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Vol. 20, Issue 8, 2254-2264, April 15, 2009
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*Smurfit Institute of Genetics and Trinity College Institute of Neuroscience, University of Dublin Trinity College, Dublin-2, Ireland Institut für Neurobiologie, Universität Münster, D-48149 Münster, Germany Department of Biological Science, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India National Centre for Biological Sciences, Bangalore 560085, India ||Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721
Submitted October 28, 2008;
Revised January 30, 2009;
Accepted February 18, 2009
Monitoring Editor: Marcos Gonzalez-Gaitan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Farsad and De Camilli, 2003; Zimmerberg and Kozlov, 2006). Recent findings that different F-BAR domain proteins form and stabilize membrane structures of different diameters, have contributed substantially to the molecular understanding of how this sequence of events occurs (Habermann, 2004b; Itoh et al., 2005; McMahon and Gallop, 2005; Tsujita et al., 2006; Shimada et al., 2007).
BAR and F-BAR/EFC proteins show in vitro liposome-tubulating activity similar to those described previously for dynamin (Stowell et al., 1999). However, the crescent-shaped F-BAR/EFC domains usually form wider lipid tubules, whose diameters are broadly consistent with intrinsic domain curvatures determined by x-ray crystallography (McMahon and Gallop, 2005; Shimada et al., 2007). Thus, F-BAR proteins FBP17 and CIP4, with an intrinsic curvature of 60 nm, form tubules whose diameter is larger than those formed by BAR domains of amphiphysin, which has a tighter intrinsic curvature of 22 nm. Therefore, F-BAR proteins may function early in the process of endocytosis to stabilize wide membrane invaginations; BAR domain proteins may help form narrower membrane necks where dynamin and membrane fission proteins function (Wigge and McMahon, 1998; Simpson et al., 1999; Habermann, 2004a; Peter et al., 2004; Itoh et al., 2005; Kessels and Qualmann, 2006).
Studies in cultured cells are consistent with the above-mentioned model. Overexpression of any of several F-BAR/EFC domain proteins including syndapin/Pacsin enhances formation of tubular intermediates of membrane endocytosis, particularly when their Src homology (SH) 3 domain interactions with dynamin or actin are inhibited (Itoh et al., 2005; Tsujita et al., 2006). These F-BAR protein-induced tubules in cultured cells 1) contain dynamin and known components of endocytosis; 2) are transient; and 3) are greatly elongated under conditions of dynamin or F-actin inhibition, suggesting a dynamic equilibrium between tubulation and membrane fission (Itoh et al., 2005; Dawson et al., 2006; Tsujita et al., 2006). Despite the elegance of existing structural, and cell biological analyses, the biological contexts in which F-BAR domain proteins function in vivo remain largely unknown. Here, we use genetic and cell biological approaches in Drosophila to analyze potential in vivo functions of syndapin, one of the best-known F-BAR proteins, conserved from insects to mammals (Kessels and Qualmann, 2004).
Syndapins/Pacsins, have C-terminal SH3 domains capable of binding to proline-rich domains of dynamin, and two actin-regulatory proteins, WASp and synaptojanin (Kessels and Qualmann, 2002). To directly address the biological function of syndapin, we used the Drosophila larval neuromuscular junctions, in which one can easily study the biogenesis of a tubulolamellar postsynaptic membrane system termed the subsynaptic reticulum (SSR) (Budnik et al., 1996; Guan et al., 1996; Albin and Davis, 2004). Our results indicate that syndapin promotes formation of an endogenous tubulolamellar membrane system through a mechanism that requires both its F-BAR and SH3 domains and provides new insight into potential mechanisms and functions of F-BAR proteins in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Dlg protein in dlgm52 mutants was severely reduced but is not a null allele (Supplemental Figure S1) (Mendoza et al., 2003). The isolation and characterization of synd mutant alleles has been described previously (Kumar et al., 2008).
Antibodies and Immunochemistry
Wandering third instars were pinned dorsally on a Sylgard dish and dissected in cold calcium-free HL3 saline (70 mM NaCl, 5 mM KCl, 20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, and 5 mM HEPES, pH 7.3) to expose the neuromuscular junctions. Dissected larvae were then fixed in 3.5% paraformaldehyde in phosphate-buffered saline (PBS) containing 0.5 mM EGTA for 30 min, or in Bouin's fixative (for glutamate receptor [GluR] IIA staining) for 5 min. Larvae were then washed in PBS containing 0.15% Triton X-100, blocked for 1 h in 5% normal goat serum, and incubated overnight at 4°C with the primary antibody. Polyclonal anti-syndapin antibodies were raised in rat or rabbit against the N-terminal (Synd
SH3, amino acids [aa] 1–377) of the protein. Affinity-purified anti-syndapin antibody was used at 1:50 dilution for immunostaining. The monoclonal antibodies anti-Dlg, anti-DGluRIIA, anti-CSP, and anti-spectrin were obtained from the Developmental Studies Hybridoma Bank (Department of Biological Sciences, University of Iowa, Iowa University, IA) and were used at 1:50 dilution. Anti-Wsp was a gift from Eyal Schejter (Weizmann Institute of Science, Rehovot, Israel) and was used at 1:200 dilution. Polyclonal anti-dPAK was a gift from Chihiro Hama (National Institute of Neuroscience, Tokyo, Japan) and was used at 1:500 dilution. Polyclonal rabbit anti-Dlg was a gift from Vivian Budnik and was used at 1:1000 dilution. Polyclonal anti-Syt was a gift from Hugo Bellen (Baylor College of Medicine, Houston, TX) and was used at 1:1000, and anti-dynamin was used at 1:200. Secondary antibodies coupled to Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen, Carlsbad, CA) was used at 1:400 dilution. Stained larval preparations were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA) and imaged with a laser scanning confocal microscope (LSM510 Meta; Carl Zeiss, Jena, Germany).
Western Blotting
Fly heads were homogenized in 1x SDS sample buffer (50 mM Tris-Cl, pH 6.8, 2% SDS, 2% β-mercaptoethanol, 0.1% bromphenol blue, and 10% glycerol), boiled for 5 min, and 2 fly head equivalents of protein was fractionated on a 13.5% SDS-polyacrylamide gel electrophoresis. The protein was transferred onto polyvinylidene difluoride membrane and blocked for 1 h in 5% fat-free milk. The anti-syndapin antibodies were used at 1:10,000 dilutions. The horseradish peroxidase (HRP)-coupled secondary antibody (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom) was used at 1:50000 dilutions. Signals were detected using enhanced chemiluminescence system.
Cell Culture and Transfection
Drosophila S2R+ cells were propagated in 1x Schneider's Drosophila media (Invitrogen) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin in 75-cm2 T-flasks (Sarstedt, Rommelsdorfer Starbe, Germany) at 25°C. Drosophila Schneider S2R+ cells (3 x 105) were transiently cotransfected with pUAST constructs (1.1 µg) and Act5C-GAL4 DNA (0.6 µg) by using FuGENE reagent (Roche Diagnostics, Indianapolis, IN) as described previously (Bogdan and Klambt, 2003). For confocal spinning-disk imaging microscopy, cells were replated on chambered coverglass (Nalge Nunc International, Rochester, NY) pretreated with concanavalin A.
Generation of Transgenic Flies
The Synd Open Reading Frame was amplified using cDNA (EST clone, LD46328) as template. The amplicon was cloned at EcoRI and NotI site in pUAST. The enhanced yellow fluorescent protein (EYFP)-syndapin constructs were generated by polymerase chain reaction (PCR) amplification of various syndapin domains (synd full-length, 1-494 aa; synd FCH, 1-150 aa; synd F-BAR, 1-300 aa; and synd SH3, 406-494 aa) and cloned into Drosophila Gateway vector (developed by Murphy laboratory, Carnegie Institution of Washington, Baltimore, MD). All constructs were confirmed by sequencing for the absence of any point mutations. For generating constructs with substituted amino acids, site-directed mutagenesis was performed (Mutagenex, Piscataway, NJ) on the wild-type syndapin construct and cloned into pUAST vector. The embryonic transformation of Drosophila was performed by Genetic Services (Cambridge, MA). Several transgenes harboring the construct were obtained and all of them expressed Synd protein at high levels.
Electron Microscopy
Third instar larval body muscles were dissected in cold Ca2+-free HL3 medium. The samples were fixed in Ca2+-free Trump's fixative (pH 7.2, 4% paraformaldehyde, 1% glutaraldehyde, 100 mM cacodylate, 2 mM sucrose, and 0.5 mM EGTA) in the dissection chamber for 
30 min at room temperature. The segments A2 and A3 were dissected out and further fixed overnight at 4°C. The samples were rinsed in 0.1 M cacodylate buffer with 264 mM sucrose, postfixed in 2% osmium tetroxide, and stained en bloc during ethanol dehydration with 2% uranyl acetate. Muscles embedded in Araldite were sectioned at 60 nm. Sections stained with 2% uranyl acetate and 1% lead citrate were examined with a 100CX transmission electron microscope (JEOL, Tokyo, Japan.
Quantitation and Morphometric Analyses
Fluorescence imaging was carried out using a laser scanning confocal microscope (LSM510 Meta; Carl Zeiss). All the control and experimental samples were processed in the same way, and the same setting was used for acquiring the images. Type I boutons were imaged at 63x, and the average florescence intensity of syndapin around boutons was calculated using MetaMorph software (GE Healthcare). For calculation of SSR width and bouton area, NMJs labeled with anti-Synd and anti-HRP were imaged at 63x with zoom 4 on the confocal microscope, and the maximal intensity projection image was obtained. Although anti-HRP labels some extra bouton epitopes, the bouton boundary could be unambiguously identified under immunofluorescence microscope. The SSR width was calculated as a difference between widths of total synapse (bouton + SSR) across the bouton and the width of the bouton. Only type I boutons at muscle 6/7 from four or more animals were used for quantitation. For quantification of SSR complexity, electron micrographs were printed at 25,000x and the number of membrane segments crossing a line of 0.5 µm was manually counted. For each bouton, four to six measurements were taken, averaged, and expressed as membrane layers/micrometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Consistent with these observations, Drosophila syndapin is predominantly postsynaptic at the Drosophila third instar larval neuromuscular junction. At the postsynapse, it is tightly localized to the tubulolamellar SSR (Figure 1, A–C). Synd strongly colocalizes with the actin-regulatory protein Wsp (Figure 1, D–F) and is encircled by spectrin immunoreactivity (Figure 1, G–I), consistent with the prior observation that spectrin surrounds the SSR (Pielage et al., 2006). Unlike Amphiphysin, syndapin is not visible in muscle T-tubules (Razzaq et al., 2001; Lee et al., 2002) (Supplemental Figure S1).
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) (Supplemental Figure S2). Quantitative analysis indicated that the width of Synd-immunoreactive regions, which correlates with SSR expansion in the vicinity of boutons is increased about twofold after muscle expression, whereas bouton size remained unchanged (Figure 2, D–K; Table 1). In addition, UAS-synd; mef2-Gal4 larvae reared at 29°C, a temperature that allows more robust Gal4-mediated expression, not only expanded syndapin-immunopositive regions at postsynaptic sites but also spread to flanking "extrasynaptic" zones that were bereft of presynaptic innervation in "flares" of unique appearance (Figure 2, D–I).
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Synaptic and Extrasynaptic Membrane Expansions Contain Native Markers of SSR
We further asked whether key components of the native SSR were present in the expanded synaptic and extrasynaptic SSR observed after syndapin overexpression. Three of these proteins are Dlg, dPAK, and WASp.
Dlg was present in both synaptic and extrasynaptic SSR, although at somewhat lower density in extrasynaptic regions (Figure 4, A–C). dPAK was also strongly recruited to the expanded SSR, being present not only in postsynaptic puncta but also in extrasynaptic regions bereft of presynaptic terminals (Figure 4, D–F). In addition, Wsp immunoreactivity was greatly increased around the boutons and strong immunoreactivity was observed in the extrasynaptic regions (Figure 4, G–I). Interestingly, postsynaptic glutamate receptor levels and distribution was not altered after SSR expansion. In the wild-type NMJ, GluR is present in a postsynaptic domain that excludes Synd (Supplemental Figure S2). Synd overexpression does not alter the GluR containing domain, but specifically expands GluR free SSR (Figure 4, J–L).
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Syndapin-mediated SSR Expansion Occurs in dlg and dpak Mutants
The observation that Synd expands Dlg- and dPAK-containing membranes suggested that Synd could recruit the signaling proteins dPAK and Dlg, which in turn induce SSR formation via a yet unknown pathway (Lahey et al., 1994; Parnas et al., 2001; Albin and Davis, 2004). An alternative model is that Synd, when appropriately targeted to postsynaptic sites, acts directly on the membrane to induce tubulation and SSR formation. This second model is more consistent with the in vitro activity of F-BAR proteins (Itoh et al., 2005; Tsujita et al., 2006).
Genetic epistasis experiments to discriminate between these two models of Synd action showed that Synd could induce postsynaptic membrane expansions even in dlgm52 and dpak11 mutants that show highly reduced SSR (Figure 5). Thus, these data argue for a mechanism in which Synd acts parallel to or downstream of Dlg and dPAK, perhaps directly on membrane as suggested by recent studies of F-BAR family proteins. We tested a prediction of this alternative model.
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To test whether the cellular properties of Synd is conserved to other F-BAR domain proteins, we expressed EYFP-tagged domains of syndapin in S2-cells and analyzed their ability to form tubules. As shown in Figure 6, whereas full-length syndapin produced only weak tubulation, consistent with Itoh et al., 2005, the F-BAR domain (1-300 aa) induced massive tubule formation in cultured cells. In contrast, the FCH domain (1–150 aa) or the SH3 domain (406-494 aa) of syndapin had no visible effect on membrane tubulation.
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; Tsujita et al., 2006). We then asked how these syndapin variants affected Synd targeting and SSR formation in vivo. We generated transgenic flies expressing deletion constructs or selected mutant forms of Synd in postsynaptic muscle. Full-length EYFP-tagged Synd, which had weak tubulation effect in S2 cells, behaved identically to wild-type Synd in its localization and robust effect on SSR expansion (Figure 7, A–C and G–I). The Synd FCH (1–150 aa) and Synd SH3 domains (406–494 aa) were not targeted to the NMJ and had no effect on postsynaptic membrane morphology (data not shown). Interestingly, the F-BAR domain (1-300 aa), although not efficiently targeted to the postsynapse, induced patches of dense membrane randomly distributed over the muscle surface (Figure 7, D–F and J–L). These data suggest that 1) membrane modeling activity of syndapin is contained in the F-BAR domain; 2) Synd targeting to the postsynapse requires both its F-BAR and the SH3 domains; and 3) in contrast to its effect on membrane tubulation in cultured cells, full-length Synd, when appropriately targeted, is capable of substantial membrane remodeling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tsujita et al., 2006; Shimada et al., 2007; Frost et al., 2008; Takano et al., 2008). However, our knowledge of these proteins in vivo, in the multicellular context, remains very limited. Here, we provide an in vivo analyses of syndapin in the context of its role in the biogenesis of subsynaptic reticulum, a unusual complex membrane system. We make three key observations on Drosophila syndapin: 1) we show that syndapin can promote formation of a tubulolamellar membrane system in vivo, 2) syndapin causes membrane remodeling that can occur without accompanying membrane fission, and 3) syndapin promotes SSR expansion by using evolutionarily conserved amino acids in its F-BAR domain. We consider these three points in turn below.
Syndapin Promotes SSR Expansion
The SSR is a unique system of tubules and lamellae formed by extensive infoldings of the postsynaptic muscle membrane; thus, in organization, they are quite different from the relatively simple F-BAR–induced membrane tubules described in cultured cells. The SSR surrounds large boutons at the Drosophila NMJ. Although, a role for signaling and scaffolding proteins such as dPAK and Dlg has been demonstrated in the formation of SSR, mechanisms that underlie biogenesis of this complex membrane system are still poorly understood.
Syndapin overexpression in muscle caused induction of synaptic and extrasynaptic membrane-dense subsynaptic reticulum, based on optical and electron microscopic analyses. In particular, EM sections of membrane structures induced by syndapin overexpression showed not only circular/elliptical profiles expected for tubules, but also longer parallel membrane profiles, suggesting sections through lamellae as indeed is seen for native SSR. To our knowledge, this is the first demonstration that an F-BAR protein can promote the formation of lamellar membrane infoldings. The mechanism by which syndapin may promote lamella formation is unclear, but it is likely that this arises from context specific interactions with other protein components of the SSR (Shibata et al., 2006; Gorczyca et al., 2007).
Syndapin seems to induce SSR through mechanisms that are either downstream of, or independent of, dPAK and Dlg function. This is indicated by three observations. First, the syndapin immunoreactivity is significantly reduced in dPAK and Dlg mutants (Supplemental Figure S1). Second, unlike Dlg that can induce SSR when expressed either pre- or postsynaptically, syndapin acts in a cell-autonomous manner in postsynaptic muscle. This could indicate either a function downstream of these signaling molecules or an entirely independent mechanism. The third observation is that although Synd-induced membrane is strikingly similar to the endogenous SSR in general appearance; it has some notable differences from the endogenous SSR in structure and composition. Synd-induced SSR has more densely packed membranes (30% more membrane layers per micrometer) and also contains lower amounts of Dlg and dPAK than the endogenous SSR. Both of these differences could conceivably arise from limiting amounts of Dlg, dPAK or some other factor(s) required for the precise organization of the SSR; however, our current data do not address this issue unequivocally.
Does SSR biogenesis induced by syndapin reflect its true physiological function rather than an interesting but physiologically irrelevant activity of the protein? The SSR remains normal in synd loss-of-function mutants. Although this could suggest that Synd has no physiological function in SSR biogenesis, an alternative possibility is that other postsynaptic F-BAR proteins compensate for the absence of syndapin. Indeed, potential functional redundancies among F-BAR proteins are suggested by reports that different F-BAR proteins can coexist on a single tubule (Frost et al., 2008).
Although alternative models are tenable, we suggest that Synd has a role in SSR biogenesis in vivo based on four arguments. First, Synd is localized to the postsynaptic SSR and would therefore most simply be expected to have an SSR-related function. The observed expansion of the SSR is consistent with this premise. Second, full-length Synd overexpression does not cause random patches of SSR to be induced all over the muscle surface but rather causes local expansions as well as flares that often seem to emanate from the existing SSR. Thus, the observed consequence of Synd overexpression seems to originate from sites to which Synd is normally targeted in vivo. Third, that Synd can promote formation of a unique, highly complex membrane system in vivo indicates that it participates in intricate processes that likely require the coordinated function of many different proteins. Finally, consistent with the previous argument, muscle expression of other Drosophila F-BAR domain proteins such as Nervous wreck (Coyle et al., 2004) (Supplemental Figure S4) or Cip4 (data not shown) does not induce SSR expansion.
Syndapin Decouples Membrane Tubulation and Fission for SSR Expansion
In cultured cells, overexpression of F-BAR proteins induces transient, dynamin-containing plasma membrane tubules that are rapidly fragmented by dynamin-mediated membrane scission (Itoh et al., 2005; Tsujita et al., 2006). Here, tubulation can be decoupled from membrane fission only if either the SH3 domain is removed or if SH3 interacting molecules (e.g., dynamin) are inhibited. A physiological decoupling of membrane tubulation and fission activities has been shown previously for the N-BAR domain proteins, mouse Amphiphysin 2 and Drosophila Amphiphysin, during T-tubule formation (Razzaq et al., 2001; Lee et al., 2002). Our observations suggest that similar physiological decoupling of the two activities—membrane deformation and membrane fission also occurs for the syndapin, an F-BAR domain protein.
In support of this, we show that unlike synd, the membrane fission protein dynamin is not enriched in the SSR (Supplemental Figure S2); this is different from strong colocalization observed between dynamin and syndapin in transient tubules in cultured cells. Furthermore, the presence of the dynamin-interacting SH3 domain does not inhibit syndapin's ability to promote SSR formation. Thus, in contrast to prior observations in cultured cells, our data show that syndapin in vivo can 1) be present without accompanying dynamin and 2) can form stable membrane infoldings without need to experimentally inhibit SH3 domain interactions.
Syndapin Uses Conserved Basic Amino Acid Residues to Promote SSR Expansion
The mechanism by which syndapin promotes SSR formation is likely to require direct membrane interactions mediated by previously identified residues on the concave face of its F-BAR domain. Mutations in key residues on the concave face of the F-BAR domain, required for phospholipid binding, block the ability of syndapin to induce SSR. Thus, mechanisms that underlie F-BAR protein's ability to tubulate membrane in vitro seem to be required for syndapin's ability to expand the SSR. However, Synd-induced SSR formation requires additional events, including correct targeting to the postsynapse, a function that requires the C-terminal SH3 domain.
Although Synd lacking its SH3 domain is extremely efficient at membrane tubulation/remodeling in S2 cells, this truncated protein is not postsynaptically targeted in muscle cells and is ineffective for SSR expansion. Thus, the SH3 domain of Synd must interact with targeting molecules that control syndapin's postsynaptic localization. By extension, the targeting of other F-BAR domain proteins, which may be mediated by analogous SH3 domain interactions, could be important for their respective in vivo functions. A simple model explaining and summarizing our observation on syndapin is presented as Figure 9.
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). It is conceivable that other F-BAR domain proteins will be found to be involved in the biogenesis of these or other complex membrane system. Further studies are required to understand the different processes involved in SSR biogenesis and also to test the relevance of these findings to other members of the F-BAR protein family.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Vimlesh Kumar (kumarv{at}tcd.ie) or Mani Ramaswami (mani{at}u.arizona.edu)
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Atwood, H. L., Govind, C. K., and Wu, C. F. (1993). Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J. Neurobiol 24, 1008–1024.[CrossRef][Medline]
Bogdan, S., and Klambt, C. (2003). Kette regulates actin dynamics and genetically interacts with Wave and Wasp. Development 130, 4427–4437.
Budnik, V., Koh, Y. H., Guan, B., Hartmann, B., Hough, C., Woods, D., and Gorczyca, M. (1996). Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg. Neuron 17, 627–640.[CrossRef][Medline]
Coyle, I. P., Koh, Y. H., Lee, W. C., Slind, J., Fergestad, T., Littleton, J. T., and Ganetzky, B. (2004). Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila. Neuron 41, 521–534.[CrossRef][Medline]
Dawson, J. C., Legg, J. A., and Machesky, L. M. (2006). Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis. Trends Cell Biol 16, 493–498.[CrossRef][Medline]
Farsad, K., and De Camilli, P. (2003). Mechanisms of membrane deformation. Curr. Opin. Cell Biol 15, 372–381.[CrossRef][Medline]
Frost, A., Perera, R., Roux, A., Spasov, K., Destaing, O., Egelman, E. H., De Camilli, P., and Unger, V. M. (2008). Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817.[CrossRef][Medline]
Gorczyca, D., Ashley, J., Speese, S., Gherbesi, N., Thomas, U., Gundelfinger, E., Gramates, L. S., and Budnik, V. (2007). Postsynaptic membrane addition depends on the Discs-Large-interacting t-SNARE Gtaxin. J. Neurosci 27, 1033–1044.
Guan, B., Hartmann, B., Kho, Y. H., Gorczyca, M., and Budnik, V. (1996). The Drosophila tumor suppressor gene, dlg, is involved in structural plasticity at a glutamatergic synapse. Curr. Biol 6, 695–706.[CrossRef][Medline]
Habermann, B. (2004a). The BAR-domain family of proteins: a case of bending and binding? EMBO Rep 5, 250–255.[CrossRef][Medline]
Habermann, B. (2004b). The BAR-domain family of proteins: a case of bending and binding? EMBO Rep 5, 250–255.[CrossRef][Medline]
Itoh, T., Erdmann, K. S., Roux, A., Habermann, B., Werner, H., and De Camilli, P. (2005). Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804.[CrossRef][Medline]
Kessels, M. M., and Qualmann, B. (2002). Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J 21, 6083–6094.[CrossRef][Medline]
Kessels, M. M., and Qualmann, B. (2004). The syndapin protein family: linking membrane trafficking with the cytoskeleton. J. Cell Sci 117, 3077–3086.
Kessels, M. M., and Qualmann, B. (2006). Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization. J. Biol. Chem 281, 13285–13299.
Kumar, V., Alla, S. R., Krishnan, K. S., and Ramaswami, M. (2008). Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction. Mol. Cell Neurosci.
Lahey, T., Gorczyca, M., Jia, X. X., and Budnik, V. (1994). The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure. Neuron 13, 823–835.[CrossRef][Medline]
Lee, E., Marcucci, M., Daniell, L., Pypaert, M., Weisz, O. A., Ochoa, G. C., Farsad, K., Wenk, M. R., and De Camilli, P. (2002). Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle. Science 297, 1193–1196.
McMahon, H. T., and Gallop, J. L. (2005). Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596.[CrossRef][Medline]
Mendoza, C., Olguin, P., Lafferte, G., Thomas, U., Ebitsch, S., Gundelfinger, E. D., Kukuljan, M., and Sierralta, J. (2003). Novel isoforms of Dlg are fundamental for neuronal development in Drosophila. J. Neurosci 23, 2093–2101.
Parnas, D., Haghighi, A. P., Fetter, R. D., Kim, S. W., and Goodman, C. S. (2001). Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix. Neuron 32, 415–424.[CrossRef][Medline]
Perez-Otano, I., Lujan, R., Tavalin, S. J., Plomann, M., Modregger, J., Liu, X. B., Jones, E. G., Heinemann, S. F., Lo, D. C., and Ehlers, M. D. (2006). Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1. Nat. Neurosci 9, 611–621.[CrossRef][Medline]
Peter, B. J., Kent, H. M., Mills, I. G., Vallis, Y., Butler, P. J., Evans, P. R., and McMahon, H. T. (2004). BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499.
Pielage, J., Fetter, R. D., and Davis, G. W. (2006). A postsynaptic spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction. J. Cell Biol 175, 491–503.
Razzaq, A., Robinson, I. M., McMahon, H. T., Skepper, J. N., Su, Y., Zelhof, A. C., Jackson, A. P., Gay, N. J., and O'Kane, C. J. (2001). Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. Genes Dev 15, 2967–2979.
Shibata, Y., Voeltz, G. K., and Rapoport, T. A. (2006). Rough sheets and smooth tubules. Cell 126, 435–439.[CrossRef][Medline]
Shimada, A. et al. (2007). Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129, 761–772.[CrossRef][Medline]
Simpson, F., Hussain, N. K., Qualmann, B., Kelly, R. B., Kay, B. K., McPherson, P. S., and Schmid, S. L. (1999). SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation. Nat. Cell Biol 1, 119–124.[CrossRef][Medline]
Stowell, M. H., Marks, B., Wigge, P., and McMahon, H. T. (1999). Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring. Nat. Cell Biol 1, 27–32.[CrossRef][Medline]
Takano, K., Toyooka, K., and Suetsugu, S. (2008). EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization. EMBO J 27, 2817–2828.[CrossRef][Medline]
Tolhurst, G., Vial, C., Leon, C., Gachet, C., Evans, R. J., and Mahaut-Smith, M. P. (2005). Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood 106, 1644–1651.
Tsujita, K., Suetsugu, S., Sasaki, N., Furutani, M., Oikawa, T., and Takenawa, T. (2006). Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol 172, 269–279.
Wigge, P., and McMahon, H. T. (1998). The amphiphysin family of proteins and their role in endocytosis at the synapse. Trends Neurosci 21, 339–344.[CrossRef][Medline]
Zimmerberg, J., and Kozlov, M. M. (2006). How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell. Biol 7, 9–19.[CrossRef][Medline]
Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N., and Benzer, S. (1994). Paralysis and early death in cysteine string protein mutants of Drosophila. Science 263, 977–980.
Zinsmaier, K. E., Hofbauer, A., Heimbeck, G., Pflugfelder, G. O., Buchner, S., and Buchner, E. (1990). A cysteine-string protein is expressed in retina and brain of Drosophila. J. Neurogenet 7, 15–29.[Medline]
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spectrin (green) antibodies. (I) Merged image of G and H showing that the spectrin immunoreactivity surrounds the syndapin reactivity. Bar, 5 µm (in C) for A–I.
