Syndapin Oligomers Interconnect the Machineries for Endocytic Vesicle Formation and Actin Polymerization
2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês
10.1074/jbc.m510226200
ISSN1083-351X
AutoresMichael M. Kessels, Britta Qualmann,
Tópico(s)Retinal Development and Disorders
ResumoSyndapins were proposed to interconnect the machineries for vesicle formation and actin polymerization, as they interact with dynamin and the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Syndapins, however, have only one Src homology 3 domain mediating both interactions. Here we show that syndapins self-associate via direct syndapin/syndapin interactions, providing a molecular mechanism for the coordinating role of syndapin. Cross-link studies with overexpressed and endogenous syndapins suggest that predominantly dimers form in vivo. Our analyses show that the N-terminal Fes/Cip4 homology domain but not the central coiled-coil domain is sufficient for oligomerization. Additionally, a second interface located further C-terminally mediated interactions with the N terminus. The Src homology 3 domain and the NPF region are not involved and thus available for further interactions interconnecting different syndapin binding partners. Our analyses showed that self-association is crucial for syndapin function. Both syndapin-mediated cytoskeletal rearrangements and endocytosis were disrupted by a self-association-deficient mutant. Consistent with a role of syndapins in linking actin polymerization bursts with endocytic vesicle formation, syndapin-containing complexes had a size of 300-500 kDa in gel filtration analysis and contained both dynamin and N-WASP. The existence of an interconnection of the GTPase dynamin with N-WASP via syndapin oligomers was demonstrated both by coimmunoprecipitations and by reconstitution at membranes in intact cells. The interconnection was disrupted by coexpression of syndapin mutants incapable of self-association. Syndapin oligomers may thus act as multivalent organizers spatially and temporally coordinating vesicle fission with local actin polymerization. Syndapins were proposed to interconnect the machineries for vesicle formation and actin polymerization, as they interact with dynamin and the Arp2/3 complex activator N-WASP (neural Wiskott-Aldrich syndrome protein). Syndapins, however, have only one Src homology 3 domain mediating both interactions. Here we show that syndapins self-associate via direct syndapin/syndapin interactions, providing a molecular mechanism for the coordinating role of syndapin. Cross-link studies with overexpressed and endogenous syndapins suggest that predominantly dimers form in vivo. Our analyses show that the N-terminal Fes/Cip4 homology domain but not the central coiled-coil domain is sufficient for oligomerization. Additionally, a second interface located further C-terminally mediated interactions with the N terminus. The Src homology 3 domain and the NPF region are not involved and thus available for further interactions interconnecting different syndapin binding partners. Our analyses showed that self-association is crucial for syndapin function. Both syndapin-mediated cytoskeletal rearrangements and endocytosis were disrupted by a self-association-deficient mutant. Consistent with a role of syndapins in linking actin polymerization bursts with endocytic vesicle formation, syndapin-containing complexes had a size of 300-500 kDa in gel filtration analysis and contained both dynamin and N-WASP. The existence of an interconnection of the GTPase dynamin with N-WASP via syndapin oligomers was demonstrated both by coimmunoprecipitations and by reconstitution at membranes in intact cells. The interconnection was disrupted by coexpression of syndapin mutants incapable of self-association. Syndapin oligomers may thus act as multivalent organizers spatially and temporally coordinating vesicle fission with local actin polymerization. The cortical actin cytoskeleton is crucial for shaping cells and for maintaining specialized morphologies that are directly related to various cellular functions. Cortical cytoskeletal structures can be extremely elaborate and, supported by experimental data in particular for exocytic membrane trafficking events, have therefore mainly been viewed as a barrier for membrane trafficking events (1Muallem S. Kwiatkowska K. Xu X. Yin H.L. J. Cell Biol. 1995; 128: 589-598Crossref PubMed Scopus (391) Google Scholar, 2Valentijn K. Valentijn J.A. Jamieson J.D. Biochem. Biophys. Res. Commun. 1999; 266: 652-661Crossref PubMed Scopus (77) Google Scholar). For endocytosis, in contrast, mounting evidence suggests that the actin cytoskeleton may also have a supporting role (for reviews, see Refs. 3Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-116Crossref PubMed Scopus (357) Google Scholar, 4Qualmann B. Kessels M.M. Int. Rev. Cytol. 2002; 220: 93-144Crossref PubMed Scopus (155) Google Scholar, 5Engqvist-Goldstein Å.E.Y. Drubin D.G. Annu. Rev. Cell Dev. Biol. 2003; 19: 287-332Crossref PubMed Scopus (494) Google Scholar, 6Orth J.D. McNiven M.A. Curr. Opin. Cell Biol. 2003; 15: 31-39Crossref PubMed Scopus (206) Google Scholar). The formation of vesicles from a donor membrane requires complex protein machinery and additional proteins to control it (7Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (426) Google Scholar, 8Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3101) Google Scholar). Among those is the large GTPase dynamin that has been observed to concentrate at constricted necks of forming endocytic vesicles and is a crucial but mechanistically still not fully understood player in the vesicle fission step (9Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar, 10Sever S. Curr. Opin. Cell Biol. 2002; 14: 463-467Crossref PubMed Scopus (114) Google Scholar, 11Sever S. Damke H. Schmid S.L. Traffic. 2000; 1: 385-392Crossref PubMed Scopus (185) Google Scholar, 12Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1129) Google Scholar). We have suggested several possibilities how the actin cytoskeleton may support endocytosis (3Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-116Crossref PubMed Scopus (357) Google Scholar). Cytoskeletal components may localize the endocytic machinery to domains of the plasma membrane, either by trapping it or by direct anchoring. This would greatly speed up the assembly of the machinery at locations of receptors and cargo to be internalized. Furthermore, actin polymerization may provide the forces to deform the membrane and to drive vesicle formation and detachment. In line with these hypotheses, short lived actin structures at sites of endocytosis coinciding with dynamin-mediated vesicle release have indeed been observed (13Merrifield C.J. Feldman M.E. Wan L. Almers W. Nat. Cell Biol. 2002; 4: 691-698Crossref PubMed Scopus (564) Google Scholar, 14Yarar D. Watermann-Storer C.M. Schmid S.L. Mol. Biol. Cell. 2005; 16: 964-975Crossref PubMed Scopus (347) Google Scholar). Most recently, the burst of actin polymerization at clathrin-coated pits has been reported to coincide with a local recruitment of the Arp2/3 complex (15Merrifield C.J. Qualmann B. Kessels M.M. Almers W. Eur. J. Cell Biol. 2004; 83: 13-18Crossref PubMed Scopus (181) Google Scholar), which promotes actin filament nucleation and polymerization (16Higgs H.N. Pollard T.D. Annu. Rev. Biochem. 2001; 70: 649-676Crossref PubMed Scopus (546) Google Scholar, 17Welch M.D. Mullins R.D. Annu. Rev. Cell Dev. Biol. 2002; 18: 247-288Crossref PubMed Scopus (385) Google Scholar). Also the Arp2/3 complex activator N-WASP 2The abbreviations used are: N-WASP, neural Wiskott-Aldrich syndrome protein; SH3, Src homology 3; GST, glutathione S-transferase; GFP, green fluorescent protein; MBP, maltose-binding protein; HA, hemagglutinin; PRD, proline-rich domain; HEK293, human embryonic kidney 293; IP, immunoprecipitation; EDC, 1-ethyl-3-[dimethylaminopropyl]carbodiimide; BAR, BIN/amphiphysin/RVS; CC, coiled-coil; FCH, Fes/Cip4 homology. (18Takenawa T. Miki H. J. Cell Sci. 2001; 114: 1801-1809Crossref PubMed Google Scholar, 19Millard T.H. Sharp S.J. Machesky L.M. Biochem. J. 2004; 380: 1-17Crossref PubMed Scopus (212) Google Scholar) appears transiently at sites of endocytosis (15Merrifield C.J. Qualmann B. Kessels M.M. Almers W. Eur. J. Cell Biol. 2004; 83: 13-18Crossref PubMed Scopus (181) Google Scholar). The hypothesis that actin polymerization during endocytosis is triggered by N-WASP, and the Arp2/3 complex was substantiated by experiments showing that an interference with N-WASP function in vivo had a strong impact on receptor-mediated endocytosis (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar). These data were recently confirmed by the analysis of cells from N-WASP knock out mice that also showed impairments in endocytosis (21Benesch S. Polo S. Lai F.P. Anderson K.I. Stradal T.E. Wehland J. Rottner K. J. Cell Sci. 2005; 118: 3103-3115Crossref PubMed Scopus (137) Google Scholar, 22Innocenti M. Gerboth S. Rottner C. Lai F.P.L. Hertzog M. Stradal T.E.B. Frittoli E. Didry D. Polo S. Disanza A. Benesch S. Di Fiore P.P. Carlier M.-F. Scita G. Nat. Cell Biol. 2005; 7: 969-976Crossref PubMed Scopus (172) Google Scholar). In order to support and not inhibit endocytic vesicle formation, actin polymerization during endocytosis requires an extremely fine control in time and space. The molecular mechanism that ensures that N-WASP and the Arp2/3 complex trigger local actin polymerization specifically at sites of endocytosis and only during and/or after vesicle fission appears to be the interaction of N-WASP with so-called accessory proteins of the vesicle formation machinery, such as syndapins (23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar, 24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). Syndapins, a family of dynamin-binding proteins also referred to as PACSINs, were suggested to be molecular links between membrane trafficking and cortical cytoskeleton dynamics (reviewed in Ref. 25Kessels M.M. Qualmann B. J. Cell Sci. 2004; 117: 3077-3086Crossref PubMed Scopus (138) Google Scholar). Both aspects of syndapin function are supported by in vivo data, the dynamin-binding syndapin Src homology 3 (SH3) domain is a potent inhibitor of receptor-mediated endocytosis, and overexpression of full-length syndapins induced a cortical actin phenotype (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). Dominant-negative effects observed upon overexpression of N-WASP and fragments thereof encompassing the syndapin-binding proline-rich domain and corresponding rescue experiments strongly suggested that N-WASP's role in endocytosis may involve the syndapin association (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar). This hypothesis was further strengthened by the observation that syndapins can recruit N-WASP to cellular membranes and that the reconstitution of such protein complexes elicited local actin polymerization in an Arp2/3 complex-dependent manner (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar). The functional duality of syndapins, however, is based on protein interactions all mediated via one binding module, the C-terminal SH3 domain of syndapins. This raised the question whether syndapins exist in a variety of different two-component complexes, which may even represent distinct functions, or whether syndapins indeed have the capability to connect endocytic, cytoskeletal, and other cellular functions represented by their SH3 domain interaction partners. We have suggested earlier that the syndapin N-terminal regions could oligomerize, since syndapins contain stretches of a high propensity for coiled-coil domains (23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar). In this study, we addressed a potential syndapin oligomerization in vitro and in vivo and examined its importance for both the cytoskeletal and the endocytic functions of syndapin. DNA Constructs and Recombinant Proteins—Constructs coding for glutathione S-transferase (GST) fusion proteins of syndapin I full-length (amino acids 1-441), syndapin I ΔSH3 (amino acids 1-382), syndapin I SH3 (amino acids 376-441), and the P434L mutant form of the syndapin I SH3 domain were described previously (23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar, 24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). GST-N-WASP PRD (amino acids 313-349) was generated by PCR and inserted into pGEX-5.1. Mammalian expression constructs encoding the corresponding full-length protein or fragments thereof fused to the C terminus of green fluorescent protein (GFP), the FLAG epitope, and the Myc epitope were generated by subcloning the corresponding DNA inserts from the pcDNA3.1/His vector (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar) into the BamHI-XbaI sites of pEGFP-C1 (Clontech) and the BamHI-EcoRI sites of pCMV-Tag2B (Stratagene) or pRK5, respectively. Constructs to express maltose-binding protein (MBP) fusion proteins of syndapin I full-length protein or parts thereof were generated by PCR using the corresponding GST-syndapin I plasmid as a template. The PCR products were cloned into the pMAL-c2 vector (New England Biolabs). GST and MBP fusion proteins were expressed in Escherichia coli BL21 cells and purified as described previously (23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar). Xpress-tagged syndapin I, GFP-N-WASP, and the plasmid encoding mitochondria-targeted syndapin I were described in Refs. 24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar and 20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar, respectively. The hemagglutinin (HA)-dynamin 1 construct was a gift of Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA). Further partial syndapin I expression constructs were generated by PCR, cloned into the appropriate expression vectors, and analyzed by DNA sequencing. The Mito-GFP vector was generated by introducing a sequence coding for GFP, which was amplified by PCR from a commercially available GFP vector (Clontech), into the mitochondrial targeting vector (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar). Subsequently, the cDNAs encoding for rat N-WASP full-length (amino acids 1-501) and the proline-rich domain (PRD) of rat N-WASP (amino acids 265-391) (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar) were subcloned into the Mito-GFP vector. Antibodies—Rabbit anti-syndapin and anti-GST antibodies as well as guinea pig anti-N-WASP antibodies were raised and affinity-purified as described previously (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar, 23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar, 24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). Monoclonal anti-FLAG antibodies (M2) were from Sigma, monoclonal anti-Xpress antibodies were from Invitrogen, and monoclonal anti-dynamin antibodies were purchased from Transduction Laboratories. Rabbit anti-MBP antibodies were from New England Biolabs, rabbit anti-FLAG antibodies were from Zymed Laboratories Inc., and rabbit anti-GFP antibodies (290) were from Abcam. Monoclonal anti-GFP antibodies (B34), anti-Myc antibodies (9E10), and antibodies against the HA tag (HA.11) were purchased from Babco. Secondary antibodies used in this study include goat anti-mouse peroxidase (Dianova), goat anti-guinea pig peroxidase (ICN Biochemicals), donkey anti-rabbit peroxidase (Dianova), Alexa Fluor™ 568 goat anti-mouse (Molecular Probes, Inc., Eugene, OR), Alexa Fluor™ 350 goat anti-rabbit (Molecular Probes), and Alexa Fluor™ 488 goat anti-mouse (Molecular Probes). Coprecipitation Assays—In order to test for a direct syndapin I/syndapin I interaction, GST fusion proteins were immobilized on glutathione-Sepharose in the presence of 5% bovine serum albumin and incubated with MBP fusion proteins overnight at 4 °C in phosphate-buffered saline containing 1.25% bovine serum albumin, 300 mm NaCl, and 1% Triton X-100. Proteins coprecipitated with the GST fusion proteins were eluted with glutathione-containing buffer and analyzed by SDS-PAGE and Western blotting with rabbit anti-MBP antibodies. Gel Filtrations—Rat brain extracts were fractionated on a Superose 6 column (30 × 1 cm). Fractions eluted with buffer A (10 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EGTA, 0.1 mm MgCl2) were resolved by SDS-PAGE and blotted to nitrocellulose membranes. After Ponceau-S staining, the distribution of syndapin I and of its direct binding partners was detected with antibodies against dynamin 1, N-WASP, and syndapin I. Preparation of Cell Extracts and Immunoprecipitation—For immunoprecipitations of epitope-tagged proteins, human embryonic kidney 293 (HEK293) cells were transfected with different GFP- and FLAG-tagged constructs using the Lipofectamine PLUS transfection reagent method according to the instructions of the manufacturer (Invitrogen). The cells were grown for an additional 40 h, harvested, and homogenized in immunoprecipitation (IP) buffer (10 mm Hepes, pH 7.4, 1 mm EGTA, 0.1 mm MgCl2, 100 mm NaCl, 1% Triton X-100) for 20 min at 4 °C. Cell lysates were cleared by centrifugation at 14,000 × g for 20 min at 4 °C. 6 μg of monoclonal anti-FLAG antibody M2 (Sigma), 3 μg of mouse anti-GFP antibody 3E6 (Molecular Probes), or nonimmune mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) prebound to protein G-Sepharose was incubated with the high speed supernatants prepared from the lysed HEK cells overnight at 4 °C. The precipitated material was washed and probed for coimmunoprecipitated proteins by SDS-PAGE and immunoblotting using rabbit and mouse monoclonal anti-FLAG antibodies, rabbit and mouse monoclonal anti-GFP antibodies, and rabbit anti-syndapin antibodies. For coimmunoprecipitations of tertiary complexes of dynamin, syndapin, and N-WASP, HEK293 cells were cotransfected with HA-dynamin 1, with GFP-N-WASP, and, when indicated, with Xpress-syndapin I as described above. Cell lysates adjusted to 30 mm NaCl and 1% Triton X-100 final were incubated with 6 μg of anti-HA antibodies or normal mouse IgG (Santa Cruz Biotechnology) for 2 h at 4 °Cand subsequently precipitated with protein A/G-agarose (Santa Cruz Biotechnology). Specifically precipitated material was analyzed for immuno- and coimmunoprecipitated proteins by SDS-PAGE and immunoblotting with mouse monoclonal anti-HA antibodies, mouse monoclonal anti-GFP antibodies, and rabbit anti-syndapin SH3 antibodies (2521). For competition experiments, HEK293 cells were cotransfected with HA-dynamin 1 and Xpress-syndapin I. Prior to the immunoprecipitation with anti-Xpress antibodies, cell lysates were incubated with GST-N-WASP PRD fusion protein for 10 min at 30 °C as indicated. For coimmunoprecipitations of syndapin I-containing protein complexes from gel filtration fractions, affinity-purified anti-syndapin I or anti-GST antibodies prebound to protein A/G-agarose (Santa Cruz Biotechnology) were incubated with syndapin I-containing fractions adjusted to 75 mm NaCl and 1% Triton X-100 overnight at 4 °C. After intense washing, the precipitated material was analyzed for the presence of syndapin I, dynamin 1, and N-WASP via immunoblot analyses. Cross-linking Experiments—For cross-link studies of endogenous proteins, rat brain extracts were prepared as described (26Qualmann B. Boeckers T.M. Jeromin M. Gundelfinger E.D. Kessels M.M. J. Neurosci. 2004; 24: 2481-2495Crossref PubMed Scopus (111) Google Scholar). Brain extracts at a total concentration of 0.7 mg/ml and a final concentration of 100 mm NaCl were incubated in the presence of increasing amounts of the heterobifunctional cross-linker 1-ethyl-3-[dimethylaminopropyl]carbodiimide (EDC) (Pierce) in 20 mm Hepes, pH 7.4, 0.2 mm MgCl2,and 2 mm EGTA for 20 min at 30 °C. The cross-linking reaction was stopped by the addition of 4× SDS-PAGE sample buffer, and the samples were analyzed by immunoblotting. Extracts from HEK293 cells transfected with different constructs encoding for different tagged syndapin proteins or fragments thereof were prepared as described above. The extracts were incubated with increasing amounts of EDC as indicated for 20 min at 30 °C and analyzed by SDS-PAGE on 8% or 5-8% polyacrylamide gels and by subsequent immunoblotting. In Silico Analyses and Predictions of Protein Structure—Software used for predictions of protein secondary structure (especially of α-helices and loop regions) is available on the World Wide Web at cmpharm.ucsf.edu/cgi-bin/nnpredict and www.predictprotein.org. Coiled-coil predictions were performed by using both the PAIRCOIL program (available on the World Wide Web at paircoil.lcs.mit.edu/cgi-bin/paircoil) and the program COILS with window settings of 14, 21, and 28 amino acids (available on the World Wide Web at www.ch.embnet.org/cgi-bin/COILS). Cell Culture and Immunofluorescence Microscopy—HEK293, HeLa, and COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For mitochondrial targeting experiments, COS-7 cells were seeded onto poly-d-lysine-coated coverslips 1 day prior to transfection. Transfections were performed with Polyfect reagent according to the manufacturer's instructions (Qiagen). Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, containing 0.9 mm CaCl2 and 0.5 mm MgCl2 for 15 min at room temperature and processed for immunofluorescence according to Ref. 27Kessels M.M. Engqvist-Goldstein Å.E.Y. Drubin D.G. Qualmann B. J. Cell Biol. 2001; 153: 351-366Crossref PubMed Scopus (194) Google Scholar. For mitochondrial staining, cells were incubated with MitoTracker® as described previously (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (165) Google Scholar). Images were recorded digitally using a Leica DMRD fluorescence microscope and a Zeiss Axioplan 2 microscope and processed using Adobe Photoshop software. Examinations of the actin cytoskeletal phenotype induced by overexpression of syndapin full-length proteins and fragments thereof were performed in HeLa cells and have been described previously (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). F-actin was stained with Alexa568-phalloidin (Molecular Probes). The presence of several or extended lamellipodial areas decorated with filopodial structures and marked by accumulated F-actin signal at the leading edge was used for scoring. The effect of each construct was determined in several independent assays, and 134-528 transfected cells were scored in systematic searches across the coverslips. The assays were conducted as blind studies to ensure that the researcher was unbiased. Induction of the cortical actin phenotype upon N-WASP overexpression and epidermal growth factor stimulation was performed essentially as established by Miki et al. (28Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar). For quantitative analysis of the phenotype and of changes induced upon cotransfection with syndapin constructs, 316-387 transfected cells from three independent assays were scored in systematic searches across the coverslips and grouped into two categories, smooth and rough/filopodia-decorated appearance of plasma membrane edges, respectively. Transferrin Uptake Assays—COS-7 cells were subjected to transferrin uptake assays 48 h after transfection as described previously (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar, 27Kessels M.M. Engqvist-Goldstein Å.E.Y. Drubin D.G. Qualmann B. J. Cell Biol. 2001; 153: 351-366Crossref PubMed Scopus (194) Google Scholar). The percentages of transfected cells showing no detectable uptake of transferrin, significantly reduced transferrin signals, and normal levels of internalized transferrin and S.D. values were calculated by scoring and counting cells in at least three independent experiments each. Syndapins have been suggested to functionally interconnect endocytosis and the actin cytoskeleton by interactions of their C-terminal SH3 domain with dynamin and N-WASP (24Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1061Crossref PubMed Scopus (253) Google Scholar). However, syndapins have only one SH3 domain for an interaction with either dynamin or N-WASP. To function as molecular links, syndapins would thus have to build multivalent platforms in a non-SH3 domain-dependent manner. Analyses of fractions of rat brain cytosol obtained by size exclusion chromatography over a Superose 6 column revealed that the size of syndapin I-containing complexes by far exceeds the calculated size of monomeric syndapin. Syndapin I has a predicted molecular mass of 50.4 kDa, but syndapin-containing complexes were detected in a range between apparent molecular masses of 300 kDa and about 500 kDa, corresponding to fractions 15-17 (Fig. 1A). Complexes with a molecular mass around 400 kDa were especially abundant. The existence of syndapin complexes with even higher molecular mass also remains possible because the analysis was performed with cytosolic extracts cleared from extremely large protein complexes by high speed centrifugation. Immunoblotting analysis of rat brain cytosol fractionated by gel filtration showed that the N-WASP and dynamin distribution both peak in the syndapin I-containing fractions (Fig. 1A). The apparent molecular weights of syndapin, N-WASP, and dynamin all exceed those of the monomeric proteins and even those of combinations of one syndapin molecule with one interaction partner. Blot overlay analysis with a GST fusion protein of syndapin I as a probe revealed that literally all direct syndapin binding partners run at molecular weights that are much larger than those of the monomeric proteins and thus correspond to protein complexes that partially cofractionate with syndapin I (data not shown). Coimmunoprecipitations from the gel filtration fractions directly demonstrated that syndapin I is complexed with N-WASP and dynamin in high molecular weight fractions. Both N-WASP and dynamin were specifically coimmunoprecipitated with syndapin I from fraction 16/17 (data not shown) as well as from fraction 15 (Fig. 1B). Based on secondary structure predictions, we have hypothesized earlier that syndapins might have the capability to interact with themselves and form oligomers (23Qualmann B. Roos J. DiGregorio P.J. Kelly R.B. Mol. Biol. Cell. 1999; 10: 501-513Crossref PubMed Scopus (250) Google Scholar). Since our gel filtration studies (Fig. 1) support such a scenario, we experimentally addressed the existence of syndapin-syndapin interactions by conducting coimmunoprecipitation experiments with differentially tagged syndapin I proteins overexpressed in HEK293 cells (Fig. 2, A-D). Both FLAG-tagged syndapin I and a GFP-tagged syndapin I fusion protein can be expressed and coexpressed in HEK cells (Fig. 2, A and B) and were successfully immunoprecipitated by anti-FLAG and anti-GFP antibodies, respectively (Fig. 2C). Analysis of the immunoprecipitates from cells double-transfected with FLAG-syndapin I and GFP-syndapin I demonstrated that FLAG-syndapin I was specifically coimmunoprecipitated with GFP-syndapin I (Fig. 2D). GFP-syndapin did not associate with anti-FLAG antibodies when transfected alone or with nonimmune mouse IgG. Consistently, FLAG-syndapin I was specifically coimmunoprecipitated with GFP-syndapin I but not with GFP alone (Fig. 2D). Control experiments demonstrate that FLAG-syndapin was not precipitated by anti-GFP antibodies upon single transfection of the construct or by unrelated mouse IgG (Fig. 2D). Our coimmunoprecipitation studies thus demonstrate that syndapin I can efficiently form oligomers in vivo. We next addressed the question of whether the syndapin oligomerization is independent of the SH3 domain, as hypothesized. For this purpose we first coexpressed different GFP-syndapin I constructs in combination with FLAG-syndapin I (Fig. 2, E and F) and conducted immunoprecipitations of the GFP fusion proteins (Fig. 2G). Similar to full-length syndapin I, both GFP-syndapin I with a mutated SH3 domain and GFP-syndapinΔSH3 effectively and specifically coimmunoprecipitated FLAG-syndapin I (Fig. 2H). In contrast, FLAG-syndapin I was not coimmunoprecipitated together with the GFP-syndapin I SH3 domain (Fig. 2H). These data clearly show that the non-SH3 part of syndapin I is sufficient for oligomerization. This leaves the SH3 domain free for further protein-protein interactions forming larger macromolecular complexes, as seen in Fig. 1. In order to firmly exclude putative posthomogenization artifacts in our coimmunoprecipitation analyses, we next reconstituted and visualized the syndapin-syndapin interactions that we observed in cellular extracts (Fig. 2, A-H) in intact cells (Fig. 2, I-O). We have developed a mitochondrial targeting system that allows for incorporating syndapin fusion proteins into the outer mitochondrial membranes in a manner that ensures that the syndapin part is facing the cytoplasm (20Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Sco
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