP65 and GFP-YAP65/−4 were selected; immunoblot analyses using YAP65 antisera indicated that the expression of chimeric protein relative to endogenous YAP65 is ∼1:1 (not shown). Neither GFP chimera was found to associate with the beads when lysates were incubated with protein A agarose in the absence of antibody, or when samples were immunoprecipitated with normal rabbit IgG . In agreement with the coimmunoprecipitation experiments from nontransfected cells, a significant fraction of GFP-YAP65 was present in EBP50 immunoprecipitates . In contrast, GFP-YAP65/−4 did not immunoprecipitate together with EBP50. Taken together, our data indicate that EBP50 and YAP65 associate in a stable complex in cells, and that the association requires an intact YAP65 COOH terminus. To determine whether the association with EBP50 is involved in compartmentalizing YAP65 at the apical membrane, we compared the distributions of GFP-YAP65 and GFP-YAP65/−4 in the stable cell lines. Like endogenous YAP65, GFP-YAP65 was concentrated in the apical compartment of 16HBE14o− cells . In contrast, the GFP-YAP65/−4 mutant protein was not accumulated at the apical cell surface, indicating that the EBP50–YAP65 interaction is required for the proper localization of YAP65 in airway epithelia. Surprisingly, the GFP-YAP65/−4 mutant protein was preferentially associated with the lateral cell surfaces of the transfected cells. These data indicate YAP65 can associate with proteins or lipids on the lateral cell surface, but that the full-length protein localizes to the apical compartment because of its high affinity interaction with EBP50. YAP65 was originally cloned based on its ability to associate in vitro with the SH3 domain of c-Yes, a nonreceptor tyrosine kinase (NRTK) of the Src family . These data raise the intriguing possibility that, by binding YAP65, EBP50 functions to recruit c-Yes or other NRTKs to the apical cell surface where they may play key regulatory roles. To begin to explore the functional significance of the EBP50–YAP65 interaction, we determined whether EBP50 protein complexes also contained NRTK activity when isolated in vitro. We immobilized GST-EBP50 fusion proteins on glutathione agarose, and incubated the affinity resins with lysates prepared from 16HBE14o− cells. After incubation, the affinity resins were extensively washed to remove unbound proteins and incubated with γ-[ 32 P]ATP in the presence of the Src family kinase–specific substrate peptide, p34 cdc2 . Under these conditions, we observed significant levels of Src family kinase activity associated with GST-EBP50 and GST-PDZ2, whereas background levels of activity were associated with GST and GST-PDZ1 . Since we previously showed that YAP65 associated with GST-EBP50, and that this interaction was mediated by the COOH terminus of YAP65, we next determined whether the association between EBP50 and Src family kinases was mediated by YAP65. We preincubated GST-EBP50 affinity resins with 400-nM YAPwt peptide, and then measured the levels of Src family kinase activity associated with the GST-EBP50 affinity resins. We found that incubation of GST-EBP50 affinity resins with the YAPwt peptide completely blocked the association between GST-EBP50 and the Src family kinase activity . These data are consistent with the hypothesis that YAP65 serves as an adaptor protein to recruit Src family kinases to EBP50 complexes. Our immunoblot analyses of whole cell lysates prepared from 16HBE14o− and CalU3 cells indicate that both c-Src and c-Yes are expressed in these cells . Although previous in vitro binding assays suggest that YAP65 preferentially associates with the c-Yes SH3 domain , binding between YAP65 and Src family kinases has not been carefully examined in cells. To determine whether the NRTK activity associated with GST-EBP50 is due to association between EBP50, YAP65, and c-Yes, we incubated 16HBE14o− cell lysates with immobilized GST-EBP50 fusion proteins. c-Yes immunoreactivity was easily visualized in the bound fraction of GST-EBP50 and GST-PDZ2 affinity resins . In contrast, no c-Yes was found to associate with GST or with GST-PDZ1 . In addition, we performed duplicate GST-PDZ pull-down experiments using antisera to both c-Yes and c-Src. As expected, a significant fraction of c-Yes was associated with GST-PDZ2 but not GST . In contrast, while c-Src was easily detected in the input sample, we observed minimal binding to GST and GST-PDZ2 . Taken together these data indicate that YAP65 specifically mediates the association between EBP50 and c-Yes, but not c-Src. Furthermore, when EBP50 immunoprecipitates were blotted with antisera to c-Yes, we found a significant fraction of the c-Yes expressed in 16HBE14o− cells in the immunoprecipitates. . By comparing the amount of c-Yes in the whole cell lysate to the amount found in the EBP50 immunoprecipitates, we conclude that ∼15–20% of the c-Yes is contained within EBP50 protein complexes in 16HBE14o− cells. Taken together, the data indicate that EBP50, YAP65, and c-Yes stably associate in cells. Previous immunohistochemical studies suggest that c-Yes is localized at the apical membrane of intestinal and tracheal epithelium . c-Yes is also enriched at adherens junctions in liver and in some cultured cells , and in lipid-insoluble complexes at the apical cell surface of MDCK cells . Our biochemical assays indicating a stable association between EBP50, YAP65, and c-Yes are consistent with these immunohistochemical studies; however, we also found that a significant fraction of the c-Yes expressed in 16HBE14o− cells was not associated with YAP65. Therefore, we used confocal microscopy to compare the distributions of EBP50, YAP65, and c-Yes in our cell culture model systems. In all cultures examined, c-Yes was visualized at the apical cell surface, although some of the protein was present in other cellular compartments and on intracellular vesicles . The degree of colocalization varied from culture to culture, suggesting that the state of differentiation or other unknown factors influence the distribution of c-Yes. The partial overlap between EBP50, YAP65, and c-Yes is consistent with our biochemical results, indicating that a significant fraction of these proteins are stably associated in cells. Although our work confirms that YAP65 and c-Yes stably associate in cells, the function of this interaction is not yet known. Many protein kinases and phosphatases are restricted to distinct subcellular compartments by association with specific targeting proteins . Therefore, we considered the possibility that one function of the interaction between YAP65 and c-Yes is to target c-Yes to the apical cell surface. We used confocal microscopy to compare the distribution of c-Yes in 16HBE14o− cells stably expressing GFP.YAP65 and GFP.YAP65/−4. Consistent with the localization in wild-type 16HBE14o− cells , c-Yes was accumulated at the apical membrane in cells stably overexpressing GFP.YAP65 . In contrast, in cells stably overexpressing GFP.YAP65/−4, a significant amount of c-Yes was redistributed to the lateral cell borders along with the mutant GFP-YAP65/−4 protein . Some c-Yes was still detected at the apical membrane of cells overexpressing GFP.YAP65/−4, and was likely due to association with endogenous YAP65 or other apical membrane components. These data build on our conclusion that YAP65 and c-Yes stably associate in cells and further suggest that this interaction functions to target c-Yes to the apical cell surface. EBP50 and the related protein E3KARP (NHE3 kinase A regulatory protein), are known to regulate the activity of the Na + /H + exchanger NHE3 . This regulation involves direct binding between an internal sequence within the cytoplasmic COOH terminus of NHE3 and residues 149–358 of EBP50 . EBP50 binds ezrin, and ezrin is known to bind the regulatory subunit of PKA in overlay assays . Therefore, one hypothesis is that EBP50 recruits PKA to the apical membrane in close proximity with NHE3 . While this model is attractive, there are no conclusive data establishing a role for ezrin as an A kinase anchoring protein in cells. In addition to NHE3, CFTR and the βAR also directly associate with EBP50 . While the function of the CFTR–EBP50 interaction is not yet known, binding of β 2 AR to EBP50 is implicated in regulation of cellular pH by modulation of NHE3. Specifically, in fibroblasts, agonist activation of βARs stimulated association of the receptor with EBP50, and prevented EBP50 from regulating NHE3 . However, these experiments have not been replicated in polarized cells, where NHE3 is known to reside at the apical cell surface . Furthermore, NHE3 is not expressed in airway epithelial cells, indicating that the functions of EBP50 may be tissue-specific. EBP50 may function to target proteins to the apical cell surface, and may also serve as a scaffold to organize apical membrane proteins into regulatory complexes. A logical approach to elucidating the function of EBP50 is the identification of additional proteins in EBP50 protein complexes. Our data establish that YAP65 associates with EBP50 via the COOH terminus, and the COOH terminus of YAP65 is necessary and sufficient for association with EBP50 . In addition, pull-down assays using the YAP65wt peptide and GST fusion proteins of PDZ1 and PDZ2, clearly demonstrated preferential binding of YAP65 to PDZ2 . Furthermore, the YAP65 COOH-terminal peptide was unable to compete for binding between GST-EBP50 and CFTR-CT . These results strongly support the conclusion that YAP65 is recruited to EBP50 complexes by association with PDZ2 of EBP50. Our localization studies clearly demonstrate that endogenous YAP65 is preferentially localized at the apical membrane of 16HBE14o− cells and in well differentiated primary cultures from human nasal epithelial cells . The expression of GFP-YAP65 chimeras in 16HBE14o− cells supports our localization studies, since GFP-YAP65 was also accumulated at the apical membrane . Furthermore, our results indicate that YAP65 is tethered at the apical membrane by association with EBP50, since GFP-YAP65/−4, which lacks the PDZ2 interaction motif, was no longer localized at the apical membrane . Since we were able to coimmunoprecipitate EBP50 and GFP-YAP65, but not GFP-YAP65/−4 , we conclude that association with EBP50 functions to recruit YAP65 to the apical compartment in polarized cells. Together with the overexpression studies, our ability to coimmunoprecipitate endogenous EBP50 and YAP65 from 16HBE14o− cells suggests that the two proteins stably associate. However, we cannot rule out the possibility that the binding of YAP65 to other apical membrane PDZ proteins is also involved in the targeting of YAP65. Two additional PDZ proteins, E3KARP and PDZK1, share significant sequence identity with the EBP50 PDZ domains , and both proteins may associate with YAP65 in vitro (Kultgen, P., and S.L. Milgram, unpublished results). Thus, it is possible that YAP65 associates with several apical membrane PDZ proteins including EBP50, and that together these proteins are responsible for the compartmentalization of YAP65 in the subapical compartment. The generation of additional reagents to carefully examine the expression and subcellular distributions of E3KARP and PDZK1 will be needed to resolve this question. YAP65 contains multiple protein–protein interaction domains, and the binding of YAP65 and EBP50 would allow for the recruitment of additional proteins to the subapical compartment. If YAP65 functions as a dimer, as suggested by the presence of the predicted coiled coil, YAP65 could facilitate the formation of EBP50 multimers, thus, generating a larger protein complex. Dimerization of YAP65 might also be involved in mediating, or regulating, its association with other proteins. Since YAP65 contains one or two WW domains it is likely that additional proteins associate with YAP65. Two novel proteins, WBP1 and WBP2, have been identified which associate with the first WW domain of human YAP65 in vitro . Thus, it will be critical to determine whether one or both of these proteins associate with YAP65 in cells, and what functional roles these interactions may play. YAP65 was cloned using an anti–idiotype antibody directed against the protooncogene c-Yes, a member of the Src family of NRTKs. Although the two proteins were clearly shown to associate in vitro, our GST-EBP50 pull-down experiments demonstrate for the first time that YAP65 and c-Yes stably associate in cells . There is considerable overlap in the distributions of YAP65 and c-Yes in our cell culture model systems . Furthermore, a significant fraction of the endogenous c-Yes expressed in 16HBE14o− cells was redistributed from the apical membrane in cells overexpressing GFP-YAP65/−4 . Collectively, these data suggest that in epithelial cells one function of YAP65 is to target c-Yes to the apical cell surface. Thus, similar to AKAPs (A kinase anchoring proteins) and RACKs (receptors for activated C kinase), which interact with protein kinase A and C, respectively ( Mochly-Rosen et al. 1991 ; Colledge and Scott 1999 ; YAP65 may restrict the activity and enhance the specificity of c-Yes in epithelia. In addition, it is possible that YAP65 may also modulate the activity of the Src family kinase by a yet unknown mechanism. Additional experiments, both in cells and in vitro, will be required to fully understand the function of these interactions. By comparing the amount of c-Yes in the input sample, and the amount found associated with GST-EBP50 affinity columns and coimmunoprecipitations, we estimate that 15–20% of the c-Yes expressed in 16HBE14o− cells was copurified together with YAP65. These results are not surprising since many other proteins are known to associate with Src family kinases, and these protein associations are regulated dynamically by extracellular stimuli . Although we clearly see colocalization of EBP50, YAP65, and c-Yes in the well differentiated primary nasal epithelial cultures and 16HBE14o− cells, we also find c-Yes in other cellular compartments . In kidney epithelial cells stably overexpressing epitope-tagged ezrin, c-Yes was found in ezrin immunoprecipitates . Since ezrin and EBP50 are known to associate , we have not ruled out the possibility that a portion of the activity associated with EBP50 was actually bound by ezrin. However, a direct association between ezrin and c-Yes has not been demonstrated. In biochemical assays, YAP65 was also shown to bind the SH3 domain of c-Src and c-Yes, although the affinity for c-Yes was greater . Since c-Src is expressed in 16HBE14o− cells , we also probed GST-EBP50 affinity resins with c-Src antisera, but did not find evidence for c-Src associated with EBP50. Therefore, our data suggest that the YAP65–EBP50 complex specifically associates with c-Yes at the apical cell surface. However, YAP65 likely associates with multiple proteins that contain SH3 domains, so it would not be surprising to find small amounts of c-Src contained within the EBP50 protein complexes. Our GFP-YAP65 cell lines will be very useful for determining whether additional proteins associate with the YAP65 proline-rich motif. Our previous work and the new results reported here identify several members of the EBP50 protein complex, including an adaptor protein (YAP65) and an NRTK (c-Yes). Although we focused on airway epithelia, EBP50, YAP65 and c-Yes are widely expressed in epithelia, and the makeup of EBP50 protein complexes will differ based on the specific genes expressed in each epithelium. One possible function for apical membrane c-Yes is modulation of ion channels. Src family kinases play a critical role in modulating ion transport through voltage and ligand-gated ion channels . Furthermore, Src family kinase–mediated regulation of ion transport may be facilitated by interactions of the kinases with submembranous scaffolding proteins. For example, the association of PSD95 and Fyn in neurons facilitates tyrosine phosphorylation of the N -methyl- d -aspartate receptor . In addition, the direct binding of p56lck to hDlg is thought to be involved in modulation of Shaker Type Kv1.3 K + channels . Src family kinases may be involved in the regulation of Na + /H + exchanger function . Since EBP50 directly binds NHE3 and indirectly associates with c-Yes, it is intriguing to speculate that these interactions are important for modulation of NHE3. Although NHE3 is not expressed in airway epithelial cells , Src family kinases may modulate the activity of other apical membrane conductances in these cells. Since exogenously applied c-Src can regulate the gating of CFTR in heterologous expression systems , it will be important to determine whether c-Yes functions as a regulator of CFTR in vivo. Src family kinases are also involved in the control of gene expression . In addition, a recent report indicates that YAP65 may serve as a transcriptional coactivator in some cells, and exogenously expressed YAP65 was observed in the nucleus of NIH 3T3 fibroblasts . Although we do not see YAP65 in the nucleus of polarized airway epithelial cells grown on permeable supports, it is possible that YAP65 is capable of translocation from the cytoplasm to the nucleus in response to extracellular stimuli. Such regulation may be analogous to that of β-catenin, a protein at the epithelial adherens junction, that translocates from the cell membrane to the nucleus to regulate gene expression . Therefore, proteins contained within EBP50 complexes may be involved in transducing signals from the apical cell surface to the nucleus in response to external stimuli. Further characterization of proteins contained within EBP50 protein complexes in different cell types and the identification of extracellular stimuli that modulate the composition of these complexes will be critical for understanding the regulation of membrane proteins present at the apical cell surface in epithelia. | Study | biomedical | en | 0.999999 |
10562289 | Rabbit anti–mouse claudin-2 pAb and rabbit anti–mouse claudin-3 pAb were raised and characterized previously . Guinea pig anti–mouse claudin-1 pAb was raised against the synthetic polypeptides CPRKTTSYPTPRPYPKPTPSSGKD, which corresponds to the COOH-terminal cytoplasmic domains of mouse claudin-1 (amino acid 186–209). These pAbs were affinity-purified on nitrocellulose membranes with GST fusion proteins with the COOH-terminal cytoplasmic domain of respective claudins. GST fusion proteins were expressed in Escherichia coli (strain DH5α) and purified with the glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). Rat anti–mouse claudin-1 mAb and rat anti–mouse claudin-2 mAb were produced as described previously . Wistar rats were immunized with GST fusion proteins with the COOH-terminal cytoplasmic domain of respective claudins, and the lymphocytes were fused with P3 myeloma cells to obtain hybridoma cells. Fusion plates were screened by immunofluorescence staining of L transfectants expressing claudin-1 or -2 or by immunoblotting of GST fusion proteins. Mouse anti-FLAG mAb (M2) was purchased from Eastman Kodak Co. To construct plasmids for the expression of GST/claudin-1 and GST/claudin-2 fusion proteins in E . coli , cDNAs encoding the COOH-terminal cytoplasmic region of claudin-1 (amino acid 188–211) and that of claudin-2 (amino acid 189–230) were amplified by PCR. Each cDNA fragment was subcloned into pGEX4T-1 (Amersham Pharmacia Biotech) to produce pGEX-IN (GST/claudin-1) and pGEX-IU (GST/claudin-2). The plasmid for the expression of GST/claudin-3 was constructed as described previously . The mammalian expression vectors for claudin-1, -2, and -3 were constructed as follows. The cDNA fragment containing the whole open reading frame of claudin-1 or -2 was excised from pGTCL-1 or pGTCL-2 by SacI or EcoRI digestion, respectively. Each DNA fragment was subcloned into pCAGGSneodelEcoRI to produce the expression vectors pCCL-1 (claudin-1) and pCCL-2 (claudin-2). Similarly the whole open reading frame of claudin-3 cDNA was subcloned into pCAGGSneodelEcoRI to construct the expression vector pCCL-3 . To construct the expression vector for the claudin-1 mutant lacking its COOH-terminal cytoplasmic domain, the cDNA fragment of claudin-1 corresponding to amino acid 148–188 was obtained by PCR using primers GGCGACATTAGTGGCCACAGCATG (sense) and CGCGGATCCTTTCCGGGGACAGGAGCA (antisense), followed by EcoRI and BamHI digestion. The cDNA fragment of claudin-1 encoding amino acid 148–211 was removed from the plasmid pSKCL-1F, which contained FLAG-tagged claudin-1 cDNA , by EcoRI and BamHI digestion and was replaced by this PCR-amplified truncated cDNA fragment. Then the FLAG-tagged COOH-terminal cytoplasmic domain-deleted claudin-1 cDNA was subcloned into the expression vector pCAGGSneodelEcoRI to make pCCL-1ΔCF. To establish L transfectants singly expressing claudin-1, -2, or -3 (C1L, C2L, and C3L, respectively), mouse L cells cultured in 35-mm dishes were transfected with 1 μg of pCCL-1, pCCL-2, or pCCL-3 in 1 ml of Opti-MEM using lipofectamine plus (GIBCO BRL). After 14–16-d selection in DME containing 500 μg/ml of G418, resistant colonies were picked up. Isolated clones were screened by immunofluorescence microscopy. C1C2L cells coexpressing claudin-1 and -2 were established by transfection of C1L cells with a mixture of 1 μg of pCCL-2 and 0.1 μg of pPGKpuro, and then selected in DME containing 8 μg/ml of puromycin. Similarly, C1C3L and C2C3L cells were produced by transfecting C3L cells with pCCL-1 and pCCL-2, respectively, together with pGKpuro. C1FC2L cells coexpressing claudin-1 with a FLAG tag at its COOH terminus and claudin-2 were established by transfecting C1FL cells expressing FLAG-claudin-1 with 1 μg of pCCL-2 and 0.1 μg of pSV2hph , followed by selection in 200 μg/ml of hygromycin B. C1ΔCFL cells expressing COOH-terminal cytoplasmic domain-truncated claudin-1 with FLAG-tag was produced by transfecting L cells with pCCL-1ΔCF, followed by G418 selection. Several stable clones were isolated for each transfection experiment. Among these, clone 16 of C1L, clone 12 of C2L, clone 17 of C3L, clone 1 of C1C2L, clone 12 of C1C3L, clone 15 of C2C3L, clone 11 of C1FC2L, and clone 16 of C1ΔCFL were recloned and used for this study, since they expressed relatively large amounts of introduced proteins. Mouse L cells and transfectants were cultured in DME supplemented with 10% FCS. SDS-PAGE was performed according to the method of Laemmli 1970 , and proteins were electrophoretically transferred from gels onto polyvinylene difluoride membranes. The membranes were soaked in 5% skimmed milk and incubated with the primary antibodies. After washing, the membranes were incubated with the second antibodies for rat, rabbit (Amersham Pharmacia Biotech), or guinea pig (Chemicon International, Inc.) IgG, followed by incubation with streptavidin-conjugated alkaline phosphatase (Amersham Pharmacia Biotech). Nitroblue tetrazolium and bromochloroindolyl phosphate were used as substrates to visualize the enzyme reaction. L transfectants (1.5 × 10 6 cells) were plated on 60-mm culture dishes with coverslips. For coculture, 7.5 × 10 5 cells of each transfectant were mixed and plated. After a 48-h culture, cells on coverslips were fixed with 1% formaldehyde in PBS for 10 min at room temperature, washed with PBS, and treated with 0.2% Triton X-100 in PBS for 10 min. Cells were then washed with PBS, soaked in 1% BSA in PBS, and incubated with primary antibodies for 30 min in a moist chamber. After washing with PBS three times, cells were incubated with the fluorescently labeled second antibodies for 30 min. FITC-conjugated goat anti–rat IgG (BioSource International), Cy3-conjugated goat anti–rabbit IgG (Amersham Pharmacia Biotech), Cy3-conjugated goat anti–mouse IgG (Amersham Pharmacia Biotech), rhodamine-conjugated goat anti–guinea pig IgG, and FITC-conjugated goat anti–rabbit IgG (Chemicon International, Inc.) were used as secondary antibodies. Cells were washed three times with PBS and then mounted in 90% glycerol-PBS containing para-phenylenediamine and 1% n- propylgalate. Frozen sections of mouse liver were stained immunofluorescently, as described previously . Specimens were observed using a fluorescence Zeiss Axiophot photomicroscope, and the images were recorded with a SensysTM cooled CCD camera system (Photometrics). For conventional freeze-fracture EM, L transfectants cultured on 60-mm dishes were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) overnight at 4°C, washed with 0.1 M sodium cacodylate buffer three times, immersed in 30% glycerol in 0.1 M sodium cacodylate buffer for 2 h, and then frozen in liquid nitrogen. Frozen samples were fractured at −100°C and platinum-shadowed unidirectionally at an angle of 45° in Balzers Freeze Etching System (BAF060, BAL-TEC). Samples were then immersed in household bleach, the replicas floating off the samples were picked up on formvar-filmed grids and examined with a JEOL 1200 EX electron microscope at an acceleration voltage of 100 kV. Immunoelectron microscopy for examining freeze-fracture replicas was performed as described by Fujimoto 1995 . Mouse liver was cut into small pieces and quickly frozen in high pressure liquid nitrogen with an HPM010 high pressure freezing machine (BAL-TEC). L transfectants were fixed with 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 5 min at room temperature, washed three times in 0.1 M phosphate buffer, immersed in 30% glycerol in 0.1 M phosphate buffer for 3 h, and then frozen in liquid nitrogen. Frozen samples were then fractured and shadowed as described. Samples were immersed and stirred in lysis buffer containing 2.5% SDS, 10 mM Tris-HCl, and 0.6 M sucrose (pH 8.2) for 12 h at room temperature, then replicas floating off the samples were washed with PBS containing 5% BSA. The replicas were incubated with primary antibodies for 2 h, washed with PBS-BSA, incubated with colloidal gold-conjugated secondary antibodies, washed with PBS-BSA, and then picked up on formvar-filmed grids. Goat anti–rabbit IgG coupled with 5-nm gold, goat anti–mouse IgG coupled with 15-nm gold (Amersham Pharmacia Biotech), and goat anti–guinea pig IgG coupled with 15-nm gold (British BioCell International) were used as secondary antibodies. We raised monoclonal and polyclonal antibodies against the COOH-terminal cytoplasmic domains of claudin-1, -2, and -3. Immunoblotting of GST fusion proteins with the COOH-terminal tails of claudin-1, -2, and -3 confirmed the specificity of these antibodies . Then, cDNAs encoding claudin-1, -2, and -3 were transfected into L fibroblasts and stable transfectants were obtained (C1L, C2L, and C3L cells, respectively). When the total cell lysates of these transfectants were immunoblotted with anticlaudin-1, -2, and -3 pAbs, bands of respective claudins with the expected molecular masses were clearly detected . Previously, we obtained L transfectants stably expressing claudin-1 or -2 that were tagged with a FLAG sequence at their COOH termini (C1FL and C2FL cells, respectively) and found that introduced FLAG-claudin-1 and -2 were concentrated at cell–cell contact planes in an elaborate network pattern . Immunofluorescence microscopy of C1L, C2L, and C3L cells with anticlaudin-1, -2, or -3 antibodies revealed that introduced claudin-1, -2, and -3 without an epitope tag were also highly concentrated at cell–cell borders to form elaborate networks . Similar to C1FL and C2FL cells, well-developed networks of rTJ strands/grooves were induced at these cell–cell contact planes of C1L, C2L, and C3L cells, as revealed by conventional freeze-fracture EM . Judging from the morphological characteristics, as well as the size of the networks detected by immunofluorescence microscopy at cell–cell contact planes , each line in these networks can be regarded to represent individual rTJ strands. These immunofluorescence observations further confirmed the specificity of anticlaudin-1, -2, and -3 pAbs and mAbs used in this study. Northern blotting revealed that several distinct species of claudins are coexpressed in individual organs, such as the lung, liver, and kidney . Since claudin-1, -2, and -3 appeared to be expressed in the liver, we first double stained frozen sections of the mouse liver with guinea pig anticlaudin-1 pAb/rabbit anticlaudin-2 pAb or guinea pig anticlaudin-1 pAb/rabbit anticlaudin-3 pAb. As shown in Fig. 3 , a–d, claudin-1, -2, and -3 were colocalized at the junctional complex regions along bile canaliculi. These findings indicated that claudin-1, -2, and -3 were evenly mixed in these cells, at least at the level of immunofluorescence microscopy. Furthermore, the freeze-fracture replicas obtained from mouse liver were double labeled with anticlaudin-1 pAb and anticlaudin-3 pAb . Although, with unknown reason, the labeling ability of guinea pig anticlaudin-1 pAb for freeze-fracture replicas was very low, the 15-nm and 5-nm gold particles representing the localization of claudin-1 and -3, respectively, appeared to be admixed along individual TJ strands. Therefore, at the level of TJ strands, it was likely that claudin-1, -2, and -3 were copolymerized into individual TJ strands as heteropolymers of claudins . To evaluate this possibility, we used L transfectants as a model system. As shown previously and in Fig. 2 C, claudin-1, -2, and -3 can form homopolymers in L transfectants. We then cotransfected claudin-1 and -2, claudin-1 and -3, or claudin-2 and -3, and obtained stable transfectants . When confluent cultures of C1C2L cells were double stained with anticlaudin-1 mAb and anticlaudin-2 pAb, both species of claudins showed the same concentration pattern at cell–cell contact planes . Also, at higher magnification, almost identical network patterns of staining were observed at cell–cell contact planes by anticlaudin-1 mAb and anticlaudin-2 pAb . Similarly, claudin-1 and -3, and claudin-2 and -3 were also colocalized in C1C3L and C2C3L cells, respectively . Next, we observed the morphology of rTJ strands in C1C2L, C1C3L, and C2C3L cells by conventional freeze-fracture EM. As previously shown in C1FL and C2FL cells , in glutaraldehyde-fixed C1L cells claudin-1–induced strands were largely associated with the protoplasmic (P) -face as mostly continuous structures with vacant grooves at the extracellular (E) -face , whereas in C2L cells claudin-2–induced strands were discontinuous at the P-face with complementary grooves at the E-face that were occupied by chains of particles . Similar to C1L cells, P-face–associated TJs were induced in C3L cells . Interestingly, C1C3L cells bore typical P-face–associated TJs , whereas C1C2L and C2C3L cells showed an intermediate morphology of rTJ strands/grooves between P- and E-face–associated TJs ; the grooves at E-face of C1C3L cells were completely vacant , whereas those in C1C2L and C2C3L cells were characterized by evenly scattered particles . These findings suggested that two distinct species of claudins were completely mixed up in individual rTJ strands in these L transfectants. To confirm this speculation, the freeze-fracture replicas obtained from confluent C1C2L cells were double immunolabeled with guinea pig anticlaudin-1 pAb and rabbit anticlaudin-2 pAb. As shown in Fig. 6 a, the 15- and 5-nm gold particles representing the localization of claudin-1 and -2, respectively, appeared to be admixed along individual rTJ strands. Similar results were also obtained in C1C3L cells . However, as described above, the labeling ability of anticlaudin-1 pAb for freeze-fracture replicas was fairly low . Therefore, we obtained L transfectants coexpressing claudin-2 and FLAG-tagged claudin-1 (C1FC2L cells), and the freeze-fracture replicas obtained from confluent C1FC2L cells were double labeled with anti-FLAG mAb (15-nm gold particles) and anticlaudin-2 pAb . In these images, numerous 15- and 5-nm gold particles appeared to distribute evenly and specifically along individual rTJ strands. Taken together with Fig. 5 , we concluded that in the model system of L transfectants, distinct species of claudins can be copolymerized to form rTJ strands as heteropolymers . As described in Fig. 1 A, in TJs each TJ strand associates laterally with another TJ strand in the apposing membrane, which is responsible for intercellular adhesion at TJs. The next question was whether this lateral association, i.e., the cell adhesion at TJs, is attributable to homophilic or heterophilic interactions of the extracellular regions of claudins. Since paired rTJ strands were induced in C1L, C2L, and C3L cells, the homophilic interaction of claudins, as described in Model A can be expected in these cells. We next examined the possibility of the heterophilic interaction of claudins between homopolymers by coculturing pairs of C1L, C2L, and C3L cells. As shown in Fig. 7 a–c, when C1L and C3L cells were cocultured and double stained with anticlaudin-1 mAb and anticlaudin-3 pAb, three types of cell–cell contact planes were distinguished in terms of immunofluorescence staining: the claudin-1–positive/claudin-3–negative planes, which would be formed between adjacent C1L cells; the claudin-1–negative/claudin-3–positive planes, which would be formed between adjacent C3L cells; and the claudin-1–positive/claudin-3–positive planes, which would be formed between adjacent C1L and C3L cells. At higher magnification, in the claudin-1–positive/claudin-3–positive planes, both claudin-1 and claudin-3 in C1L and C3L cells, respectively, were concentrated in an elaborate network pattern , and their networks were mostly overlapped . The same results were obtained in cocultures of C2L and C3L cells . These findings indicated that claudin-3–based homopolymers (rTJ strands) can associate laterally with claudin-1– and claudin-2–based homopolymers through the heterophilic interactions of claudin-3 with claudin-1 and claudin-2, respectively. In contrast, when C1L and C2L cells were cocultured and double stained with anticlaudin-1 mAb and anticlaudin-2 pAb, the claudin-1–positive/claudin-2–positive planes were not formed . Therefore, it was likely that, at least in L transfectants, claudin-1 cannot interact with claudin-2 in a heterophilic manner. In conclusion, in L transfectants, paired strands can be formed not only by homophilic interactions, but also by heterophilic interactions of claudins, except for some combinations of claudins. To further confirm this conclusion, confluent cocultures of C2L and C3L cells were fixed with glutaraldehyde and examined by conventional freeze-fracture EM . As described above, induced TJs in C2L and C3L cells were E- and P-face–associated, respectively, i.e., the fracture planes at C2L/C2L contact planes showed discontinuous strands on the P-face and grooves occupied with chains of particles on the E-face , while the fracture planes at the C3L/C3L contact planes showed continuous strands on the P-face and vacant grooves on the E-face . In the C2L/C3L coculture, in addition to these C2L/C2L and C3L/C3L fracture planes, fracture planes, which would be derived from the contact regions between adjacent C2L and C3L cells, were occasionally identified. In these planes, as expected, E-C2L and P-C3L or E-C3L and P-C2L (data not shown) were seen to be paired. Interestingly, as exemplified in Fig. 8 c, when the fracture plane jumped from the E- to the P-face, the continuity of the network pattern of grooves of E-C2L and strands of P-C3L was completely maintained, conclusively indicating that in these planes individual claudin-2 homopolymers in C2L cells always associated laterally with claudin-3 homopolymers to form TJs in adjacent C3L cells. In this study, we discussed the formation of TJ strands from the viewpoint of claudin–claudin interaction. However, we should consider the possible involvement of peripheral membrane proteins in TJ strand formation. Since the COOH-terminal cytoplasmic domain of claudins was thought to be responsible for their interactions with peripheral membrane proteins, we constructed a claudin-1 mutant lacking its COOH-terminal cytoplasmic domain (claudin-1ΔC), and obtained L transfectants expressing FLAG-tagged claudin-1ΔC . These claudin mutants were concentrated at cell–cell borders as shown immunofluorescently in Fig. 9 B. Freeze-fracture replicas obtained from C1ΔCFL cells revealed that this claudin-1 mutant still bore well-developed network of rTJ strands , and interestingly, these rTJ strands were largely associated with the P-face as mostly continuous structures with vacant grooves at the E-face. These findings suggested that the interaction between claudins and peripheral membrane proteins was not required for the formation of TJ strands, per se, as well as their P-face association. Previous studies indicated that claudins are polymerized within plasma membranes to constitute the backbone of TJ strands . Furthermore, it is accepted that each TJ strand laterally associates with another TJ strand in apposing membranes to form paired strands. Therefore, there would be two methods of interaction between claudins; the side-by-side interaction for polymerization to form TJ strands, and the head-to-head interaction between each of the paired strands for cell adhesion. Since claudins comprise a multigene family , each of the above methods of interaction can be further subdivided into homo- and heterophilic interactions . Therefore, the possible molecular organizations of paired TJ strands can be subclassified into four models . In this study, we evaluated these models using L transfectants expressing claudin-1, -2, and -3 singly or in combination. First, a single species of claudins was sufficient to reconstitute TJ strands, indicating that homopolymers were formed in these cells . Furthermore, these homopolymers were associated laterally, both in a homophilic , as well as heterophilic manner . Therefore, TJs in L transfectants can adopt both organizations represented in Model A and B . On the other hand, heteropolymers were also induced in L transfectants . Models C and D were not distinguished in the L transfectant system, but since both Model A and B were possible, both Model C and D would also be possible . The thickness of TJ strands is ∼10-nm in freeze-fracture replica images . Interestingly, the gap junction channel consisting of six connexins is also ∼10-nm in diameter . Since connexin also has four transmembrane domains with the same membrane topology as claudins, it is tempting to speculate that oligomers of claudins are also unit structures in TJs, and that these unit structures are arranged linearly to form individual TJ strands. This raises a new question as to whether these unit structures themselves are homomeric or heteromeric, further subdividing the above four models; heteropolymers can be formed by linearly aligned heteromeric unit structures, as well as distinct homomeric unit structures. This type of discussion has been reported in detail for gap junctions, in which the unit structures have been determined , but in TJ strands the clarification of unit structures is a prerequisite for further discussion. Northern blotting showed that most tissues expressed more than two species of claudins . Immunofluorescence microscopy revealed that TJs in situ contained claudin-4 and -8 in the kidney and claudin-1, -2, and -3 in the liver . Therefore, taking the results obtained from the cotransfection experiments , it is likely that in situ most TJ strands are heteropolymers of claudins, although specialized TJ strands in myelin sheaths of oligodendrocytes and in Sertoli cells in the testis appeared to be mainly composed of a single species of claudin, claudin-11 . As shown in this study, not only homophilic, but also heterophilic interactions of claudins between each of the paired TJ strands are allowed. Thus, in the paired TJ strands of heteropolymers in situ homophilic interactions of various claudin species, as well as heterophilic interactions between various combinations of claudin species, are expected to occur, as shown in Model D . It is reasonable to postulate that the strength of these interactions varies depending on the claudin species involved and their combinations, and that the tightness of each TJ strand is determined as a whole by the number/type of species of claudins and their mixing ratio in the strand. For example, MDCK I cells have a fairly tighter TJ barrier than MDCK II cells, but no difference was detected in the number of TJ strands between these two distinct clones of MDCK cells . These observations suggested a qualitative difference of the paired TJ strands in their tightness between these two clones, and this difference could be explained as postulated above. To avoid further complexity, we have not discussed occludin. Immunoreplica analyses indicated that occludin was also incorporated into TJ strands in situ in most types of epithelial cells . When occludin was cotransfected into L cells, together with claudin-1, they were coincorporated into rTJ strands . Occludin singly introduced into L cells was concentrated at cell–cell borders in a punctate manner, indicating that occludins interact with each other in a homophilic manner between adjacent cells . Furthermore, when L transfectants expressing occludin were cocultured with C1L cells, neither occludin or claudin-1 was concentrated at cell–cell borders, suggesting that occludin does not interact with claudin-1 in a heterophilic manner between adjacent cells (Furuse, M., and S. Tsukita, unpublished data). Therefore, at present it is very difficult to postulate how occludin is incorporated into the paired heteropolymers of claudins (TJ strands) in situ. Occludin may be incorporated into claudin-based TJ strands in the same manner as claudins, and in the paired TJ strands occludin may be positioned opposite to occludin, which allows occludin–occludin interaction . Alternatively, it may be positioned opposite to a claudin which may result in the formation of small pores. It is also possible that occludin is involved in TJ strand formation differently from claudins. Finally, we should discuss the possible role of peripheral membrane proteins, such as ZO-1 , ZO-2 , and ZO-3 in the formation of TJ strands. ZO-1, but not ZO-2 or ZO-3, was expressed endogenously in L cells, and this endogenous ZO-1 was coconcentrated with claudin-1 and -2 at cell–cell borders as an elaborate network in C1L and C2L cells, respectively (Itoh, M., M. Furuse, K. Morita, and S. Tsukita, unpublished data). In our previous study , we reported that claudin-1 and -2 with a FLAG tag at their COOH-termini also formed a well-developed network of rTJ strands in L transfectants. Interestingly, however, endogenous ZO-1 was not recruited to these FLAG-claudin-1 or FLAG-claudin-2–based rTJ strands, probably because the FLAG sequence affected the claudin/ZO-1 interaction (Furuse, M., and S. Tsukita, unpublished data). These findings indicated that ZO-1 (and also ZO-2 and ZO-3) was not required for the formation of rTJ strands in L transfectants. Furthermore, Fig. 9 showed that a claudin-1 deletion mutant lacking almost all of its COOH-terminal cytoplasmic domain still formed a well-developed network of rTJ strands in L transfectants, favoring the notion that the peripheral membrane proteins are not involved in the polymerization of claudins within plasma membranes. Therefore, we interpreted the data presented in this study without considering the possible involvement of peripheral membrane proteins. The model system of L transfectants used in this study was very useful to analyze the properties of each claudin species. To date, 15 members of the claudin family have been identified, but it remains unclear how many claudins will be identified in the future. It is thus very difficult, by the use of epithelial cells, to clarify the nature and function of each claudin species, partly because we cannot determine exactly all the types of claudins expressed in certain epithelial cells. Therefore, to further understand the structure and functions of claudins and TJ strands, the L transfectant system will continue to be used towards the reconstitution of TJs as a complementary technique to the knockout of each claudin gene. Note Added in Proof. Positional cloning has identified a new member of the claudin family (paracellin-1/claudin-16) in which mutations cause hereditary renal hypomagnesemia in humans . This finding favors the idea that claudins possibly are involved in the formation of aqueous pores within TJ strands. | Study | biomedical | en | 0.999996 |
10562313 | p53 −/− mouse embryonic fibroblasts (MEFs; from T. Jacks, Massachusetts Institute of Technology, Cambridge, MA) were sequentially infected with pWZL-Blast-rtta, a blasticidin selectable retroviral vector expressing the reverse transactivator of the tetracycline inducible system 22 , and pBabe-puro-tet-GFP-p53-sin, a self-inactivating retrovirus expressing a GFP-p53 fusion protein under the control of the tetracycline inducible promoter. Cells were drug selected, and a clone (TGP53-4) was isolated that showed observable GFP-p53 expression, and growth arrest of the cells after addition of 1 μg/ml doxycycline to the media. EcoRI and SalI sites were introduced immediately 5′ and 3′ to the open reading frame of human MIF by PCR, and this EcoRI-SalI fragment was cloned into EcoRI-XhoI sites of pMal-C2 (New England Biolabs). A maltose binding protein (MBP)–MIF fusion was expressed in BL21 Escherichia coli cells, affinity purified by amylose chromatography, and cleaved using factor Xa. MBP was removed after cleavage by amylose chromatography. Since MBP had no effect in any of the assays used, some experiments were performed using rMIF immediately after cleavage. TGP53-4 cells were infected with a pHygroMarx I–derived provirus containing MIF cDNA or empty vector control. After hygromycin selection, cells were plated at ∼5,000 cells/plate. 1 μg/ml doxycycline was added to induce p53 expression in appropriate plates. Media were replaced every 3 d, containing fresh doxycycline where necessary. After 10 d, cells were fixed in 1% glutaraldehyde and stained with 0.25% crystal violet. For experiments using soluble rMIF, TGP53-4 cells were plated at ∼10,000 cells/plate in the presence or absence of 150 ng/ml of rMIF added to the growth media. 24 h later, doxycycline was added to induce p53 expression. Media were replaced every 3 d containing fresh doxycycline and/or rMIF. After 9 d, cells were fixed and stained as above. MEFs were prepared from 14-d CD1 mouse embryos, and were repeatedly passaged. Where necessary, cells were infected in passage 2 with pMARXIV-p53αs, pWZLneo-MIF, or control viruses, and selected by drug resistance for the selectable marker. One passage before the onset of senescence (usually around passage 4–5), cells were split and plated at ∼300,000 cells/plate in the presence or absence of rMIF. Fresh tissue culture media (containing rMIF where appropriate) were replaced every 3 d. After 15–17 d, cells were fixed in 1% glutaraldehyde and stained with crystal violet. To determine cell concentration, crystal violet was resolubilized in 10% acetic acid and absorbance at 595 nm was analyzed using a Bio-Rad 550 microplate reader. Rat-1/mycER cells were infected with retroviruses expressing LacZ, MIF, or Bcl2 cDNAs. After drug selection, cells were plated onto acid-washed coverslips at low density and shifted to media containing 0.1% fetal bovine serum (FBS) plus 0.1 μM estradiol to induce apoptosis. After 24 h, cells were stained with 4 mg/ml Hoechst 33342 for 10 min, then washed and scored by fluorescent microscopy. Cells containing condensed or fragmented DNA cells were scored as apoptotic cells. At least 100 fields/slide were analyzed by two independent observers. RAW264.7 macrophages were pretreated with varying concentrations of MIF for 24 h, and then treated with 0.25–1.0 mM sodium nitroprusside (SNP) or 0.5–1 mM S -nitrosoglutathione (GSNO) for 8 h to 2 d. Cells containing condensed or fragmented DNA after very brief fixing with paraformaldehyde and staining with Hoechst 33258 were scored as apoptotic cells. TGP53-4 cells were split onto coverslips in the presence or absence of 150 ng/ml MIF. 24 h later, 1 μg/ml doxycycline was added to the media. 16 h after doxycycline addition, cells were washed in PBS and fixed in 2% paraformaldehyde, and GFP-p53 was visualized with a Zeiss Axiophot fluorescent microscope using a standard FITC filter set. Cells were washed in PBS, harvested in PBS, centrifuged, and lysed. Equal amounts of total protein (30–300 μg) were heat-denatured, separated on a 10% SDS-polyacrylamide gel, and blotted to nitrocellulose. Blots were probed with antibodies that recognize p53 (DO-1, FL-393; Santa Cruz Biotechnology), MDM2 (SMP-14; Santa Cruz Biotechnology), Bax (BAX Δp21; Santa Cruz Biotechnology), or p21 23 followed by a horseradish peroxidase–conjugated anti–mouse antibody, and detected using enhanced chemiluminescence. Total RNA was prepared from TGP53-4 cells after induction of GFP-p53. 10 μg was separated in a 1% formaldehyde gel and blotted to Hybond-N + membranes. Blots were probed with random primed radiolabeled probes corresponding to the full-length coding sequence of mouse p21 and cyclin G. Radioactive signals were quantified using a Fuji FLA-200 phospho/fluorescent imager, and normalized to loaded RNA by quantification of fluorescence of ethidium bromide–stained ribosomal RNA bands in the RNA gel, or after blotting to the membrane. TGP53-4 cells were cotransfected with PG13, a plasmid which carries firefly luciferase under the control of three tandem copies of a p53-responsive consensus sequence, and pCDNA3-β-gal, a plasmid which carries β-galactosidase under the control of the CMV promoter. 1 d after transfection, cells were split, pooled, and replated at ∼500,000 cells/plate. 150 ng/ml rMIF was added to half of the plates. The next day, 1 μg/ml doxycycline was added to the media to induce GFP-p53. At 0 and 10 h after induction, extracts were prepared, and luciferase and β-galactosidase activities were assayed using Promega kits. Luciferase reporter activities were normalized to β-galactosidase expression levels. To identify novel regulators of p53 activity, we undertook a screen to identify genes that, when expressed at high level, were capable of bypassing p53-mediated growth arrest. A p53 −/− MEF cell line was engineered to express a GFP-p53 fusion protein under the control of a tetracycline (doxycycline)-inducible promoter ( 22 ; TGP53-4 cell line). GFP-p53 fusion proteins are localized normally and can transactivate target genes ( 24 ; and data not shown). After addition of doxycycline to the media, the p53 fusion protein was induced, and cells became growth arrested and failed to form colonies. We used the TGP53-4 cell line in a phenotype-based screen to identify negative regulators of p53 activity. These cells were infected with an A431 epidermoid carcinoma–derived cDNA library in a Moloney murine leukemia virus (MMLV)–based retroviral vector, pHygroMarx I 25 . pHygroMarx I contains a bacterial origin of replication, zeocin resistance marker between the LTRs, and a loxP site in the 3′ LTR, which is duplicated upon integration, to facilitate provirus recovery by Cre-mediated excision after integration into the genome. LinX 25 ecotropic retrovirus producer cells were transiently transfected with this library, and after 3 d, supernatant was used to infect TGP53-4 cells. Approximately 4 × 10 6 cells were infected. After drug selection for the library vector, cells were split at varying dilutions, and 1 μg/ml doxycycline was added to the media to induce expression of the GFP-p53 fusion protein. When necessary, cells were split again to improve colony discrimination. Cells that were no longer inhibited by p53 induction gave rise to colonies in the presence of doxycycline. These clones were infected with pBabe-puro-Cre, a Moloney murine leukemia virus–based virus that strongly expresses Cre recombinase to excise the provirus. Proviruses containing cDNAs from positive clones were recovered by Hirt extraction. Proviruses were recovered from a total of 50 positive colonies. Nucleotide sequencing and database analysis revealed that cDNAs recovered from five different colonies encoded the same protein, human MIF, a cytokine that was shown previously to exert both local and systemic proinflammatory activities 26 . All cDNAs encoding MIF were full length and in the sense orientation. The complete upstream regions were sequenced from three of these recovered cDNAs. Two differed in the precise 5′ terminus, indicating that they were derived from independent clones. A cDNA-encoding MIF was also independently isolated in a similar phenotype-based screen to identify negative regulators of myc-dependent apoptosis in rat fibroblasts. Rat-1 fibroblasts expressing a c-myc–estrogen receptor fusion protein (Rat-1/mycER) were infected with pools of a cDNA library prepared from Rat-1/mycER cells committed to apoptosis in pHygroMarx II. After drug selection, cells were induced to undergo apoptosis by shifting to low serum media (DMEM + 0.1% FBS) plus 0.1 μM estradiol (to induce c-myc activity) for 3 d, followed by 2 d of serum starvation without estradiol. Cells that were protected from apoptosis were recovered in media containing 10% FBS. Rescued cells were subjected to three additional cycles of apoptotic induction. Proviruses were recovered from apoptosis-resistant cells by Cre-mediated excision of genomic DNA line 27 . Since this screen was carried out in a cell line expressing wild-type p53, and myc-driven apoptosis is largely p53 dependent 28 , inhibitors of p53 function were expected to be recovered from this screen. To confirm that MIF was capable of bypassing p53-mediated growth arrest, a provirus containing MIF or a control provirus was transduced into TGP53-4 cells. Doxycycline was added to induce p53 expression. Numerous colonies formed on plates containing MIF-expressing cells, but few or no colonies formed on plates containing control cells . Since MIF was originally identified as an extracellular cytokine, we tested whether MIF protein could overcome p53-mediated growth arrest upon addition as a recombinant protein to the culture medium. MIF protein was produced as an MBP fusion protein, and cleaved to separate MIF from MBP . TGP53-4 cells were grown in the presence or absence of recombinantly produced MIF (rMIF) and doxycycline. Colony formation was observed in the absence of doxycycline, or in the presence of doxycycline, and rMIF, but not in the presence of doxycycline alone. Therefore, MIF was capable of bypassing p53-mediated growth arrest when added as a soluble factor . p53 might be inactivated by altering its subcellular localization, by decreasing protein levels, or by suppressing its ability to function as a transcriptional activator. Since GFP-p53 can be visualized directly in cells and shows normal subcellular localization, we analyzed whether p53 showed altered subcellular localization in the presence of MIF. No obvious difference in the subcellular localization of GFP-p53 was observed; p53 showed nuclear localization irrespective of MIF treatment . p53 can also be regulated by altering protein abundance; however, p53 protein levels were not reduced after MIF treatment . p53 primarily functions via its ability to transactivate gene expression. Therefore, we tested whether MIF treatment interfered with this activity. After induction of p53, RNA was prepared from TGP53-4 cells grown in the presence or absence of MIF. The abundance of two p53 transcriptional targets, p21 29 30 31 and cyclin G 32 , was assessed by Northern blot . Levels of p21 and cyclin G in MIF-treated cells were decreased to ∼50 and 40% of control levels . In addition, p53-dependent induction of MDM2, another p53 target which acts in a feedback loop to negatively regulate levels of p53 33 34 was decreased in MIF-treated cells . The effect of MIF treatment on the activity of a p53-dependent reporter was also assayed. TGP53-4 cells were transfected with PG13-luc, a plasmid which carries firefly luciferase under the control of tandem copies of a p53-responsive consensus sequence 35 , in the presence and absence of MIF, and luciferase activity was assayed after induction of GFP-p53. Treatment with rMIF suppressed p53-dependent luciferase expression . Considered together, these data suggest that MIF treatment bypassed p53-mediated growth arrest by suppressing p53-dependent transcriptional activation. In addition to its ability to induce growth arrest, p53 functions to induce apoptosis in response to cellular stress in susceptible cells 5 7 8 . As described above, we isolated a cDNA-encoding MIF in a screen designed to identify inhibitors of myc-dependent apoptosis, a process which is largely p53-dependent. To formally confirm that MIF expression could suppress this phenotype, Rat-1/mycER cells were infected with an MIF-expressing virus and control viruses, and apoptosis was induced by serum starvation and estradiol treatment. Cells that expressed MIF were partially protected from apoptosis under these conditions, though not as efficiently as cells that expressed Bcl2 . Since MIF regulates numerous functions of macrophages in in vitro assays and in vivo, we also tested whether MIF treatment was capable of inhibiting apoptosis in macrophages. After activation, macrophages release nitric oxide (NO) as part of their antimicrobial repertoire. However, high levels of NO can, in turn, cause macrophage apoptosis. For example, apoptosis is induced by treatment of RAW264.7 macrophages with cytokines that induce endogenous production of NO, or with chemical releasers of NO. Apoptosis is associated with induction of p53 and is inhibited by expression of antisense p53 constructs, indicating that NO-induced macrophage apoptosis is p53 dependent 36 37 . To test whether MIF treatment was capable of suppressing NO-induced apoptosis, we treated RAW264.7 macrophages with NO-releasers, SNP, or GSNO, in the presence of various concentrations of rMIF. MIF treatment suppressed NO-induced apoptosis in a dose-dependent manner . p53 also plays a role in controlling the onset of cellular senescence 12 13 14 . Normal primary mouse fibroblasts are capable of a finite number of divisions in culture, and ultimately arrest with a senescent morphology 11 . Loss of p53 allows primary mouse cells to extend their division potential. Thus, in a colony formation assay, cells lacking p53 are capable of forming colonies at passages at which wild-type cells are not. Therefore, we tested whether MIF was capable of elongating the potential life span of primary MEFs. At one passage before the onset of senescence (passage 4–5), primary MEFs were plated in the presence or absence of rMIF. After 15 d, numerous colonies had formed on plates treated with MIF, whereas none were observed in the absence of MIF. This indicated that MIF treatment, like loss of p53, was capable of inducing elongated life span . Colony formation occurred at a frequency of ∼10 −4 colonies/cell (the frequency of colony formation observed with cells expressing an antisense or dominant negative p53 under identical conditions is 2–3 × 10 −4 and 1–3 × 10 −3 with fibroblasts prepared from a p53 −/− mouse; Carnero, A., and D. Beach, unpublished). To determine the concentration of MIF that was optimal for colony-forming activity, we repeated the experiment in the presence of 0–600 ng/ml rMIF. Elongation of life span was dose dependent, with 150 ng/ml giving the most pronounced effect . Since MIF treatment does not completely negate p53-mediated gene expression, we sought to test whether the ability of MIF to induce a p53-related biological activity correlated with the relative suppression of p53-mediated gene expression. Primary MEFs were infected with a virus expressing MIF, an antisense construct directed against p53 or control virus in passage 2. Infected cells were selected for drug resistance, cultured, and plated on duplicate plates in passage 5. At the same time, MEFs in passage 5 were plated in the presence or absence of rMIF. 15 d after plating, one of each duplicate plate was fixed and stained with crystal violet . Protein extracts were prepared from the other duplicate plate, and the levels of p53 and two p53 targets, p21 and Bax 38 , were assayed by Western blot . In each case, the number of colonies observed roughly correlated with the relative suppression of p53 target gene expression, consistent with the hypothesis that suppression of p53 activity is largely responsible for this MIF-induced biological activity. We have demonstrated that MIF treatment was capable of overcoming p53 activity in three distinct biological assays. The ability of a secreted factor to overcome a growth-inhibitory pathway that has been associated with cellular mortality and with the response of cells to genotoxic stress may have an important physiological role. At sites of inflammation, MIF is released from T cells and from macrophages 26 . High local concentrations of MIF contribute to T cell activation and enhance the antimicrobial activity of macrophages 39 40 . When activated, macrophages release NO and other oxide radicals 41 . However, NO can also induce macrophage apoptosis. Since MIF can partially negate the p53 response and can protect macrophages from NO-induced apoptosis, this factor may normally act to protect macrophages from the destructive machinery they use to kill invading organisms. Inflammatory loci are characterized by high rates of cell death and compensatory proliferation in adjacent cells 42 . At the same time, upregulation of p53 is often observed 43 44 . Overcoming p53 activity through MIF action may help to limit the damage response, and therefore to limit the loss of host cells and to permit local cell proliferation for tissue repair. After cessation of the inflammatory state, local levels of MIF decrease, allowing restoration of the normal damage response. However, chronic bypass of p53 function by MIF could contribute to the development of tumors. Loss of p53 function is one of the most common events in human cancer. Cells that lack p53 function have enhanced proliferative potential and display extended life span. In addition, cells lacking functional p53 are deficient in responding to chromosome damage 9 10 . During inflammation, release of highly reactive oxidants by activated phagocytes has been implicated in the induction of DNA damage in neighboring cells 20 21 . In the chronic presence of MIF, cells with attenuated p53 function might continue to proliferate in the presence of DNA damage, and eventually accumulate multiple oncogenic mutations. Several chronic inflammatory conditions are strongly associated with eventual tumor formation 18 19 . For example, ulcerative colitis or Crohn's disease is associated with the eventual development of bowel cancer, whereas reflux esophagitis or Barrett's syndrome has been linked to the development of esophageal cancer. Schistosomiasis infection predisposes to the development of urinary bladder cancer, and long term Helicobacter pylori infection has been implicated in the development of gastric cancer. In some cases of H. pylori infection, ablation of the infectious agent is correlated with reversal of the inflammatory state and with regression of the associated tumor. This suggests that, in this model, at least one tumorigenic event requires continued presence of the inflammatory state, and is reversible 45 . The observation that MIF can interfere with p53 function may provide insight into the mechanisms by which certain chronic inflammatory conditions predispose individuals to tumor formation. | Study | biomedical | en | 0.999999 |
10562314 | Transgenic mice expressing a TCR specific for peptide p33 in association with H-2D b 29 and CD2- 26 and intercellular adhesion molecule (ICAM) 1 -1–deficient 31 mice have been described previously. LCMV-WE was grown on L cells at a low multiplicity of infection. Recombinant vaccinia virus expressing LCMV-GP (Vacc-LCMV-GP) 32 was originally obtained from Dr. D.H.L. Bishop (Oxford University, Oxford, UK) and was grown on BSC cells at a low multiplicity of infection. Vacc-LCMV-GP was inactivated with UV light using an XL-1500 UV cross-linker (Spectronics Corp.). To produce recombinant LCMV-GP for the cross-priming experiments, Vacc-LCMV-GP was inactivated by UV light and used to infect BSC cells at a multiplicity of infection of 10. 24 h later, cells were harvested and sonicated. Cell debris corresponding to 5 × 10 6 cells was injected per recipient. Peptides p33 (KAVYNFATM) and A4Y (KAVANFATM) were generated at the Amgen Institute (Boulder, CO) by a solid phase method using the Fmoc/tBu-based protocol on an ABI-431 instrument. The crude product was purified by HPLC. p33 defines the major CTL epitope on LCMV-GP in the H-2 b haplotype 33 . To prevent disulphide bonds, the COOH-terminal cysteine (C) has been replaced by methionine (M) 34 . Spleen cells from TCR-transgenic control or CD2-deficient mice (10 5 cells/well) were stimulated with thioglycollate-elicited macrophages (5 × 10 4 cells/well) derived from control or ICAM-1–deficient mice pulsed with graded doses of peptide p33 or A4Y in flat-bottom 96-well plates. Macrophages were pulsed for 1 h at 37°C and subsequently washed two times. Proliferation was assessed 36 h later by pulsing cultures with [ 3 H]thymidine for 12 h. Production of IFN-γ was assessed in supernatants as described 35 . Induction of Ca 2+ flux and presence of T cells forming specific conjugates was assessed as described using INDO-1–pulsed T cells 2 . Spleen cells from TCR-transgenic mice (10 5 /well) were mixed with peptide pulsed macrophages (2 × 10 5 /well) from control or ICAM-1–deficient mice, centrifuged, and incubated at 37°C (5% CO 2 ) in IMDM supplemented with 10% FCS in round-bottom 96-well plates. After 4 h, cells were harvested and stained for CD8 (PE; PharMingen) and Vα2 (FITC; PharMingen); Vα2 expression is shown for CD8 + T cells . TCR-transgenic control or CD2-deficient mice were injected intravenously with various doses of peptide p33 in saline. 24 h later, spleen cells were harvested and stained for expression of CD8 (FITC; PharMingen), Vα2 (PE; PharMingen), and CD44 (biotin; PharMingen) followed by streptavidin–allophycocyanin (PharMingen) and analyzed by flow cytometry. Spleen cells from TCR-transgenic CD2-deficient (CD45.2) or control (CD45.1) mice (10 6 cells) were adoptively transferred into normal C57BL/6 recipient mice. 1 h later, mice were challenged with live LCMV, Vacc-GP, UV light–inactivated Vacc-LCMV-GP, or recombinant LCMV-GP. 6 or 8 d later, spleen cells were harvested and stained with anti-CD2 antibodies (FITC), anti-CD8 (allophycocyanin), and anti-Vα2 (PE) or with anti-CD45.1 (FITC), anti-CD8 (allophycocyanin), and anti-Vα2 (PE). We crossed CD2-deficient mice with transgenic mice expressing a TCR specific for LCMV-derived peptide p33 to study activation of CD2-deficient T cells. To this end, splenocytes of CD2-deficient and control TCR-transgenic mice were stimulated with thioglycollate-elicited macrophages pulsed with various concentrations of peptide p33. As shown in Fig. 1 , the absence of CD2 shifted the dose–response curve by a factor of 3–10. To additionally analyze the role of the LFA-1–ICAM-1 interaction in this context, we compared ICAM-1–deficient and control macrophages as APCs for CD2-deficient and CD2-competent transgenic T cells. As reported previously 2 , the absence of LFA-1–ICAM-1 interaction shifted the dose–response curve of T cells upon stimulation with graded doses of peptide by a factor 10. Interestingly, the interference with both CD2 and LFA-1 pathways shifted the dose response by a factor of ∼100. Thus, CD2 and LFA-1 facilitated T cell activation in an additive manner . As expected, if the low-affinity ligand A4Y was used for the experiments, a similar albeit more pronounced shift in the dose–response curve could be observed . Measurement of IFN-γ production showed results similar to the proliferative response. Both CD2 and LFA-1 enhanced IFN-γ production at low peptide concentrations, with the effects being more dramatic upon stimulation with the low-affinity ligand A4Y . Thus, CD2–CD48 and LFA-1–ICAM-1 regulate T cell responses similarly by reducing the minimal amount of antigen required for activation and act in an additive manner. Functionally triggered TCRs are internalized shortly after stimulation 4 36 . TCR downregulation can therefore be used to assess the number of functionally triggered TCRs and thus the amount of signal 1 3 37 . To assess whether CD2 altered the intensity of signal 1, T cells from TCR-transgenic control or CD2-deficient mice were incubated with peptide-pulsed control or ICAM-1–deficient macrophages. Expression levels of TCRs were assessed 4 h later . TCR downregulation was reduced in the absence of either CD2 or ICAM-1 at low peptide densities . Moreover, as observed for the proliferative responses, ICAM-1 and CD2 acted in an additive fashion, and the dose–response curve of TCR downregulation was similar to the dose–response curve of the proliferative response . One of the earliest signals induced in T cells upon antigenic stimulation are increased intracellular free Ca 2+ levels ([Ca 2+ ] i ). To test whether the enhanced TCR triggering at low peptide concentrations in the presence of LFA-1–ICAM-1 or CD2–CD48 interactions would translate into increased Ca 2+ fluxes, CD2-deficient and control T cells were loaded with INDO-1 and stimulated with peptide-pulsed ICAM-1–deficient or control macrophages, and [Ca 2+ ] i was assessed . As suggested by the data on TCR downregulation, both CD2 on T cells and ICAM-1 on APCs promoted increased [Ca 2+ ] i at low antigen concentrations . Moreover, CD2–CD48 and LFA-1–ICAM-1 interactions had an additive effect. Importantly, as previously observed for T cell clones 14 , CD2 facilitated T cell–APC conjugate formation at low antigen concentrations, indicating that CD2 primarily promotes adhesion of T cells to APCs . Thus, the primary function of CD2 seems to be to enhance adhesion of T cells to APCs at low antigen concentrations, facilitating the generation of a T cell–APC contact site required for sustained signaling 38 . To assess the role of CD2 in T cell activation in vivo, TCR-transgenic CD2-deficient and control mice were injected with various doses of peptide p33 in saline, and expression of the activation marker CD44 was assessed 1 and 3 d (not shown) later. As expected from the in vitro experiments, presence of CD2 decreased the minimal amount of peptide required for the upregulation of CD44. To assess the role of CD2 in viral infections, an adoptive transfer system was employed 2 39 . Spleen cells (10 6 ) from TCR-transgenic CD2-deficient and control mice were mixed at a 1:1 ratio and adoptively transferred into nonirradiated C57BL/6 mice and immunized with LCMV (200 PFU) or a recombinant vaccinia virus expressing LCMV-GP (Vacc-GP; 2 × 10 6 PFU). To enable selective identification of the control versus CD2-deficient TCR-transgenic T cells, TCR-transgenic control mice on a CD45.1 background were used. Expansion of transferred TCR-transgenic control and CD2-deficient T cells was subsequently assessed 6 or 8 d (not shown) later. No significant difference between CD2-deficient and control T cells was observed. These results are in agreement with an earlier report, in which CD2-deficient mice were found to mount normal LCMV-specific CD8 + T cell responses 27 . Surprisingly, even the absence of both functional CD2 and LFA-1 together also failed to interfere with the response, as CD2-deficient T cells transferred into ICAM-1–deficient mice expanded normally upon infection with LCMV or Vacc-GP (not shown). The experiments performed so far suggested that CD2 plays a major role in T cell activation at low antigen densities. However, viral antigens are usually expressed at high densities, and it may therefore not be surprising that CD2-deficient T cells are able to respond normally to viral infections. To assess the role of CD2 in a situation where antigen is less abundant, we used a recombinant vaccinia virus expressing LCMV-GP (Vacc-GP), which was inactivated with UV light before infection. This treatment prevents the virus from undergoing full replication cycles, and endogenously produced antigens will therefore only reach low densities. For the experiment, a 1:1 mixture of CD2-deficient TCR-transgenic T cells obtained from CD45.2 mice and control CD2-competent transgenic T cells obtained from CD45.1 mice (in a total of 10 6 spleen cells) was transferred into C57BL/6 mice, which were subsequently immunized with UV light–inactivated Vacc-GP (2 × 10 6 PFU before inactivation). Control and CD2-deficient T cells could be conveniently distinguished by assessing CD45.1 versus CD45.2 and CD2 expression. As expected, the expansion of the transferred T cells was dramatically reduced compared with a challenge infection with live virus . Moreover, CD2-deficient T cells were clearly less efficiently proliferating than the control T cells. Note that CD45.1 + and CD2-deficient Vα2 + CD8 + T cells only account for ∼60% of the cells. This is due to the presence of endogenous CD8 + Vα2 + T cells. Thus, CD2 expression on T cells becomes critical in vivo in a situation where virus derived antigens are limiting. CTLs are usually primed by endogenously produced antigens reaching the class I pathway. However, MHC class I molecules may under some conditions also be loaded by exogenous antigens, leading to activation of specific T cells in a process called cross-priming. We have previously shown that exogenous LCMV-GP is able to reach the class I pathway if associated with cellular debris 40 . To test whether CD2 may be required for optimal CTL induction upon cross-priming, a 1:1 mixture of CD2-deficient (CD45.2) and control (CD45.1) TCR-transgenic T cells (total of 10 6 spleen cells) was transferred into C57BL/6 mice, which were subsequently immunized with recombinant LCMV-GP in association with cellular debris. 6 d later, the presence of CD45.1 + control and CD2-deficient TCR-transgenic T cells was assessed in the spleen . Although the CD2-deficient T cells were activated and proliferated upon cross-priming, the expansion was less dramatic than that observed for the control cells. This indicates that CD2 participates in regulation of T cell expansion upon cross-priming. This study demonstrates that CD2–CD48 and LFA-1–ICAM-1 interactions enhance T cell activation in an additive fashion by similar mechanisms. Both interactions facilitate TCR triggering by increasing T cell–APC interactions at low antigen densities, fine-tuning T cell responses in vitro and in vivo. T cell activation may be described in terms of the two-signal model, where signal 1 describes TCR-mediated signals and signal 2 refers to signals delivered by costimulatory molecules, which facilitate full T cell activation and prevent the induction of T cell tolerance 2 41 42 43 ; we have operationally discriminated these as signal 2c and 2t, respectively 2 . As TCRs productively triggered by MHC–peptide complexes are rapidly internalized 4 36 , the rate of TCR internalization may serve as a quantitative measure for the amount of signal 1 a T cell is receiving at a given time point 2 . Thus, a true costimulatory molecule would enhance T cell activation without changing signal 1, i.e., TCR internalization (unless it modulates the ratio of TCR engagement versus internalization) 2 . This is the case for CD28, which does not affect TCR internalization but nevertheless enhances T cell activation, apparently by increasing TCR-mediated signals intracellularly 2 3 30 . It has recently been suggested that rearrangement of membrane rafts rich in glycosphingolipids may be critical in this process 44 45 . In contrast, CD2 does not seem to affect T cell activation other than by increasing signal 1 (i.e., the number of triggered TCRs) at low antigen densities. In fact, the dose–response curves of T cell–APC conjugation, TCR internalization, Ca 2+ flux, and T cell proliferation are similarly shifted toward higher antigen concentrations in the absence of CD2, indicating that CD2 enhances T activation by facilitating T cell–APC interactions at low antigen densities. Thus, CD2 may be viewed as an adhesion molecule rather than a costimulatory molecule. This view is compatible with the recent observation that CD2 recruits an adapter molecule (CD2AP) to the T cell–APC contact site, helping to rearrange the cytoskeleton. Such a rearrangement is presumably required for a firm and stable T cell–APC interaction 28 . Our observations also fit the hypothesis that CD2 may bring T cells and APCs into close proximity, helping to exclude large molecules such as CD45 from the contact site 16 . In particular, the finding that CD2 is dispensable at high antigen concentrations may be explained by the notion that (a) the sizes of the TCR and CD2 are similar and (b) the CD2–CD48 interaction exhibits an affinity that is on the order of the TCR MHC–peptide interaction. Thus, large numbers of TCR MHC–peptide interactions may be able to substitute for CD2. We have previously argued that LFA-1 effects activation of CD8 + T cells primarily by promoting T cell–APC adhesion 2 . This may be different for CD4 + T cells, as it has been reported that LFA-1 specifically promotes Th1 development 46 . Thus, it remains possible that CD2 may affect activation of CD4 + T cells in a similarly qualitative fashion. However, we recently found that LFA-1 shifts the Th1/Th2 cytokine balance by shifting the dose response of CD4 + Th cells. In fact, absence of LFA-1 increased the minimal antigen concentration required for activation of TCR-transgenic Th cells by a factor of ∼100 (Ruedl, C., M.F. Bachmann, and M. Kopf, manuscript submitted for publication). Because induction of Th1 cells was also shifted by a factor of 100, this indicated that absence of LFA-1 shifted the response from Th1 to Th2 by globally shifting the dose response of CD4 + Th cells. Thus, although we cannot exclude the possibility that CD2 affects CD4 + T cell responses distinctly from CD8 + T cell responses, there is no data supporting such an assumption at this point. CD2-deficient mice have been found to mount largely normal T cell responses upon infection with LCMV 27 . In the light of our observation that CD2 is dispensable for T cell activation at high antigen concentrations, this earlier finding may not be surprising, as viral infection usually leads to high local antigen density. Using CD2-deficient T cells from TCR-transgenic mice specific for a peptide derived from LCMV, we could confirm the CD2 independence of the anti-LCMV CTL response 27 . Moreover, using a recombinant vaccinia virus expressing LCMV-GP, we could demonstrate that the CTL response elicited by vaccinia virus was also CD2 independent. More surprisingly, T cell responses were still unaffected in the absence of both LFA-1–ICAM-1 and CD2–CD48 interactions (not shown). Thus, antiviral immune responses may often be generated in the absence of CD2 and/or LFA-1. However, CD8 + T cell activation was clearly impaired in vivo in the absence of CD2 if limited amounts of antigen were used for immunization, as, for example, with immunization regimens involving low amounts of peptide or UV light–inactivated recombinant vaccinia virus. The latter results may be particularly interesting because they indicate that viruses do not target a particular APC in vivo that can prime CTLs in the absence of CD2 but rather suggest that CD2 dependence is dictated by antigen quantity. Thus, viruses that replicate intracellularly to high titers can prime T cells in the absence of CD2, whereas abortive viral infections that do not reach high levels of intracellular protein require CD2 for full T cell activation. This latter class of immunization may therefore be representative for infections with attenuated viruses that induce only abortive infections. Moreover, antigens introduced to the immune system by cross-presentation also did not reach high densities of class I molecules and therefore required the presence of CD2 for optimal T cell responses. Thus, T cell responses against abundant antigens occur in the absence of CD2, whereas T cell responses against rare and cross-presented antigens require the presence of CD2 for optimal responses. | Study | biomedical | en | 0.999997 |
10562315 | The P . falciparum lines FCR3S/b (K − ) and FCR3S1/b (K − ) were selected from parasites FCR3S (K − ) and FCR3S1 (K − ), respectively, for cytoadherence to the CD36 receptor on C32 melanoma cells. FCR3S/a (K − ) was obtained from FCR3S by repeated selection for nonrosetting parasites as described elsewhere 18 . FCR3S1 was cloned by limiting dilution from FCR3S, which originated from the FCR3 strain isolated in The Gambia, West Africa. The subclones FCR3S1.2 (K − ) and FCR3S1.6 (K − ) were obtained by micromanipulation from FCR3S1. Clones TM284S2 (K + ), TM284S3 (K + ), TM284S11 (K + ), TM284S12 (K + ), TM284S20 (K + ), TM284S7 (K − ), TM284S9 (K − ), and TM284S19 (K − ) were derived by micromanipulation from the strain TM284 (K + ), which, along with strain TM180 (K − ), were isolated from malaria patients in Thailand. The F32 strain was isolated in Tanzania. The parasite 3D7 was obtained by limiting dilution cloning of the isolate NF54, which was derived from a patient who acquired malaria in the airport area in Amsterdam, The Netherlands. R29 (K + ) was cloned from ITOR, a parasite from the ITO strain selected for the rosetting phenotype. The parasite Dd2 was originally cloned from the W2-MEF line of the Indochina III isolate. The P . falciparum isolates 186, 199, 341, 347, 352, and 354 were part of a larger panel of field parasites collected from African children infected with malaria. Upon collection, the blood samples were immediately frozen according to standard techniques. For their analysis, the frozen blood samples were thawed and maintained in culture in their own blood for 24–30 h until parasites developed into the mature trophozoite stage, at which time they were harvested and further processed. Sera were collected from (a) adults living in Yekepa, Liberia, an area characterized by high perennial malaria transmission (denoted 022, 102, 119, 142, 163, 164, 169, 174, 179, 198, 241, and 368), (b) adults from Fajara, The Gambia, a region with seasonal malaria transmission (denoted 072, 100, and 136), and (c) children 1–15 yr old living in Saradidi, an area in western Kenya holoendemic for malaria (denoted 011, 039, 080, 118, and 209). All donors had had repeated malaria attacks; none had symptoms of clinical malaria at the time of sampling. In all cases, informed consent was obtained from the patients and/or their parents. The sera were stored at −70°C and heat inactivated at 56°C before the assays. Control sera were obtained from healthy Swedish blood donors. All of the laboratory-adapted parasites used in this study were cultured in human group O Rh + erythrocytes at 5% hematocrit with 10% AB + serum added to the malaria culture medium containing RPMI 1640, 25 mM Hepes, 25 mM sodium bicarbonate, and 50 mg/ml gentamycin, pH 7.4. In some cases, cultured parasites were enriched for the rosetting phenotype (R + ) as previously described 18 . Surface iodination of pRBCs was performed by the lactoperoxidase/Na 125 I/H 2 O 2 method under conditions of minimal intracellular labeling. In brief, 2 × 10 9 cells of a culture at 7–15% parasitemia with a majority of parasites in the trophozoite stage were gently washed in PBS and resuspended to 1 ml in PBS plus 1 mM KI. 1 mCi of Na 125 I (Amersham) and 100 μl of 2 mg/ml lactoperoxidase (Sigma Chemical Co.) was added, and the reaction was initiated by the addition of 25 μl of 0.03% H 2 O 2 . Four subsequent additions of 25 μl of 0.03% H 2 O 2 were administered at 1-min intervals. Radioiodinated cells were washed four times with ice-cold PBS containing 5 mM KI and resuspended in 1 ml of RPMI 1640 plus 5% sorbitol. Labeling of intracellular hemoglobin accounted for <2% of total acid-precipitable incorporated radiolabel. To disrupt rosettes/agglutinates, 100 IU/ml of heparin (Løvens) was added to the cell suspension, and this was passed five times through a 23-gauge (0.6 mm internal diameter) needle using a 1-ml syringe. The cell suspension was overlaid on top of a four-step (40, 60, 70, and 80%) Percoll gradient in RPMI/5% sorbitol and centrifuged in a JA 20 rotor (Beckman Instruments, Inc.) at 10,000 rpm for 30 min at room temperature. Cells floating between the 40 and 60% Percoll layers (>95% mature parasite–containing erythrocytes) were recovered and gently washed with PBS. Enriched pRBCs were first extracted with 1% Triton X-100/PBS containing a cocktail of protease inhibitors (1 mM EDTA-N 2 , 20 μg/ml leupeptin, 0.7 μg/ml pepstatin, 0.2 mM PMSF, and 50 μg/ml aprotinin) and subsequently extracted in a solution of 2% SDS and protease inhibitors in PBS. The polypeptides in the Triton-insoluble fraction were solubilized in 2% SDS-PAGE sample buffer and separated by a gradient of 5–8.5 or 7.5–17.5% SDS-PAGE. The dried gels were scanned and analyzed using a PhosphorImager and ImageQuant analysis software (Molecular Dynamics). SDS or Triton X-100 extracts of surface-radioiodinated pRBCs (25 μl) were mixed with 375 μl of NETT (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM disodium EDTA, 20 μg/ml leupeptin, 50 μg/ml aprotinin, 0.02% sodium azide, and 0.5% Triton X-100) buffer/10 mg/ml IgG-free BSA (Sigma Chemical Co.) and 100 μl of 10% Triton X-100/PBS. 15 μl of serum was added to 500 μl of reconstituted extract, and the samples were incubated for 12 h at 4°C with rotation. Protein A–Sepharose (100 μl of a 50% suspension in NETT buffer/10 mg/ml IgG-free BSA) was added, and the samples were further incubated for 1 h at room temperature with rotation. The adsorbent was then washed twice with 1 ml of NETT, twice with 1 ml of NETT/0.35 M NaCl, and twice with 1 ml of NETT. The Sepharose beads were boiled for 4 min in the presence of 5% SDS-PAGE sample buffer, and the solubilized polypeptides were separated and analyzed as described above. Intact or radiolabeled pRBCs purified on Percoll gradients were digested with trypsin (Sigma Chemical Co.) at the indicated concentrations for 10 min at 37°C. The reaction was terminated by the addition of 1 ml soybean of trypsin inhibitor (Sigma Chemical Co.) at 1 mg/ml in RPMI 1640/10% AB + serum. Cells were washed with PBS and either resuspended in RPMI–Hepes/10% AB + serum for binding and agglutination assays or extracted with Triton X-100/SDS and analyzed by SDS-PAGE as described above. Agglutination of parasitized red blood cells was assayed as previously described 19 with some modifications. pRBCs cultured to trophozoite and late schizont stages were washed three times in malaria culture medium and then resuspended at 20% hematocrit. Aliquots of 25 μl were mixed with an equal volume of prediluted or nonprediluted serum to a final dilution of 1:2, 1:5, or 1:10. The pRBC–serum mixture was incubated at 37°C for 1 h under a constant rotation of 3 rpm. A 10-μl aliquot was mixed with 5 μl of acridine orange and mounted on a glass slide, and 50 consecutive fields of vision were examined diagonally using a 40× lens in an incident UV light microscope. Control sera from healthy Swedish blood donors were used. Semiquantitative scoring was used for analysis. The agglutination assay was scored negative when no agglutinates of 4 pRBCs were detected; 1+, 1–5 agglutinates of 4–10 pRBCs; 2+, >5 agglutinates of 4–10 pRBCs or 1–5 agglutinates of 11–20 pRBCs; 3+, >5 agglutinates of 11–20 pRBCs or 1–5 agglutinates of >20 pRBCs; 4+, >5 agglutinates of >20 pRBCs. Rosetting of uninfected red cells and autoagglutination of parasitized erythrocytes was assessed by direct staining of cultures with acridine orange (Sigma Chemical Co.) and examination using an epifluorescence microscope. Rosetting rates were measured as previously described 20 . Spontaneous autoagglutination of pRBCs in the P . falciparum cultures was measured as described elsewhere 18 . Adherence of pRBCs to unfixed C32 melanoma cells, human umbilical vein endothelial cells (HUVECs), chinese hamster ovary (CHO) cells transfected with CD36 or intercellular adhesion molecule 1, or L cells transfected with platelet/endothelial cell adhesion molecule (PECAM)1/CD31 was performed as described 21 . In some cytoadherence assays on HUVECs, C32, and L cells, rosettes were first disrupted by adding 100 IU/ml of heparin (Løvens) to the culture and passage (five times) through a 23-gauge (0.6 mm internal diameter) needle using a 1-ml syringe. The cell suspension was overlaid on a four-step (40, 60, 70, and 80%) Percoll gradient, and the trophozoite-bearing red cells were selected as described above, washed twice in RPMI, and used for the assays in serum-free medium. The assay of the binding of human nonimmune normal Swedish serum Igs to pRBCs was performed by direct labeling of the cells using FITC-conjugated sheep anti–human Ig antibodies (State Bacteriology Laboratory, Sweden) as described elsewhere 22 . Frozen blood samples from P . falciparum malaria patients were thawed, and the cells were maintained in culture for 30 h. The intact cells were mildly radioiodinated under conditions ensuring >98% isotope incorporation to surface-exposed molecular moieties, and the labeled polypeptides were analyzed by protein gel electrophoresis. In all six isolates that developed into the mature stages, we found, in addition to PfEMP1 bands (>200 kD), several polypeptides with an estimated molecular mass between 33 and 39 kD that were readily labeled on the surfaces of erythrocytes containing parasites at the trophozoite/schizont stages, but not on the ring-infected or uninfected RBCs from the same blood sample . The number, size, and degree of radiolabel incorporation of these polypeptides was variable and essentially unique for each isolate. Typically, the largest of these bands (39 kD) comigrated with the heavily radioiodinated monomer chains of erythrocytic glycophorin A and was therefore often indistinguishable from the host protein. One to three additional labeled polypeptides 33–37 kD in size formed a characteristic cluster of bands with isolate-specific patterns. We then proceeded to study the pRBC surfaces of 17 strains, lines, and clones of P . falciparum and found that radioiodinatable polypeptides in the range of 30–45 kD were commonly exported and surface expressed by a majority of these in vitro–propagated parasites . The number of bands and their relative radiolabel intensities was specific for each parasite. As determined by radioiodination experiments with highly synchronized cultures, the expression of the 30–45-kD polypeptide cluster on the pRBC surface occurred from 16 to 18 h after invasion until the end of the erythrocytic cycle (data not shown), matching the time course of expression of antigenic and adhesive phenotypes and the onset of PfEMP1 on the pRBC surface. These polypeptides were partially solubilized in Triton X-100, in contrast to the extreme insolubility of PfEMP1 in nonionic detergents. Additional parasite-specific radiolabeled bands were detected on pRBCs infected with some of the patient isolates or laboratory-cultured parasites . A 20-kD band was observed in isolate 199, whereas a polypeptide of 22 kD was radiolabeled in isolate 347 and the strain Dd2. Faint bands of 76–80 kD were visible in several of the in vitro–propagated parasites, and a polypeptide of 140 kD was detected in TM180, a strain also expressing several smaller (33–39 kD) polypeptides. Most prominent was the strongly radioiodinated band of 170 kD found in two of the clinical isolates (199 and 341) and strain F32, as well as strain TM284 and several of its clones (only TM284S2 is shown). None of these polypeptides were detected on pRBCs bearing trophozoite/schizont stages when the erythrocytes were surface 125 I-labeled before parasite invasion (data not shown). Upon examination of the panel of clones derived by micromanipulation from the TM284 strain , we observed that all five rosetting (R + ) clones (TM284S2, S3, S11, S12, and S20) had an identical pattern of surface radiolabeled polypeptides with bands detected at 39, 80 (faint), and 170 kD and a PfEMP1 polypeptide of 340 kD (TM284S2 is shown; gels optimized for PfEMP1 separation are not shown). In contrast, each of the three nonrosetting (R − ) clones obtained (TM284S7, S9, and S19) showed unique combinations of bands in the range of 33–45 kD and no detectable PfEMP1 proteins in the gels. This result was consistent with the reported role of PfEMP1 as a rosetting ligand 14 15 . Moreover, the data strongly suggested that the variation observed in the small surface antigens clustered in the range of 30–45 kD had its origin in a set of clonally variant genes. By comparing the patterns of surface-radiolabeled polypeptides expressed by the sibling parasites FCR3S1.2 (R + ) and FCR3S1.6 (R − ), sharing an isogenic background after their cloning by micromanipulation from the previously cloned FCR3S1 (R + ), it was confirmed that the expression of the small surface antigens (31–39 kD) of this parasite is subjected to clonal genetic switching . To investigate the stability of expression of the small surface antigens in parasite populations that were or were not exposed to selective pressures other than those inherent to in vitro growth, we 125 I-labeled pRBCs periodically collected from year-long continuous cultures of FCR3S1, maintained with regular enrichment for the rosetting phenotype, and the Dd2 strain, cultured without selection but displaying rosetting rates consistently >65%. The results showed no change in the type of radioiodinated polypeptides of these two parasites grown during at least 150 generations, as assessed in at least 10 separate experiments of surface labeling of pRBCs (data not shown). Although similar stability in the small surface antigen types is a characteristic of other parasite strains undergoing long-term culture, a decrease in the expression of these radiolabeled bands has been sporadically observed in some cultures. We noticed that some radiolabeled bands in the 30–45-kD cluster appeared characteristically dispersed . This observation prompted us to investigate possible molecular heterogeneity not discerned in regular SDS-PAGE gels. A two-dimensional electrophoresis analysis of an SDS extract of FCR3S1.2 pRBCs revealed the existence of charge and size microheterogeneity in the surface-radioiodinated polypeptides . The parasite-specific bands of 35 and 36 kD resolved into several molecular species or isoforms in the pI range of 5.5–6.5, displaying a charge composition distinct from the major proteins of the human erythrocyte surface, including the heavily glycosylated anion transporter Band 3 and the sialoglycoproteins, glycophorins A and B. Polyclonal sera to total erythrocytic antigens and mAbs to Band 3 and glycophorins A, B, and C were used in immunoprecipitation and Western blot for the identification of the normal RBC proteins undergoing radioiodination (results not shown). None of these antibodies recognized the parasite-specific surface-radioiodinatable antigens of FCR3S1.2 or any other strain/clone tested. As part of the P . falciparum genome sequencing project, a number of intact transcriptional units sharing homology with the sequence originally called RIF (repetitive interspersed family; reference 23 ) were identified in the subtelomeric regions of chromosome 2 24 . This large family of rif genes encoded potential transmembrane proteins of 30–40 kD termed rifins. We proceeded to investigate whether the variable 30–45-kD surface antigens characterized here were identical to predicted rif gene products. Rabbit antisera raised against the highly conserved 3′ end of the deduced amino acid sequence of rif genes PFB1040w, PFB1035w, PFB0040c, and PFB0955w were used to immunoprecipitate SDS extracts of surface- or metabolically labeled pRBCs of the FCR3S1.2 clone. Antibodies in three of the tested sera specifically recognized the small surface polypeptides of this parasite, in particular anti- rif PFB1035w, which immunoprecipitated bands of 35, 36 (doublet), and, faintly, 39 kD . The results proved that the small variant antigens expressed on the pRBC surface are products of the rif loci. We next investigated whether the expression of rifin proteins on the pRBC surface was associated with rosetting binding to uninfected erythrocytes. For this purpose, we analyzed surface radiolabeled pRBCs in R + and R − parasites cloned by limiting dilution and subcloned by micromanipulation. The rosetting clone FCR3S1, derived from the strain FCR3S by limiting dilution, consistently showed a radioiodinated polypeptide of 35 kD. The highly rosetting, autoagglutinating, and cytoadherent subclone FCR3S1.2, obtained from FCR3S1 by micromanipulation, expressed radiolabeled bands of 31, 35, and 36 kD. In contrast, the nonrosetting, noncytoadherent sibling subclone FCR3S1.6 only exhibited the 39-kD band, a polypeptide always found on pRBCs infected with parasites of the FCR3S lineage . These preliminary results were suggestive of an association between the rosetting capacity of the parasite and the expression of particular rifin variants on the host cell surface. Such an association was not observed when a similar clonal analysis was performed in another P . falciparum strain. Parasite clones with phenotype R + derived from the rosetting strain TM284 (only TM284S2 is shown here) did not display radioiodinatable polypeptides of low molecular mass other than the 39 kD polypeptide, whereas nonrosetting clones (TM284S7, TM284S9, and TM284S19) showed multiple radioiodinated polypeptides in the range of 33–45 kD . However, polypeptides outside the cluster of small variant antigens were associated with rosetting binding in TM284 parasites. A prominent surface-labeled band corresponding to a polypeptide of 170 kD and an additional 82-kD polypeptide were detected in TM284 and all of its R + clones but were not or were very faintly detected in R − clones . In FCR3S and TM284 parasites, changes in binding phenotype always concurred with changes in the expression of PfEMP1 polypeptides (data not shown). Controlled proteolytic digestion of intact radioiodinated pRBCs was used to further explore possible associations between the expression of surface antigens and binding phenotypes. Upon incubation of surface-radioiodinated, trophozoite-infected erythrocytes with trypsin, complete deletion of the 35-kD rifin band of FCR3S1 occurred at concentrations of the protease of 100 μg/ml or higher . The 39-kD polypeptide of this parasite showed a reduced sensitivity to the protease. In comparison, the 285-kD PfEMP1 polypeptide expressed by FCR3S1 was cleaved at concentrations of trypsin <1 μg/ml, in agreement with our and others' data on the high trypsin sensitivity of most PfEMP1 polypeptides. The proteolytic treatment left the bulk of Triton X-100– and SDS-soluble polypeptides unaltered, with the exception of the glycophorins. The sensitivity of the rosetting phenotype to trypsin digestion of intact pRBCs closely matched that of the PfEMP1 protein, but not the removal of the 35- or 39-kD rifins from the pRBC surface. Similarly, the trypsin sensitivity of other adhesive phenotypes of erythrocytes bearing FCR3S1 or its clone FCR3S1.2, such as autoagglutination of infected RBCs, binding to the blood group A trisaccharide, adherence to the CD36 receptor, and binding of normal human Igs paralleled the cleavage of PfEMP1 (data not shown). In contrast, an association was found between the deletion of the 35-kD rifin polypeptide and the abrogation of pRBC binding to PECAM1/CD31, expressed constitutively on HUVECs or L cells transfected with the corresponding gene . The lack of association between the expression of non-PfEMP1 antigens on the pRBC surface and adherent phenotypes of the parasite, with the exception of binding to PECAM1/CD31, was further evidenced when we panned the rosetting FCR3S and FCR3S1 parasites on C32 melanoma cells expressing the CD36 receptor. The resulting highly CD36-cytoadherent, nonrosetting parasite lines expressed different patterns of surface-radioiodinatable polypeptides, 36 and 39 kD in FCR3S/b and 39 kD in FCR3S1/b. Polypeptides of 39 kD were also detected in noncytoadherent, nonrosetting parasites . To assess the natural antigenicity of rifin polypeptides, we initially tested two human hyperimmune sera in immunoprecipitation of SDS extracts of surface 125 I-labeled pRBCs of the FCR3S1.2 clone. Antibodies in these two sera recognized radioiodinated rifins of 35, 36 (doublet), and 39 kD in addition to PfEMP1 . Rabbit antisera raised against GST fusion proteins comprising the semiconserved DBL1 and the highly conserved ATS domains of the var /PfEMP1 expressed by FCR3S1.2 immunoprecipitated PfEMP1 polypeptides but did not react with rif gene products. Having obtained the first evidence that rifin polypeptides were naturally immunogenic, we next carried out an expanded analysis of the antigenicity of these parasite products in natural infections. For this purpose, we used a panel of 18 sera from malaria-experienced individuals living in different geographical regions of Africa (Kenya, Liberia, and The Gambia) in assays of immunoprecipitation and agglutination of FCR3S1.2 and TM284 parasites . Most of the sera immunoprecipitated one or more radioiodinated rifin polypeptides in the range of 31–39 kD, both in FCR3S1.2 and TM284 parasites. Qualitative variation was observed in the antigenic pattern recognized by different sera in each parasite, a result consistent with the extent of the rif gene repertoire and the switching in expressed rifin types. At the same time, sharing of epitopes between parasite-specific variant forms of rifins was suggested by the corresponding reactivities of a majority of the individual sera to the 36-kD doublet of the FCR3S1.2 parasite and the 39-kD band of the TM284 strain, a result anticipated if conservation at the 5′ and 3′ ends is a widespread feature of rif products. The major radioiodinated 170-kD polypeptide of TM284 was not immunoprecipitated by immune sera. The PfEMP1 polypeptides of the two parasites studied were recognized by all of the sera except 72, 100, and 136 (data not shown). The agglutination of trophozoite-bearing pRBCs by the immune sera was generally, but not strictly, correlated with their capacity to immunoprecipitate the small surface antigens. To study the relative contribution of surface-exposed epitopes on rifin and PfEMP1 antigens to the agglutination reaction, we took advantage of the differential sensitivity that members of these two protein families display to controlled trypsin proteolysis of intact pRBCs. Rifin polypeptides of the clone FCR3S1 (35 and 39 kD) are at least 100-fold less sensitive to the protease than the radioiodinated PfEMP1 of this parasite, which is no longer detected after digestion with 1 μg/ml trypsin. Antibodies in immune sera agglutinated trophozoite-bearing pRBCs digested with 1, 10, or 100 μg/ml trypsin before incubation with antibody ( Table ), suggesting the presence of epitopes in rifins targeted by surface-reactive, agglutinating antibodies raised during P . falciparum infections. In this study, we show that the vast structural and antigenic variability created by P . falciparum on the surface of the host erythrocyte has its molecular basis not only in the PfEMP1 polypeptides, encoded by the var family of genes, but also in multiple additional parasite-derived products, including a prominent group of relatively small antigens encoded by the rif family of genes. These polypeptides are clonally variant and may undergo posttranslational modifications resulting in molecular microheterogeneity, a conceptually new source of antigenic diversity on the pRBC surface . Besides the variant adhesin PfEMP1, several undefined polypeptides of 20–55 kD have reportedly been detected on the surfaces of erythrocytes infected with mature stages of P . falciparum 16 17 . In each case, the detection of these polypeptides was performed after lactoperoxidase-catalyzed iodination of intact infected erythrocytes, a procedure used for the radiolabeling of exofacially exposed proteins on cell plasma membranes 25 . In our analysis of parasite-derived molecules exported to the host cell surface, with a focus on products different from var /PfEMP1, we have studied 23 clinical isolates, laboratory-adapted strains, and lines or clones selected for binding phenotypes, including rosetting and adhesion to the CD36 receptor. At least 12 polypeptides, ranging in size from 20 to 170 kD, were identified by mild radioiodination of intact trophozoite-bearing erythrocytes. The most common and prominent of these products, both in fresh clinical isolates and long-term–cultured parasites, were detected as a distinct group or cluster of bands in the range of 30–45 kD. The large family of rif genes and its purported subfamily stevor , encoding potential transmembrane proteins (rifins) with a predicted size of 30–40 kD, has been recently identified in P . falciparum 24 26 . The rif genes have a two-exon structure, with the first exon coding for a predicted signal peptide and the second for polypeptides containing an extracellular domain with conserved cysteine residues and a highly variable region, a transmembrane segment, and a short cytoplasmic tail that is highly conserved. According to preliminary estimations, there may be 200–500 rif genes in the P . falciparum genome. At least some of the rif genes are transcribed in blood stages 24 . Our finding that rabbit antibodies raised to the conserved basic COOH terminus of rifin-deduced amino acid sequences specifically immunoprecipitate surface- or metabolically labeled polypeptides of 30–45 kD indicates that these antigens are the products of the presumably most abundant gene family in P . falciparum . A salient feature of rifin polypeptides is their variability, in size as well as in the number of distinct components expressed in different parasites. The capacity of P . falciparum to undergo clonal antigenic variation has been established 27 28 , and the switching in the expression of different var genes, or their product PfEMP1, is correlated with clonal changes in surface antigenic determinants of the pRBC 3 . Rifin antigens are clonally variant, as the analysis of clones and subclones of TM284 and FCR3S1.2 parasites demonstrate. These variable products, which are partially solubilized by neutral detergents (e.g., Triton X-100) but require treatment with 1–2% SDS, conditions that disrupt the host erythrocyte cytoskeleton for complete dissolution, are transported to the plasma membrane of the infected RBC and exposed on the surface according to several criteria. They are (a) readily and consistently labeled by the lactoperoxidase/Na 125 I/H 2 O 2 method under conditions that fail to label hemoglobin and (b) cleaved by trypsin treatment of intact infected erythrocytes under conditions that do not cleave any other RBC or malarial proteins, except glycophorin polypeptides and the PfEMP1 antigen. It is unknown how rif gene expression is regulated. The clusters with varying numbers of radiolabeled bands detected in freshly isolated as well as in recently cloned parasites may reflect, besides several other possible explanations, (a) the nonclonal composition of the parasites 29 30 , (b) posttranscriptional processing of the product of a single gene, or (c) the simultaneous expression of several rif genes. Implicit in this last alternative would be a partial lack of allelic restrictive processes (e.g., allelic exclusion) controlling the expression of products from the rif loci. This is in contrast to the “one parasite–one gene” modality of surface protein expression in the var /PfEMP1 multigene family 31 . The finding of microheterogeneity in the rifins could be explained by the concurrent expression of several rif genes encoding polypeptides of similar molecular mass. Alternatively, superimposed to the variation created at the pRBC surface by the display of proteins comprising semivariable and hypervariable regions, e.g., var /PfEMP1, additional layers of antigenic variation could be generated in asexual stages of P . falciparum by the addition of carbohydrates, phosphate groups, or perhaps unusual modifications of polypeptides targeted to the erythrocyte surface. Examples of posttranslational modifications modulating the surface architecture, and likely the survival/virulence of pathogenic microorganisms, are the glycosylation and glycerophosphorylation of antigenically variable pilins in the prokaryote Neisseria species 32 33 or the glycosylation and palmitoylation of the variant surface proteins of the eukaryotic parasite Giardia species 34 . Analysis of the rif gene amino acid sequences available to date reveals numerous and relatively conserved potential sites for O - and N -glycosylation. Allowing for the limited number of clinical isolates included in this study, it is interesting to note that rif gene expression is detected as prominent bands in each of the wild parasites examined but absent or faint in some of the strains or parasite lines that were long-term cultured in the laboratory. Chromosomal truncations, resulting in gene deletions as well as gametocytogenesis and cytoadherence phenotype losses, commonly occur in P . falciparum propagated mitotically in vitro 35 36 37 38 . The function(s) of the variant rifin antigens being unknown, it is likely to be essential for survival of the parasite confronted with the host's environment and defence mechanisms but not indispensable for in vitro growth. The variant nature of the rifin antigens further suggests that presence at the pRBC surface is a compelling need. Our data do not support a direct role of rif products in rosetting binding. An accessory function in conjunction with the rosetting ligand PfEMP1 14 15 cannot be, however, completely disregarded. We have previously shown that the trypsin sensitivity of other binding phenotypes of multiadhesive parasites, i.e., binding to blood group A, autoagglutination of infected erythrocytes, binding of normal Igs, and adhesion to the CD36 receptor correlate with the enzymatic digestion of PfEMP1 18 . In contrast, here we show that cytoadherence to the endothelial receptor PECAM1/CD31 is a phenotype associated with the presence on the pRBC surface of at least one radioiodinatable polypeptide, the 35-kD rifin in FCR3S1, but apparently not to the radioiodinated domain(s) of the PfEMP1 in this parasite. It remains a possibility that a trypsin-resistant domain of PfEMP1 contains the CD31 binding specificity. The 35-kD rifin may have, in conjunction with PfEMP1 or not, either a direct or an accessory role in the binding of pRBCs to CD31 and other receptors. Although binding to PECAM1/CD31 is a feature of only some laboratory-adapted strains 39 , the extent of this binding trait in natural P . falciparum populations and its relationship to severe forms of malaria remains to be investigated. The prominent labeling of a 170-kD polypeptide in some isolates and laboratory strains and its paucity in nonbinding clones has not escaped our attention. Its labeling pattern may indicate molecular abundance at the pRBC surface. In immunoprecipitation and Western blot assays, this band is neither recognized by antibodies to the highly conserved COOH-terminal segment of PfEMP1 nor by immune sera that immunoprecipitate PfEMP1 and components of the 30–45-kD rifin cluster from the same parasite. It remains to be determined whether the 170-kD polypeptide is too antigenically diverse to be detected by heterologous antibodies or is not naturally immunogenic. Regardless of the function of rifins as well as the origin and function of the other novel surface-exposed polypeptides described here, their diversity, stability of expression, and multiplicity of combinatorial patterns (19 distinct types in 23 different parasites) suggests a potential for use as an isolate typing tool, conceivably in assays where the expressed PfEMP1 variant is simultaneously assessed. Antigens exposed on the pRBC surface undergo clonal variation at rates of up to 1–2% per generation 28 . The variant adhesin PfEMP1, which mediates binding of infected erythrocytes to vascular endothelial cells and to uninfected erythrocytes, plays a major role in this antigenic variation 3 27 31 . Adults living in areas of high malaria endemicity have antibodies in their sera capable of reacting with antigenic determinants of many P . falciparum isolates from distinct geographic regions 8 19 . These antibodies recognize conserved and polymorphic parasite antigens, including PfEMP1 9 40 . Our data indicate that rifin proteins are located on the surface of the pRBC and, moreover, that these parasite-derived components are antigenic in the course of a P . falciparum infection, eliciting a substantial humoral immune response. Furthermore, we show here that human immune sera agglutinate infected erythrocytes under conditions such that most, if not all, PfEMP1 products have been removed from the pRBC surface. The presence in sera of agglutinating or opsonizing antibodies reactive with the infected erythrocyte surface has been shown to correlate with protection from lethal disease 8 41 . This preliminary data opens a new avenue for studies conducive to the eventual elucidation of targets for immune protection against P . falciparum malaria. The relative contribution of the rifins, PfEMP1, and other surface antigens on the pRBC to the triggering of the antibody response, Ig classes, and immunity buildup have yet to be determined. This represents the first report of natural immune responses to surface molecules of intraerythrocytic P . falciparum distinct from PfEMP1. The results indicate that more than one family of genes encoding variable surface-targeted proteins, perhaps with additional epigenetic generation of submolecular variation, accounts for the remarkable diversity generated by the parasite in its host cell. | Study | biomedical | en | 0.999998 |
10562316 | SIPCs were generated by three successive inoculations (on days 0, 60, and 120) of silicone gel into the peritoneal cavity of BALB/c or BALB/c.DBA/2N-Idh1-Pep3 congenic mice 8 . The latency times for first generation tumors were SIPC3301 (227 d), SIPC3308 (190 d), SIPC3282 (152 d), SIPC3336 (220 d) and SIPC3385 (225 d). Tumors were transplanted either with or without priming into syngeneic mice, and several generations (g) were examined, including: SIPC3301 (g1, g2), SIPC3308 (g0, g1, g2), SIPC3282 (g0, g1, g3), SIPC3336 (g1, g2, g3, g4), and SIPC3385 (g0, g2, g3). SIPCs were screened for Ig secretion and Ab activity against actin, myosin, tubulin, dsDNA, and ssDNA 5 . In brief, polystyrene flat-bottomed plates were coated with different Ags. After incubation with serial dilutions of each sample in duplicate, peroxidase-conjugated anti–mouse Ig was added; dilution medium alone was included as the negative control. Each assay was done in triplicate. Approximately 10 μg of EcoR1- or BamH1-digested genomic DNA was size separated on 0.7% agarose gels. After electrophoresis, the gels were acid (HCl) treated, alkaline treated, and neutralized in 3 M NaCl, 0.5 M Tris-Cl, pH 7.6. Southern transfers were made to Nytran Plus filters (Schleicher and Schuell), UV cross-linked, and hybridized to 32 P-labeled DNA probes at 65°C with a final wash stringency of 0.2× SSC at 65°C. The probes used to detect light chain κ rearrangements were either the 1.1-kb HindIII-XbaI (intervening sequence [IVS]) or the 1.8-kb HindIII-XbaI (J κ ) fragments. For heavy chain Ig rearrangements, a 0.8-kb EcoR1 J H probe was used. Translocation to c-Myc or Pvt 1 was assayed by screening EcoR1- or BamH1-digested filters with a 1.7-kb exon 2 PstI ( c-Myc ) or a 1.7-kb cDNA ( Pvt 1 ) probe. For slot blot hybridizations, ∼1 μg of RNAs from BALB/c. DBA/2N congenic mouse liver and spleen, as well as from each of the SIPC tumors used in this study, were applied to Hybond (Nycomed Amersham plc) filters and hybridized to Ly1 , recombination activating gene 1 ( RAG-1 ) cDNA, V λ , V k -24, Vκ-1, or C κ -specific probes. After hybridization, the filters were washed at 0.2× SSC at 65°C for 30 min. Although detailed methods have been reported elsewhere 9 , metaphase cells were equilibrated in 2× SSC, digested with RNase A and pepsin, fixed in 1% formaldehyde, denatured in 70% formamide/2× SSC, and then dried. The fluorochromes, spectrum orange (dUTP conjugate; Vysis), rhodamine 110 (Perkin-Elmer), and Texas red (12-dUTP conjugate; Molecular Probes) were used for direct labeling, and the haptens biotin-16–dUTP and digoxigenin-11-dUTP (Boehringer Mannheim Corp.) were used for indirect labeling. The probes were precipitated in an excess of mouse DNA (COT-1 DNA; GIBCO BRL), and hybridized at 37°C for 72 h in 50% formamide/SSC/dextran sulfate. After hybridization and washing (4× SSC/Tween 20), the biotin was visualized by avidin-Cy5 (Nycomed Amersham plc), and the digoxigenin-11–dUTP was visualized by mouse antidigoxigenin (Sigma Chemical Co.) followed by sheep anti–mouse Cy5.5 (Nycomed Amersham plc). Chromosomes were counterstained with 4,6-diamino-2-phenylindole (DAPI) and embedded in antifade solution (1,4-phenylenediamine, Sigma Chemical Co.). Spectral images were obtained on a Leica DMRBE epifluorescence microscope equipped with an SD200 SpectraCube ® (Applied Spectral Imaging) and a customized triple bandpass optical filter. Spectrum-based classification of the raw spectral images was performed using the software SkyView (Applied Spectral Imaging). Approximately 100 μg of genomic DNA from SIPC3336 was restricted with EcoR1 and subjected to low-melting (1.0%) gel electrophoresis. Bands corresponding to the rearranged V(D)J genes were excised, extracted with phenol, and precipitated with ethanol. Inserts were then ligated to either EMBL4 or λgt10 arms (EcoR1 digested). After ligation, the phage DNA was in vitro packaged and plated to densities of 125,000 per 20 × 20 cm LB plate. Benton-Davis transfer of the phage to nitrocellulose filters was followed by hybridization to 32 P-labeled IVS or J H probes. Positive phage were mapped by restriction endonucleases, and inserts were subcloned into pGEM-4Z. Total cellular RNA was extracted from tumor tissues by RNA PLUS solution (Bioprobe Systems), and first-strand synthesis was carried out as described previously 5 . PCR amplifications were set up in 50-μl volumes containing 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl 2 , 200 mM of each dNTP, 2 U of Taq DNA polymerase (GIBCO BRL), 50 pmol of each primer, and a 3 μl aliquot of the cDNA reaction. The primer sequences used to amplify specific heavy chain or light chain V regions are shown in Table . The primers for amplification of Ly1 , RAG-1 , and RAG-2 are as follows: CCTAGATATCCAGGTGGT (Ly1 5′), GTGTCCTGGCACCAGCTG (Ly1 3′), AGGATCTGCATTCTCAGA (RAG-1 5′), TCAGCTTGCCTGTGCTCA (RAG-1 3′), GTCTCCAACAGTCAGACA (RAG-2 5′), and GCTCTTGCTATCTGTACA (RAG-2 3′). Each PCR amplification product was purified by agarose gel electrophoresis, extracted, and then subcloned into the TOPO-TA vector following the manufacturer's protocol (Invitrogen). Single bacterial colonies were isolated for direct sequencing. Sequencing was carried out according to the manufacturer's protocol (Promega). The nucleotide sequence data were analyzed, and comparisons were made with MacVector or Genetics Computer Group (GCG) software packages. The ascites fluid of 33 different SIPC tumors was assessed for binding activity against actin, myosin, tubulin, ssDNA, and dsDNA by ELISA 10 . We have focused on purified Abs from five of these tumors, each of which binds to at least one of the Ags from the panel. As far as heavy chain class, four out of the five SIPCs examined in this study expressed IgA . Although most Abs show higher reactivity to myosin, several are particularly reactive to dsDNA and ssDNA. The tumor SIPC3301 displays low reactivity with essentially all Ags on this panel. The SIPC3282, SIPC3308, SIPC3336, and SIPC3385 Abs were found to display a polyreactive binding activity ( Table ). Cytogenetic studies have revealed that a majority of PCs contain reciprocal T(12;15) translocations 1 . Alternatively, several SIPC tumors exhibit a reciprocal T(6;15) translocation (considered to be the variant translocation), as demonstrated by the SKY™ analysis performed on two representative SIPC tumors . While SKY™ cytogenetics is a good indicator of T(12;15) or T(6;15) translocations, it is not entirely certain whether c-Myc or Pvt 1 is targeted by these translocations, as we have only found two SIPC tumors with c-Myc rearrangements and no tumors with Pvt 1 rearrangements at the Southern hybridization level . The assay for molecular rearrangement is based on utilization of a series of hybridization probes surrounding the major breakpoints of Pvt 1 or c-Myc , and by incorporating large restriction fragments into the analyses 11 . The absence of detectable rearrangements with Pvt 1 or c-Myc in such a large number of SIPC tumors ( Table , and data not shown) suggests that, in general, SIPC-associated translocations could reside outside the usual or more common breakpoint locations, and may not be detectable with the probes used in this study. Initially, Ig heavy chain–specific rearrangements were identified in each tumor by Southern blot analysis using a J H probe . Heavy chain-specific rearrangements were found in all tumors, including apparent rearrangements of both alleles in SIPC3385, SIPC3282, and SIPC3301. We also found shared rearrangements with both BamH1 (not shown) and EcoR1 digestions in SIPC3308, SIPC3336, and SIPC3282, suggesting the same V H gene may be expressed in these tumors. We cloned and sequenced the 3.5-kb EcoR1 fragment from SIPC3336, and established that the rearrangement consists of a member of the V H -J558 family (H13-3; reference 12 ) rearranged to D H SP2-9 and J H 3. To more specifically determine V H gene usage in the SIPC tumors, we examined the expressed sequences by reverse transcription (RT)-PCR . Indeed, three of the SIPC tumors that share the 3.5-kb EcoR1 rearrangement all use the same H13-3 gene from the V H -J558 family. We verified that this sequence was germline (nonmutated) by sequencing eight clones derived from the PCR product of BALB/c genomic DNA which was amplified by primer pairs specific for H13-3 (see Table ). Interestingly, these same three tumors have also rearranged to J H 3, and use the same D H region (D H SP2-9) encoding the amino acid residues Trp-Phe. Although rearranged to J H 3 and D H SP2-9 as well, SIPC3301 has a longer nucleotide addition sequence, and is in fact encoded by another member (26.4.1-α; reference 13 ) of the V H -J558 family. The RT-PCR product of SIPC3385 was also found to use a member of the V H -J558 family, but in this case the nitrophenyl-binding 4m3 V H gene 14 . In SIPC3385, the rearrangement involves J H 4 and another member of the D H SP2 family with N sequence additions. Analysis of the V H region sequences reveals somatic mutational activity with high replacement to silent (R/S) ratios, since all the SIPCs that express H13-3 exhibit four to six replacement changes with no accompanying silent base changes . In the case of SIPC3385, we only find a single replacement in V H . We examined the light chain–specific rearrangements of the SIPC tumors at the Southern hybridization level. The tumors SIPC3308, SIPC3336, and SIPC3282 all shared identical rearrangements with both EcoR1 and BamH1 (data not shown) digestions. By cloning and sequencing the 18-kb EcoR1 fragment , we obtained a V κ 24C-J κ 4 15 sequence suggesting that SIPC3308, SIPC3336, and SIPC3282 may all share the same rearrangement. When we performed PCR amplifications in each of these tumors with primers specific for V κ 24C and J κ 4, we obtained positive products that were identical as well as nonmutated . To test whether the V κ 24C-J κ 4 rearrangement is productive (expressed), we used V κ 24C and C κ –specific primers in RT-PCR assays, and again, found positive products in each of these tumors (data not shown). Although the nature of the common 9.5-kb EcoR1 band found in SIPC3308, SIPC3336, and SIPC3282 is uncertain at the moment, we do know it must be related to the rearrangement to V κ 24C-J κ 4, since they are always found in association with the productive rearrangement in each of these tumors. Southern blots with a C κ only (minus IVS) probe result in no hybridization to the 9.5-kb EcoR1 band, proving this fragment to be nonproductive and probably a byproduct of the rearrangement process (data not shown). Both SIPC3385 and SIPC3301 exhibit different rearrangements at the Southern blot level. Therefore, we performed RT-PCR amplifications to determine the expressed V κ gene for these two tumors as well. The tumor SIPC3385 was found to express a nonmutated V κ 21G-J κ 2 sequence 16 , whereas SIPC3301 expressed a nonmutated V κ 34C-J κ 2 sequence 17 . In the process of verifying the expressed V κ sequences discussed above, we subjected all the SIPC tumors to RT-PCR amplification using sets of primers ( Table ) that were cross-reactive to each of the V κ and V λ gene families. Unexpectedly, we found that in addition to expression of V κ 24C-J κ 4, V κ 21G-J κ 2, and V κ 34C-J κ 2, each of the SIPC tumors expressed an additional Ig κ or Ig λ light chain gene, a finding in violation of allelic exclusion normally associated with Ab gene expression 18 19 20 . All the SIPC tumors examined in this study were found to express either V κ 1A or V κ 1C 21 22 rearranged to either J κ 1, J κ 2, or J κ 4 segments . Furthermore, we also found that both V κ 1C sequences and V κ 1A sequences exhibited some level of somatic mutation (as found in the V H above). With a 1:1 R/S ratio for the whole V region (and a higher R/S ratio in CDR2) in V κ 1A, only minimal levels of Ag selection are evident. We also found three tumors, SIPC3282, SIPC3301, and SIPC3336, that express V λ 1-C λ 1 sequences . In this instance, these sequences are mutated (all changes are replacements), and there is evidence of clonal divergence. Interestingly, no V λ sequences were found in SIPC3385 or SIPC3308. This later result is important, as it is a critical distinction between SIPC3308 and SIPC3336. Levels of Ig κ and Ig λ were compared by slot blot hybridization using specific probes for V κ 1, V κ 24, V λ , and C κ . Interestingly, SIPC3301 expressed high levels of Ig λ , whereas SIPC3385 expressed high levels of V κ 1. Consistently, both SIPC3282 and SIPC3336 expressed high levels of both V κ 1 and V κ 24. Since there is evidence that peritoneal cavity B cells are enriched in B-1 cells, we also tested for Ly1 expression by RT-PCR amplification of total RNA from thymus, spleen, two PCs, (ABPC18, MOPC104E) and the SIPC tumors. Although expression of Ly1 was found in thymus, spleen, and the two conventional PCs, no expression of Ly1 was evident in the SIPC tumors (data not shown). RAG-1 (data not shown) and RAG-2 activity, both of which have recently been found in the peripheral B-1 population 23 , were also independently assayed by RT-PCR amplification among similar panels of RNAs. Both RAG-1 and RAG-2 expression were found in thymus, spleen, SIPC3301, SIPC3308, SIPC3385, ABPC18, and MOPC104E, but not in SIPC3282 or SIPC3336. Since spontaneous PCs occur rarely in mice, studies in plasmacytomagenesis rely on induction models. All PCs arise in the peritoneum, where the presence of nonmetabolizable paraffin oils (pristane) or plastic implants induces chronic inflammation, granuloma formation, and finally development of the PC 1 . The SIPC may differ by having fewer numbers of atypical foci 8 . Binding studies of pristane-induced PCs showed that PCs can display binding activities against phosphorylcholine, various dextrans, and fructofuranans 2 . The SIPCs, as we show in this study, possess a set of binding specificities against cytoskeletal proteins and DNA. In fact, in the initial panel of ascites from 33 SIPC tumors, binding activity against at least 1 of the Ags of the panel, and most often polyreactive binding activity, is commonly observed. To ascertain whether the SIPC tumors possess a characteristic set of binding properties and whether these binding specificities could assist in identifying a precursor cell population, we focused on the binding activity of purified proteins from five representative SIPC tumors. We have found that three independent SIPC tumors, each derived from different generations , share identical V H domains including D H and J H regions ( Table ). Several facts argue strongly in favor of the independent derivation of these tumors: (a) each tumor was harvested at different times as a result of variable latency periods (see Materials and Methods); (b) reactivity patterns differ between each tumor ( Table ); (c) SIPC3282 shows an additional rearrangement (J H ) not found in other tumors, as well as additional somatic mutations not found in other tumors; (d) SIPC3385 and SIPC3308 both lack V λ 1 rearrangements; and (e) no RAG-1/2 expression is found in SIPC3282 or SIPC3336. These results also support the existence of a strong restriction in V gene usage for the SIPCs. These results are in contrast with reports for human myeloma, where no particular selection for V genes has been observed 24 . In contrast, Waldenström macroglobulinemia patients exhibiting rheumatoid or cold agglutinin anti-I specificities exhibit a strong restriction for V genes, as the V1-69 V H 1 gene member is almost constantly expressed in the case of cryoglobulins expressing the WA recurrent idiotype (60% of cases), and the V4-34 (V H 4-21) gene is constantly expressed by cold agglutinins with anti-I specificity (95% of cases; 25 ). Although the D H region is not identical among these Abs, a report by Fais et al. 26 shows that among 50% of CLL patients expressing the 1-69 gene, the D H 3-3 region is used. In addition, a report from the same group indicated that five different cases of CD5 + IgG + CLLs expressed virtually identical Ag receptors, by recombining (unmutated) the V H -4-39 gene to D6-13-J H 5b and the V κ 012 gene to J κ 1 27 . Ig gene assembly is an ordered process that begins with heavy chain gene rearrangement 28 under the regulation of recombinase genes (RAG-1/RAG-2). As the Ig rearrangement process is error prone, only a successful V(D)J assembly leads to pre-B cell receptor assembly 29 30 and eventual downregulation of RAG-1/RAG-2 activity 31 . Further differentiated and proliferating B cells reactivate the recombinases to rearrange the light chain genes, which once again are error prone, i.e., wherein only successful rearrangement leads to maturation rather than cell death 29 30 . Therefore, allelic exclusion, typically the hallmark of Ig gene expression and of an active process, may also stem more from the low frequency of productive or successful rearrangement. With the elucidation of V gene receptor editing 32 33 34 35 36 37 38 , we now know that lymphocytes expressing self-reactivity in the bone marrow, germinal center (GC), or even in the periphery can be rescued from cell death by exchanging already rearranged V regions with other available V genes through reactivation of the RAG-1/RAG-2 recombination pathway 23 38 39 40 . It has been shown that recombinase activity is stimulated by low-affinity Abs and is inhibited by high-affinity Abs 41 , and accordingly, secondary Ig rearrangements actually occur quite frequently 42 43 44 45 . RAG-1/RAG-2 activity is also found in GC B cells bearing low-affinity receptors, or in peripheral B-1 cells 23 . Indeed, there is increasing evidence that productive and successful light chain rearrangements do not effectively arrest further rearrangement 46 47 48 49 50 51 52 . Thus, one would then expect frequent violations of allelic exclusion in that κ:λ dual expressors 53 , as well as dual expressors of Ig κ 54 55 subtype, should be quite common. In this study of SIPC tumors, we have observed only a single productive rearrangement of the light chain genes at the Southern hybridization level for V κ 24C-J κ 4. For SIPC3385 (expressing V κ 21G-J κ 2) and SIPC3301 (expressing V κ 34C-J κ 2), we find rearrangements in both EcoR1 and BamH1 Southern blots that are, in fact, consistent with predicted sizes from germline restriction maps for both V κ and J κ gene segments 16 17 . We only detected secondarily expressed alleles (κ or λ) by RT-PCR at levels below the Southern detection levels, i.e., subthreshold. We can eliminate contaminating host tissue as contributing to this observation by several lines of argument. (a) A restricted subset (V κ 1 or V λ 1) of V genes are found, rather than simply random usage. Furthermore, since V λ 1 is rarely expressed in the mouse, finding V λ 1 in 3/5 tumors is highly unlikely. (b) We have recently colinked V κ 24 and V λ 1 expression at the single cell level by RT-PCR amplification (our unpublished results). A more likely explanation, therefore, is that we must be observing only a subset of B cells in these tumors expressing both alleles. Thus, the presence of dual expressing κ:λ or κ:κ alleles indicates that secondary rearrangements are occurring in the SIPC tumors, but whether this rearrangement occurs at significant levels to alter the tumor reactivity patterns must be considered further. Interestingly, it is conceivable that the V κ 24C-J κ 4 rearrangement that is shared by three of the tumors may already be an example of secondary rearrangements after a highly selectable process, as secondary rearrangements of the same allele are often characterized by upstream V κ segments associated with downstream J κ segments. An alternative explanation, that the observed biallelic expression arises from an outgrowth of a subset of tumor cells, cannot formally be discounted. Indeed, when we compare the reactivities ( Table ) of two SIPC tumors that exhibit identical V κ 24C-J κ 4 and V(D)J rearrangements, including amino acid substitutions, we find greater reactivity to ssDNA with SIPC3308. Thus, a perceived difference in reactivity between these tumors may stem from the “subthreshold” levels of V κ 1 or V λ . These “subthreshold” rearrangements do not necessarily have to occur in the bone marrow or GC, but could occur in the periphery where RAG-1/2 can be reactivated 56 . However, as recent studies suggest that RAG-1/2 activity may not actually be reinduced, but may reflect differing levels of B cell development 57 , we may be observing a small self-renewing B cell population in the tumors presented here. Interestingly, we find RAG-1/2 still active in most of the SIPCs, with the exception of two tumors , both of which also express IgV λ . The precursor to PCs has long been thought to be the B-1 cell through two lines of evidence: (a) B-1 cells are most abundant in the peritoneum, and are associated primarily with IgA in the lamina propria 58 ; and (b) in addition to dextran, phosphorylcholine is one of the more common Ags associated with the gut flora and is dominated by the T15 idiotype 1 . It has been shown by X-irradiation and failure to regenerate the T15 idiotype by bone marrow reconstitution 59 60 that the T15 idiotype can only be restored by peritoneal B cells (i.e., B-1 cells). While B-1 cells express Ly1, it is uncertain as to whether Ly1 is activated as a consequence of immortalization, or whether this represents a distinct B cell lineage. We have determined that several pristane-induced PCs (including M104E and ABPC18) express Ly1 by RT-PCR amplification. Conversely, we have found that the SIPC tumors do not express Ly1, but further studies will be needed to rigorously determine whether the SIPCs are truly B-1a, B-1b, or even B-2 cells. We now show that the SIPC tumors exhibit secondary Ig light chain gene rearrangements (and accompanying RAG-1/2 activity), exhibit low levels of somatic mutation in V κ or V λ , and show some evidence of clonal heterogeneity. While B-2 cells are mutated and most frequently found in the GC, the B-1 population is most often found in the mantle zone with few somatic mutations 61 . The fact that we find evidence of tumors bearing somatic mutations with intraclonal heterogeneity and with high R/S ratios suggests Ag selection is occurring. V region analysis in several tumor systems besides SIPCs, including Burkitt's lymphoma 62 63 , diffuse large cell lymphoma 64 , mantle cell lymphoma 65 , and follicular lymphomas 64 , demonstrates clonal heterogeneity in that somatic mutations appear to be ongoing during the progression of the tumor. Temporally, many of these tumors arise at different stages of lymphoid maturation and in different lymphoid compartments. In contrast, more mature tumors such as multiple myeloma 66 67 and pristane-induced mouse PCs 2 have traditionally been found to exhibit homogeneous Abs, suggesting that these transformed cells must have been immortalized post-GC. Based on these findings, we propose that the SIPC tumors may have become an immortalized B cell population in the periphery. | Study | biomedical | en | 0.999996 |
10562317 | Tissue samples obtained at surgery were immediately embedded in OCT and then frozen and stored at −80°C until further processed. All tumor samples were received as coded specimens, and for patient confidentiality, numbers corresponding to the order received in the laboratory were assigned. Nine normal breast specimens were processed in the same way. The material was collected under the auspices of Institutional Review Board protocol 97-79. The 32 studied cases of breast carcinoma, graded according to Bloom and Richardson, were distributed as follows: ductal carcinoma, high grade III (DC III), n = 17; ductal carcinoma, intermediate grade II (DC II), n = 9; ductal carcinoma, low grade I (DC I), n = 1; mixed lobular carcinoma, n = 3; carcinoma in situ, n = 2. Approximately one-half of the cases presented with metastases to regional lymph nodes. Patients' ages at the time of diagnosis ranged from 23 to 75 yr. See Table for clinicopathological parameters of each case, including age, tumor histological grade, tumor size, lymph node involvement, lymphatic and vascular invasion, estrogen and progesterone receptor status, proliferation index using Ki67 antibody MIB-1, DNA ploidy, and S phase. Samples were snap frozen, and serial 5-μm-thick sections were cut. Immunohistochemical staining of acetone-fixed sections was performed by incubation with mAbs recognizing the following molecules: CD1a (IgG2a) and HLA-DR (IgG2a) (DAKO Corp.), Langerin/DCGM4 (IgG1) and DC-Lamp (IgG1) (generated at Schering-Plough Lab.; reference 26), and CD83 (IgG2b), CD80 (IgG1), CD11c (IgG1), CD3 (IgG1), CD4 (IgG1), CD8 (IgG1) (all from Coulter-Immunotech), and CD86 (IgG1; Binding Site), followed by biotinylated goat anti–mouse IgG (DAKO Corp.) and streptavidin–peroxidase (Vector Labs.). The peroxidase was developed by diaminobenzidene tetrahydrochloride (brown color; Vector Labs.), and Mayer hematoxylin (Sigma Chemical Co.) was used as counterstain. In double-step immunohistochemical staining, after an initial blocking with goat serum and BSA, primary antibodies to CD83 or CD11c were followed by goat biotinylated anti–mouse IgG2b, IgG1 (Coulter Immunology), and streptavidin–peroxidase. After another blocking for endogenous biotin with avidin–biotin (Vector Labs.) and BSA, in a secondary step, mAbs to CD4 or DC-Lamp were followed by biotinylated anti–mouse IgG1 (Coulter Immunology) and streptavidin–alkaline phosphatase (Vector Labs.). Rabbit anti–human CD3 (DAKO Corp.) was followed by swine biotinylated anti–rabbit IgG (DAKO Corp.) and streptavidin–alkaline phosphatase. Peroxidase was developed by 3-amino-9 ethylcarbazole (red color; Vector Labs.), and alkaline phosphatase was revealed using Fast Blue as a chromogen (blue color; Vector Labs.). Isotype-matched antibodies (DAKO Corp.) were used as control. For the evaluation of macrophage inflammatory protein (MIP)3α staining, goat polyclonal antibody to MIP3α (R & D Systems, Inc.) was used, followed by biotinylated goat anti–mouse IgG (DAKO Corp.) and biotinylated rabbit anti–goat IgG (DAKO Corp.) and developed using streptavidin–peroxidase (Vector Labs.). For immunofluorescence, incubation with primary antibodies to CD1a and DC-Lamp was followed by FITC-conjugated goat antibodies to mouse Igs (Molecular Probes, Inc.). In a subsequent secondary step, the rabbit polyclonal anticytokeratin antibody (DAKO Corp.) was followed by biotinylated anti–rabbit IgG (DAKO Corp.) and revealed by streptavidin–Texas Red (Molecular Probes, Inc.). Isotype-matched antibodies were used as a control. Confocal laser scanning microscopy was performed along the x–y axis with a confocal laser scanning microscope (TCS-SP; Leica Inc.) equipped with 20, 40, and 100× oil objectives. Each slide was examined on at least two separate occasions by at least two individuals, including two pathologists. All cell counts were performed using an Olympus Ax-70 epifluorescence photomicroscope at a magnification of 400 (40× objective and 10× eyepiece). Cells displaying membrane staining, cytoplasmic staining, nuclear counterstain, and appropriate morphology were included. The area counted in each section was chosen randomly from a representative field of tumor. For each section, three areas were assessed, and the counts are expressed as the mean number of cells per high power field. In each case, a serial hematoxylin and eosin section was examined for orientation and confirmation of the histological diagnosis. Each case was scored blindly with respect to patient history, presentation, and previous scoring. DCs were generated from CD34 + hematopoietic progenitors (HPCs) or from CD14 + blood precursors as described previously 6 . In brief, cord blood CD34 + HPCs were cultured with GM-CSF (50 ng/ml; Schering-Plough Lab.), stem cell factor (20 ng/ml; Amgen) and TNF (12.5 ng/ml; Genzyme Corp.). DCs were harvested at the stage of immature precursor and used either as a total population or after sorting of CD1a + CD14 − cells on day 7 of culture. Monocyte-derived DCs were generated by culturing adherent fraction of PBMCs with GM-CSF (100 ng/ml; Schering-Plough Lab.) and IL-4 (50 ng/ml; Genzyme Corp.) 27 28 29 . DCs were used on day 7 either as immature CD1a hi CD83 − HLA-DR int or after 48 h of activation with CD40 ligand as mature CD1a lo CD83 + HLA-DR hi cells. We have used an adapted version of the Stamper-Woodruff assay measuring binding of the lymphocytes to endothelium . Immature DCs labeled with CD1a–FITC or mature DCs labeled with HLA-DR–FITC were overlaid onto frozen sections of breast carcinoma and allowed to adhere for 1 h. After stringent washing, tissue sections were indirectly stained for cytokeratin expression. Raji cells were used as a negative control. Binding of DCs to breast carcinoma sections was quantitated in several ways to establish the pattern of cell adherence. First, we determined the density of immature and mature DCs adhering to the breast cancer section at the tumor and stromal site, respectively. Density values were based on the number of FITC-labeled DCs that adhered per 0.2 mm 2 (40× objective, high power field). For each section, three areas were assessed (see Table ). Counting the number of DCs that were collected during the washing procedure permitted us to estimate that ∼15% of DCs remained on the tissue section. RNA was prepared from fragments of tumor samples that were snap frozen and stored at −80°C until use using acidified phenol procedure according to the manufacturer's instructions (GIBCO BRL). RNA preparations were treated with RNase-free DNase (37°C for 30 min; Boehringer-Mannheim), digested with proteinase K in 1% SDS, extracted with phenol-chloroform, and precipitated with ethanol. RNA from tonsils was used as positive control. The following primers were used: β-actin, 5′-CTCCTTAATGTCACGCACGATTC-3′ forward and 5′-GTGGGGCGCCCCAGGCACCA-3′ reverse; MIP3α, 5′-TTGCTCCTGGCTGCTTTG-3′ forward and 5′-ACCCTCCATGATGTGCAAG-3′ reverse; MIP3β, 5′-CTGCTGGTTCTCTGGACTTC-3′ forward and 5′-CACACTCACACTCACAACAACAC-3′ reverse. PCR was performed in 35 cycles: 94°C for 1 min, 55°C for 1.5 min, and 72°C for 1.5 min. We have analyzed the differentiation/maturation status of DCs in frozen sections obtained from 32 patients with adenocarcinoma of the breast using immunohistochemistry. The patients' characteristics and clinicopathological parameters are given in Table . Breast carcinoma cells were identified by morphology and cytokeratin expression. Immature DCs were characterized by CD1a expression, and the LC subset was characterized by expression of Langerin/DCGM4 (Lang), a recently identified Ag uniquely expressed within LCs 30 . In all samples, CD1a + cells with dendritic morphology were found to infiltrate the tumor beds . The number of CD1a + immature DCs per high power field (400×) ranged from 1 to 48 cells, with mean and median numbers 12 and 7, respectively ( Table ). Although a majority of DCs were CD1a + Lang + LCs, CD1a + Lang − cells were also found. The immaturity of CD1a + DCs present within the tumor was further demonstrated by the presence of MHC class II intracellular compartments, as assessed by serial sectioning in confocal microscopy in all CD1a + DCs examined . Normal breast tissue, i.e., that not infiltrated by tumor, rarely displayed CD1a cells, and five of nine sections were negative, whereas four of nine sections showed less than three CD1a + cells within the epithelial duct over the entire section . Thus, the presence of CD1a + cells within the breast cancer tissue was not solely due to its epithelial origin. We next sought to determine if mature DCs were also infiltrating tumor tissue. Mature DCs were characterized by expression of CD83 and DC-Lamp. Although no CD83 + cells were found in 9/9 samples of normal breast tissue, CD83 + mature DCs could be identified in 20/32 breast cancer samples (the threshold for positivity was established arbitrarily for a density greater than or equal to two cells per high power field). Most interestingly, mature DCs were located in the peritumoral areas surrounding but not penetrating the beds of carcinoma cells . The number of CD83 + mature DCs per high power field (400×) ranged from 0 to 28 cells, with mean and median numbers 5 and 6, respectively ( Table ). Staining for DC-Lamp, a highly specific marker of mature DCs 26 , confirmed the peritumoral localization of mature DCs in breast cancer tissue . Compared with immature DCs residing within the tumor bed, the mature DC-Lamp + DCs found in the peritumoral area displayed intense MHC class II labeling mostly located at the cell periphery, attesting to cell surface expression . The antibodies recognizing HLA-DR, CD80, and CD86 did not reveal DC compartmentalization because their expression pattern also includes macrophages that are scattered throughout the breast cancer tissue in all studied samples. Similarly, CD11c + labeling, found earlier to define DCs within germinal centers 9 , was present in all 32 tumor samples without any apparent compartmentalization. Because mature DCs can normally be found only in secondary lymphoid organs where they closely interact with Ag-specific T cells, we next analyzed the T cell distribution and activation pattern in breast carcinoma samples. In 19 evaluated samples, CD3 + T cells infiltrated peritumoral areas, where they could be seen either scattered throughout the area in close proximity to tumor cells or clustered . Double stainings for CD4 and CD83 demonstrated, in three out of five studied cases, T cells clustered around mature DCs in the peritumoral areas . The majority of infiltrating T cells (70–75%) were CD3 + CD8 + , and a fraction of them (<10%) displayed the early activation markers CD69 and CD25. No T cell proliferation could be seen, as judged by labeling with Ki67 antibody (not shown). We assessed the capacity of fluorochrome-labeled DCs to adhere to frozen sections of breast cancer tissue in an adapted version of the Stamper-Woodruff assay measuring binding of lymphocytes to endothelium 31 . As illustrated in Fig. 3 , immature DCs adhere selectively to the tumor cells and spread upon binding. In contrast, mature DCs, generated by CD40 ligation, adhere selectively to the peritumoral areas, thus confirming the pattern of in situ DC localization . Both immature and mature DCs adhere to tissue sections that contain infiltrating DCs, as detected immunohistochemically ( Table ). Labeled Raji cells did not to bind to any of the tumor sections, further demonstrating the specificity of DC binding. Recent studies demonstrated the expression of CCR6 on immature DCs 32 , and the DC ligand MIP3α was identified in epithelia, the site of residence of immature LCs 33 . We thus analyzed the expression pattern of MIP3α within breast tumors. First, reverse transcriptase (RT)-PCR analysis of whole breast cancer samples indicated the presence of MIP3α mRNA, whereas no MIP3α mRNA could be amplified from normal breast tissue . PCR analysis of MIP3β expression showed lack of mRNA in the breast cancer tissue, whereas MIP3β was expressed in the tonsillar tissue . Immunohistochemical analysis of seven breast cancer samples ( Table ) further demonstrated the expression of MIP3α protein by the majority of tumor cells and colocalization of immature DCs and MIP3α-producing cells . Two normal breast specimens did not express the MIP3α protein. Thus, accumulation of immature DCs within the tumor bed may be dependent on the expression of MIP3α by tumor cells. Herein we demonstrate the unique compartmentalization of immature and mature DCs within breast carcinoma tissue. 32/32 tumor samples displayed variable immature DC infiltration. This compartmentalization was further confirmed by the binding of in vitro–generated immature DCs to tumor beds. Tumor-infiltrating immature DCs were heterogeneous, and two populations could be identified, both CD1a + Langerin + LCs and non-Langerhans, CD1a + Langerin − cells. Because dermal (interstitial) DCs were found in vitro and in vivo to express CD1a but not Langerin, we currently conclude that CD1a + Langerin − tumor-infiltrating DCs may be intDCs. The pathophysiological significance of this heterogeneity remains to be established. Earlier studies revealed that intDCs generated in vitro from CD34 + HPCs display (a) more potent phagocytic activity than LCs and (b) a unique ability to induce differentiation of naive B cells 6 7 . As a hallmark of immature phenotype, DCs infiltrating the tumor bed expressed compartmentalized intracellular MHC class II molecules 34 35 36 . This subcellular distribution of MHC class II molecules changed dramatically in mature peritumoral DCs, where most of the class II molecules were relocated to the cell surface. The observed numbers of immature CD1a + DCs in the tumor microenvironment appeared much higher than in normal breast epithelium, suggesting increased homing and infiltration. This may be best explained by the high levels of intratumoral MIP3α, a chemokine that was recently shown to specifically attract immature DCs 33 37 38 39 and is now found to be expressed within the tumor epithelium. The immunohistochemistry analysis with anti-MIP3α is not, unfortunately, precise enough to allow a correlation between the amount of MIP3α within the tumor cells and the numbers of infiltrating immature DCs. The increased numbers of immature DCs could reflect a transient stage due to the high in and out migration or the sequestering of immature DCs within the tumor tissue. Perhaps the most important finding is the presence, in 20/32 breast carcinoma samples, of mature DCs that were located specifically within the peritumoral areas. This striking compartmentalization was further confirmed by the binding of in vitro–generated mature DCs to peritumoral areas. This indicates that stromal factors are determining the DC adherence. Because mature DCs are only observed in lymphoid organs, where they closely interact with T cells, it is tempting to consider that their presence within the tumor tissue reflects an ongoing immune response, possibly tumor specific. These mature DCs present within the tumor tissue could derive from any of the currently defined DC subsets discussed above. Unfortunately, the tools available now do not allow us to determine the subset origin. Because of recent data showing the role of DC subsets in Th1/Th2 polarization and the induction of different classes of immune response 8 , the determination of the origin and immunocompetence of mature, tumor-associated DCs will be of great importance to our understanding of the development of tumor immunity. The limited number of relatively heterogeneous breast cancer tissue samples analyzed to date does not allow us to establish a prognostic significance of the infiltration of tumor with immature or mature DCs. Such determination will require the analysis of a large number of samples in prospective or retrospective studies. Unfortunately, currently available CD1a and DC-Lamp antibodies do not permit analysis of paraffin-embedded archival material. Therefore, we are now evaluating alternative methodologies for the determination of DC phenotype in archival material and initiating a prospective analysis using frozen tissue samples. Our observations open a new avenue in tumor immunology. A thorough analysis of the DC system within tumors will provide clues critical to understanding the development of tumor immunity. | Study | biomedical | en | 0.999995 |
10562318 | For construction of the Txk transgenic vector, most of the 3′ untranslated txk cDNA sequence was first eliminated by digestion of the Bluescript SK plasmid containing the txk cDNA 1 with BspM1 and XhoI and resealing of the plasmid incorporating an Sal1 linker to create a Sal1 site at the 3′ end of the txk cDNA. Then the EcoR1-Sal1 txk cDNA fragment was cloned into a plasmid containing the CD2 promoter and enhancer 28 and sequences from TCR-ζ genomic DNA 29 . The CD2/Txk/TCR-ζ sequences were separated from vector sequences by digestion with Not1 and were isolated for pronuclear injection as described previously 29 . Txk transgenic mice were backcrossed to C57BL/6 (B6) mice for >10 generations. TCR transgenic mice used in these studies included H-Y and AND TCR transgenic mice. H-Y mice express an MHC class I–restricted TCR for the male antigen H-Y 30 , and AND mice express an MHC class II–restricted TCR specific for pigeon cytochrome C 31 . All TCR transgenic mice were in the H-2D b background. itk knockout mice were kindly provided by D. Littman (New York University Medical Center, New York, NY). Thymocytes were cultured when necessary in RPMI complete medium as described previously 1 . The resting period for thymocytes was 5 h at 37°C, 5% CO 2 . Polyclonal antiserum against Txk peptide 1 (CKPLPPLPQEPPDER), found near the NH 2 terminus of Txk, conjugated to KLH was raised in rabbits by Covance, Inc. Peptide synthesis was performed by Biosynthesis, Inc. Anti-CD3 (145-2C 11 ) and anti-CD28 (37.51) were prepared in our laboratory by protein G column purification (Sigma Chemical Co.). Polyclonal anti-Lck serum and anti–ZAP-70 ascites were provided by L. Samelson (National Institutes of Health). T3.70-FITC was prepared in our laboratory. Commercial antibodies used include anti-CD3–FITC (PharMingen), anti-CD4–PE (PharMingen), anti-CD8a–Quantum red (Sigma Chemical Co.), anti-Vα11–FITC (PharMingen), H57-597–biotin (PharMingen), polyclonal anti–bovine PLC-γ1 (Upstate Biotechnology), mixed monoclonal anti–bovine PLC-γ1 (Upstate Biotechnology), and antiphosphotyrosine (4G10; Upstate Biotechnology). 10 8 thymocytes were incubated for 30 min at 37°C in 5 ml complete medium containing 5 mCi TRAN 35 SLABEL (ICN Biomedicals). For immunoprecipitation, cell lysates were prepared by incubating cells in NP-40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM Hepes, 1 mM EDTA, 1 mM Na 3 VO 4 , 1% NP-40, and 1× Complete™ protease inhibitor cocktail; Roche) for 30 min on ice. Lysates were cleared by microcentrifugation for 15 min at 12,000 g . After incubation of lysates with appropriate antibodies, complexes were collected with protein A–Sepharose (Sigma Chemical Co.) and washed three times with NP-40 wash buffer (same as NP-40 lysis buffer except with 0.2% NP-40). Samples were boiled in reducing sample buffer (0.175 M Tris, pH 6.8, 30% glycerol, 3% SDS, 15% β-mercaptoethanol, 0.1% bromophenol blue) and subjected to PAGE. 10% polyacrylamide gels were used in all cases except for immunoprecipitation with the ZAP-70 antibody, in which case 12% gels were used. In the case of metabolic labeling, gels were treated with DMSO and 20% 2,5-diphenyloxazole (PPO) in DMSO, dried, and exposed to BioMax MR film (Eastman Kodak Co.) and a BioMax TranScreen LE intensifying screen. In cases of Western blotting, gels were transferred by standard methods 32 . For in vitro TCR cross-linking , 10 8 thymocytes were resuspended in RPMI 1640 at 10 7 cells/ml. 100 μg of biotinylated H57-597 and 100 μg of biotinylated GK1.5 (both prepared in our laboratory) were added for 10 min on ice. After centrifugation, the cells were resuspended in RPMI 1640 containing 20 μg/ml streptavidin (Life Technologies) for various times at 37°C. Extracts were then prepared in Triton X-100 lysis buffer (same as NP-40 lysis buffer except with 1% Triton X-100 instead of 1% NP-40) as described above. After immunoprecipitation with anti-Txk antiserum or anti-Txk antiserum with Txk peptide 1 and washing with NP-40 wash buffer minus EDTA, complexes were incubated with kinase buffer (20 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM MnCl 2 , 5 mM MgCl 2 , 2 μM NaATP, 10 μCi [γ- 32 P]ATP, and 10 μg enolase) for 15 min at room temperature. Complexes were then boiled in reducing sample buffer and subjected to electrophoresis on 10% polyacrylamide gels. The gels were then fixed, Coomassie blue stained 32 , and exposed to x-ray film (X-OMAT AR; Eastman Kodak Co.). Standard flow cytometry was performed as described previously 33 using a Becton Dickinson FACScan™ and CELLQuest™ software. For calcium flux studies, cells were washed and resuspended in HBSS/FBS (HBSS with calcium and magnesium, 10 mM Hepes, and 1% FBS). The calcium probe indo-1 and the detergent Pluronic (Molecular Probes) were added at final concentrations of 10 μM and 300 μg/μl, respectively. After washing, the cells were also stained with anti-CD8α–FITC (PharMingen) and anti-CD4–PE (PharMingen). Cells were analyzed on a FACS Vantage™ (Becton Dickinson) equipped with an argon laser tuned to 488 nm and a krypton laser tuned to 360 nm. Indo-1 fluorescence was analyzed at 390/20 and 530/20 for bound and free probe, respectively. The signals for bound and unbound indo-1 were collected in linear mode. The FACS Vantage™ was equipped with two modifications necessary for reproducible measurement of TCR-induced calcium flux: a Time Zero injection module (Cytek) and ratio offset, so that the gain of the fluorescence ratio could be increased while keeping the baseline signal in the lower channels. For each stimulation, an aliquot of cells was warmed at 37°C for 3 min before stimulation with TCR cross-linking antibody. Data were collected for 30 s, at which time varying amounts of biotinylated H57-597 (0.2–10 μg; PharMingen) were added in a total volume of 50 μl. At 60 s, 20 μg of streptavidin (Life Technologies), also in a total volume of 50 μl, was injected. Cells were collected at a rate of 1,000–2,000 cells/s. The kinetic data shown in Fig. 3 and Fig. 8 were generated using the software programs WinList (Verity Software House, Inc.) and Canvas (Deneba Software). The percentage of cells that responded by an increase in intracellular calcium after stimulation with TCR cross-linking antibody was determined using Multitime Software for Analysis of Kinetic Flow Cytometry Data (Phoenix Flow Systems). A threshold of fluorescence ratio was determined in the window of time before antibody addition. This threshold was then applied to the time window after the addition of antibody and streptavidin. The maximum number of cells above the threshold averaged over a 6-s time interval was expressed as a percentage of all of the cells in the same interval. From this number, we subtracted the percentage of cells above threshold in the resting time window to obtain the percentage of cells responding. CD4 + cells were isolated from total LN cells using positive selection magnetic cell sorting (MACS; Miltenyi Biotec) according to the manufacturer's recommendations. CD4 + cell populations were >95% pure as measured by standard flow cytometry. Measurements for levels of IL-2, IL-4, and IFN-γ were performed by ELISA using the reagents and protocol from PharMingen. To examine the role of Txk in T cell development and TCR signal transduction, we overexpressed Txk in transgenic mice. Transgene expression was driven by the human CD2 promoter/enhancer . The CD2 promoter was chosen to drive Txk expression because it directs expression early and throughout T cell development, i.e., in CD4 − CD8 − (double negative [DN]), CD4 + CD8 + (double positive [DP]), and CD4 + CD8 − and CD4 − CD8 + (single positive [SP]) thymocytes and in mature peripheral T cells ( 29 ; and Love, P.E., unpublished observations), approximating the expression pattern of endogenous txk 1 . Five founder lines were established, and two founder lines showing the highest levels of expression were analyzed. Fig. 1 shows Txk protein overexpression in the founder line showing highest expression. Extracts from hemizygous and homozygous transgenic thymocytes were immunoprecipitated with anti-Txk antibody after metabolic labeling . In vitro immune complex kinase assays were also performed using anti-Txk antiserum to show the functionality of Txk in Txk transgenic mice. As shown in Fig. 1 c, the tyrosine kinase substrate enolase was phosphorylated to a greater extent in extracts immunoprecipitated from txk transgenic thymocytes compared with extracts from wild-type thymocytes. Greater enolase phosphorylation was also seen for txk transgenic LNs, as was expected from the expression pattern of the CD2 promoter 28 . Data averaged from several experiments show that Txk protein overexpression was ∼10-fold in txk homozygous transgenic mice. Increases in in vitro kinase activity were also ∼10-fold in txk homozygous transgenic mice. txk transgenic mice exhibited normal development and fertility. T cell maturation appeared unaffected as assessed by normal numbers of thymocytes and T lymphocytes. In addition, percentages of thymocyte subsets in txk transgenic mice (DN, DP, and SP thymocytes) were normal, as were the ratios of CD4 + to CD8 + cells in thymus and in LNs as determined from several experiments . Staining for CD3 and the following markers (data not shown) showed similar profiles for txk transgenic and nontransgenic thymocytes (TCR-α/β, TCR-γ/δ, CD5, CD69, and CD24) and lymphocytes (TCR-α/β, TCR-γ/δ, CD5, CD44, and CD62L). Because the Tec family kinases Itk and Btk have been shown to regulate calcium flux in T and B cells, respectively 26 27 , we analyzed calcium mobilization in response to TCR cross-linking in txk transgenic thymocytes and lymphocytes. Cells were preloaded with the calcium dye indo-1, stained for CD4 and CD8, and then stimulated with biotinylated anti–TCR-β antibody. At low concentrations of anti–TCR-β (1.0 μg/ml), the calcium response was enhanced in txk transgenic DP thymocytes relative to nontransgenic DP thymocytes as assessed by: (a) the percentage of cells responding (18.6 vs. 3.2%, respectively); (b) the accelerated kinetics of the early calcium response; and (c) the increased amplitude of the kinetic response . However, with higher concentrations of anti–TCR-β (10 μg/ml), the calcium response was equivalent in txk transgenic and nontransgenic thymocytes (data not shown). In addition, the kinetics of the delayed calcium response were similar in txk transgenic and nontransgenic mice . SP thymocytes and LN T cells from txk transgenic mice exhibited similarly enhanced responses compared with equivalent populations of nontransgenic cells when stimulated with submaximal concentrations of antibody . Again, in txk transgenic mice the percentage of CD4 SP cells responding was increased (75.3 vs. 37.6% cells responding for CD4 + thymocytes, and 41.0 vs. 33.2% cells responding for CD4 + lymphocytes), as were the kinetics and amplitude of the early calcium response . One outcome of an enhanced TCR-mediated calcium response should be increased IL-2 production. We measured cytokine production in CD4 + LN cells from txk transgenic mice after stimulation by plate-bound anti-CD3 or anti-CD3 plus anti-CD28 ( Table ). CD4 + LN cells from txk transgenic mice produced significantly more IL-2 than nontransgenic CD4 + T cells. Moreover, the effect of Txk overexpression was dose dependent, since lymphocytes from txk homozygous transgenic mice produced more IL-2 than lymphocytes from txk hemizygous transgenic mice. However, no enhancement in proliferation was observed either in lymphocytes or thymocytes from txk transgenic mice in response to TCR cross-linking using similar conditions to those used for analysis of cytokine production (data not shown). It has been previously reported that txk is preferentially expressed in Th1 versus Th2 T cell clones 2 . To investigate whether Txk overexpression would direct CD4 + lymphocytes down either a Th1 or Th2 developmental pathway, we also measured levels of IL-4 and IFN-γ in response to TCR cross-linking. Table shows that both IL-4 and IFN-γ secretion were increased in txk transgenic CD4 + LN cells, indicating an overall increase in cytokine production in txk transgenic mice but no bias toward either the Th1- or Th2-specific cytokine profile under these conditions. To further investigate where in the TCR-mediated signal transduction pathway Txk exerts its effect, we next examined phosphorylation levels and activities of several signal transduction molecules known to be activated upon TCR engagement. Early events after engagement of the TCR are phosphorylation of the ζ chain and CD3 subunits of the TCR by Lck, and recruitment of ZAP-70 to the TCR complex. Recruitment of adapter molecules to the TCR complex then results in activation of two more distal pathways of signal transduction. The first pathway involves PLC-γ1–dependent phosphoinositol lipid metabolism and leads to the release of calcium from intracellular stores and the activation of protein kinase C. Another major signal transduction pathway involves activation of Ras and the mitogen-activated protein (MAP) kinases (for a review, see reference 34). Tyrosine phosphorylation levels of early effectors of TCR signal transduction (TCR-ζ, ZAP-70, Lck) did not vary between txk transgenic and B6 control thymocytes either before or after TCR engagement . Similarly, tyrosine phosphorylation (Vav) or kinase activity (Erk-2) of signal transduction molecules in the MAP kinase pathway was identical in txk transgenic and nontransgenic thymocytes (data not shown). Notably, tyrosine phosphorylation of PLC-γ1 was reproducibly greater in ex vivo thymocytes from txk transgenic mice than from nontransgenic thymocytes . Since ex vivo thymocytes are known to be activated to some extent due to contact with thymic epithelial cells 35 , we also determined the tyrosine phosphorylation levels of PLC-γ1 in thymocytes that had been “rested” by incubation at 37°C for 5 h in the absence of thymic epithelial cells. In rested thymocytes, the level of PLC-γ1 phosphorylation was low and appeared equivalent in control and txk transgenic thymocytes . In addition, PLC-γ1 phosphorylation was equivalent in thymocytes from txk transgenic and nontransgenic mice that had been activated for 5 min with anti-TCR plus anti-CD4 antibodies . However, a time course analysis revealed that at early time points after TCR engagement (0.5–1 min), PLC-γ1 phosphorylation was greater in thymocytes from txk transgenic mice . This effect was transgene dose dependent, as the level of PLC-γ1 phosphorylation was greater in homozygous transgenic than in hemizygous transgenic mice . Interestingly, at later time points (>1 min) the extent of PLC-γ1 phosphorylation was equivalent in nontransgenic, hemizygous transgenic, and homozygous transgenic mice . The amount of inositol trisphosphate generated in thymocytes from nontransgenic, hemizygous transgenic, and homozygous transgenic mice was also equivalent at 1, 2, and 5 min after TCR cross-linking (data not shown); however, inositol trisphosphate levels at earlier (<1 min) time points were not measured. Together these results reveal that within a certain window, txk transgenic T cells appear hyperactivated as measured by enhanced calcium flux kinetics and by the level of PLC-γ1 tyrosine phosphorylation. A sensitive measure of the effects of T cell activation on thymocyte development can be obtained using TCR transgenic mice to examine thymocyte positive and negative selection. To determine if positive selection was affected in txk transgenic mice, we used the H-Y transgenic TCR 30 and the AND transgenic TCR 31 . The H-Y transgenic TCR is class I restricted and H-2D b specific. The antigen for the H-Y transgenic TCR is from the male-specific protein H-Y, and T cells expressing the transgenic TCR undergo positive selection in female mice and negative selection in male mice. In female H-Y transgenic mice, both the percentage and total number of CD8 SP thymocytes expressing high levels of the transgenic TCR (as detected by staining with the H-Y TCR clonotype-specific antibody T3.70) were reduced in txk transgenic mice compared with non- txk transgenic littermates . This effect was observed consistently within multiple individual experiments comparing age-matched mice . Moreover, in all experiments, the percentage and total number of transgenic TCR hi (T3.70 hi ) DP thymocytes (the direct precursors of T3.70 high CD8 SP thymocytes) were increased in txk transgenic mice . Again, the effect of the txk transgene was dose dependent, since the reduction in T3.70 hi CD8 SP thymocytes was greater in homozygous txk transgenic mice than in hemizygous txk transgenic mice ( Table ). Thus, overexpression of Txk inhibited thymocyte positive selection in H-Y females, resulting in a specific reduction in the number of T3.70 hi CD8 SP thymocytes. Similar results were obtained in mice expressing the class II–restricted transgenic TCR, AND. The number of transgenic TCR hi CD4 SP thymocytes (as detected by staining with antibody specific for Vα11) was decreased in txk × AND mice relative to non- txk transgenic AND littermates . As with the H-Y transgenics, total thymocyte numbers were similar between AND and txk × AND mice, and the number of transgenic TCR hi (Vα11 hi ) DP thymocytes was increased in txk × AND relative to AND mice . The decrease in SP transgenic TCR hi thymocytes in txk × H-Y and txk × AND mice suggests that either fewer DP transgenic TCR hi thymocytes are positively selected in these mice, or that the transgenic TCR hi SP thymocytes normally generated by positive selection are undergoing negative selection. Since Txk and Itk are related kinases that appear to have similar functions in the calcium signaling pathway, we also examined whether Txk could substitute for Itk. The efficiency of positive selection is markedly reduced in itk − / − mice as measured in both H-Y TCR transgenic and AND TCR transgenic mice. Significantly, introduction of the txk transgene into the itk − / − background partially restored the generation of transgenic TCR hi SP thymocytes and peripheral T cells (data not shown) in both H-Y TCR and AND TCR transgenic mice. Moreover, the unusual transgenic TCR hi population of DN thymocytes, which is expanded in itk − / − TCR transgenic mice 21 , was reduced to levels comparable to those seen in itk +/− or itk +/+ mice upon expression of the txk transgene. In addition, both the TCR-induced calcium response and TCR-induced PLC-γ1 tyrosine phosphorylation were normalized in itk − / − txk transgenic mice. These results demonstrate that, in spite of the structural differences between Txk and Itk, overexpression of Txk can at least partially reverse the developmental and signal transduction defects in itk − / − mice. By overexpressing the Tec family kinase Txk in transgenic mice, we observed specific effects on TCR signaling, which include selective augmentation of the calcium signaling pathway, enhanced cytokine production, and alterations in positive selection. These effects were not due to a chance integration effect of the transgene, since a second founder line with lower Txk expression had similar but reduced effects (data not shown). Engagement of the TCR results in the activation of at least two major intracellular signaling pathways (for a review, see reference 34). One pathway leads to the activation of Ras and MAP kinases, the other to the initiation of phosphoinositol lipid metabolism. Activation of phosphatidylinositol 3′-hydroxyl kinase results in the generation of phosphatidylinositol 3,4-bisphosphate in addition to other phosphoinositol lipids. The products of phosphatidylinositol 3,4-bisphosphate breakdown by PLC-γ stimulate calcium release from intracellular stores 36 and activate the protein kinase C pathway 34 . Several Tec family members have been implicated in phosphoinositol lipid metabolism pathways. Phosphatidylinositol 3′-hydroxyl kinase has been shown to activate the kinase activities of both Btk and Itk 37 38 probably through the generation of inositol phosphates, which can bind the PH domains of Btk and Itk, targeting them to the cell membrane 39 40 41 . Btk and Itk are then phosphorylated by Src superfamily kinases, resulting in their activation 37 38 42 43 44 45 46 . Since Txk lacks a PH domain, either another mechanism mediates its cell membrane localization or this step is not essential for its activation. Btk action is thought to be mediated, at least in part, by the downstream activation of PLC-γ2, the major PLC-γ isoform expressed in B cells 25 26 . In cells mutant for Btk, PLC-γ2 phosphorylation was reduced, leading to a defect in B cell receptor–mediated phosphoinositol hydrolysis and calcium influx. In T cells, absence of Itk results in decreased phosphorylation of PLC-γ1 and decreased calcium flux in response to TCR stimulation 27 . Txk appears to function similarly to Itk in T cells as evidenced by the further impairment in PLC-γ1–mediated signaling responses in mice made deficient for both itk and txk 24 , and by the increase in tyrosine phosphorylation of PLC-γ1 and in intracellular calcium responses observed here in txk transgenic thymocytes. Moreover, Itk and Txk function specifically within the calcium signaling pathway, as TCR proximal signaling events such as phosphorylation of TCR-ζ and ZAP-70 are unaffected in itk − / − 27 , itk − / − × txk − / − 24 , and txk transgenic mice . Activation of Vav and Erk-2 is also unaffected in txk transgenic mice; however, decreased MAP kinase activation was noted in itk − / − × txk − / − mice 24 . Although Txk and Itk appear to perform similar functions in terms of enhancing PLC-γ1 phosphorylation and the calcium response to TCR cross-linking, endogenous Txk cannot replace Itk, since significant defects in PLC-γ1 phosphorylation and calcium flux are readily observed in itk − / − mice 27 . However, we have shown that overexpression of Txk can, at least partially, restore the defect in thymocyte selection and calcium mobilization in itk − / − mice. One explanation for these results could be that Txk and Itk are functionally redundant but that endogenous Itk is expressed at significantly higher levels than endogenous Txk, such that Txk overexpression is required to correct the itk − / − defect. Alternatively, Txk may function inefficiently as a substitute for Itk, perhaps due to its lack of a PH domain. A similar example of partially redundant Src family tyrosine kinases in T cells was observed when expression of a constitutively active Fyn transgene in lck − / − mice resulted in partial rescue of thymocyte development 47 . It is also important to note that our results do not exclude the possibility that Txk indirectly enhances PLC-γ1 phosphorylation through some as yet unknown intermediary. We also addressed whether overexpression of Txk could correct the defect in positive selection in itk − / − mice. In the absence of Itk, positive selection of thymocytes bearing a defined transgenic TCR (H-Y or AND) is severely reduced 21 . Overexpression of Txk in itk − / − mice partially reversed this defect, suggesting that Txk could substitute for Itk. An additional observation made by Liao and Littman 21 and borne out here is the presence of an increased number of transgenic TCR hi DN thymocytes in H-Y and AND transgenic itk − / − mice. Although the identity of these cells is unknown, their number decreased with Txk overexpression, suggesting a return toward a normal phenotype and a redundancy in function between Txk and Itk. Therefore, these experiments reveal a similar role for Txk and Itk in the signaling pathway required for positive selection. Txk overexpression also influenced the efficiency of positive selection in the wild-type ( itk +/+ ) background. This effect was seen only in TCR transgenic mice and therefore did not represent a generalized inhibition of positive selection, but rather seems to be due to the alteration in the signaling response of the TCR. In contrast to the rescuing effect of Txk overexpression in itk − / − mice, in wild-type ( itk +/+ ) mice Txk overexpression resulted in the generation of fewer transgenic TCR hi SP thymocytes in both H-Y TCR transgenic and AND TCR transgenic mice. If Txk overexpression enhances TCR signaling as suggested by our biochemical and functional analysis, it might have been predicted that the efficiency of positive selection would increase in TCR transgenic mice, resulting in the generation of more rather than fewer transgenic TCR hi SP thymocytes. Other genetic alterations that enhance TCR signaling have been shown to increase the efficiency of positive selection in TCR transgenic mice as assessed by criteria similar to those employed here. For example, in the absence of CD5, which exerts an inhibitory effect on TCR signaling, thymocytes are hyperresponsive to TCR cross-linking, and the efficiency of positive selection is enhanced in H-Y TCR transgenic mice 48 . Similar effects on positive selection were observed in CD45 +/− mice as assessed in P14 TCR transgenic mice 49 . However, negative selection was also enhanced in CD45 +/− mice as assessed by lymphocytic choriomeningitis virus infection 49 . In our experiments, we observed fewer transgenic TCR hi SP thymocytes in txk transgenic mice. This result could be explained by the failure of txk transgenic thymocytes to signal appropriately for positive selection, perhaps due to the delivery of an unbalanced TCR signal resulting from selective augmentation of the PLC-γ/calcium pathway relative to other downstream pathways such as the Ras pathway. Alternatively, the reduction in transgenic TCR hi SP thymocytes in txk transgenic mice could be attributed to induction of cell death (negative selection) as a result of increased TCR signaling. In fact, negative selection has been shown to be particularly sensitive to alterations in calcium flux 50 51 . Elevated calcium responses have been correlated with negative selection in H-Y transgenic mice 50 and in TCR transgenic fetal thymic organ cultures treated with altered peptide ligands 52 . In addition, in B cells, qualitatively different calcium signals can result in differential transcriptional responses 53 54 , suggesting that the kinetics of the calcium response can selectively influence the expression of specific genes. Although negative selection was unaffected by Txk overexpression in H-Y transgenic male mice (data not shown), increased cell death may still account for the apparent decrease in positive selection seen in H-Y female and AND transgenic mice. Negative selection occurs early in H-Y male thymocytes, before the DP stage 55 , and enhancement of negative selection has been difficult to demonstrate using this particular system. Since we observed normal thymocyte cellularity and increased numbers of transgenic TCR hi DP thymocytes in txk transgenic × TCR transgenic mice, enhanced cell death may be occurring at a later stage (e.g., the early SP stage 56 ). Consistent with this idea, a higher percentage of thymocytes from txk transgenic mice undergoes apoptosis when cultured either in the absence or presence of stimulating antibodies (anti-TCR plus anti-CD28) compared with thymocytes from non- txk transgenic mice (data not shown). Although these findings are subject to other interpretations, we favor the hypothesis that increased apoptosis of txk transgenic thymocytes cultured in the absence of stimulating antibodies reflects the outcome of signals generated in vivo. Indeed, PLC-γ1 was found to be hyperphosphorylated in ex vivo thymocytes from txk transgenic mice, indicating enhanced signaling in vivo. In conclusion, our results identify a specific role for Txk in the TCR signaling pathway leading to calcium mobilization, and demonstrate that Txk can influence the efficiency of positive selection. In addition, these findings reveal a similarity in function for the Tec family kinases Txk and Itk in T cells. | Study | biomedical | en | 0.999997 |
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