Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation.

The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin-2 (Amph2) SH3 domain to 2.2 A. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.     

SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity

Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.     

Importance of the pleckstrin homology domain of dynamin in clathrin-mediated endocytosis

The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamin's function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamin's interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.     

Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.     

An InCytes from MBC Selection: Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.     

Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.     

The role of dynamin and its binding partners in coated pit invagination and scission

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.     

Expression and purification of dynamin II domains and initial studies on structure and function

Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His)(6) peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.     

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.     

Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p 相似文献   

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.     

Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481-486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.     

Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains

Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission.     

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.     

Neural Wiskott-Aldrich syndrome protein is recruited to rafts and associates with endophilin A in response to epidermal growth factor

Neural Wiskott-Aldrich syndrome protein (N-WASP) has been implicated in endocytosis; however, little is known about how it interacts functionally with the endocytic machinery. Sucrose gradient fractionation experiments and immunofluorescence studies with anti-N-WASP antibody revealed that N-WASP is recruited together with clathrin and dynamin, which play essential roles in clathrin-mediated endocytosis, to lipid rafts in an epidermal growth factor (EGF)-dependent manner. Endophilin A (EA) binds to dynamin and plays an essential role in the fission step of clathrin-mediated endocytosis. In the present study, we show that the Src homology 3 (SH3) domain of EA associates with the proline-rich domain of N-WASP and dynamin in vitro. Co-immunoprecipitation assays with anti-N-WASP antibody revealed that EGF induces association of N-WASP with EA. In addition, EA enhances N-WASP-induced actin-related protein 2/3 (Arp2/3) complex activation in vitro. Immunofluorescence studies revealed that actin accumulates at sites where N-WASP and EA are co-localized after EGF stimulation. Furthermore, studies of overexpression of the SH3 domain of EA indicate that EA may regulate EGF-induced recruitment of N-WASP to lipid rafts. These results suggest that, upon EGF stimulation, N-WASP interacts with EA through its proline-rich domain to induce the fission step of clathrin-mediated endocytosis.     

The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting.

Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 domains that interact with dynamin and synaptojanin. The amphiphysins may function in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions with clathrin and AP2. We have recently identified and cloned several amphiphysin II splice variants that differentially incorporate clathrin-binding domains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphysin II variants to clathrin-coated pits on plasma membranes purified from transfected COS-7 cells. Full-length amphiphysin II targets to the plasma membrane where it partially co-localizes with clathrin. However, splice variants and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not affect amphiphysin II distribution. Surprisingly, plasma membrane targeting was dependent on the presence of a 31-amino acid alternatively spliced sequence at the N terminus of amphiphysin II, a result confirmed using subcellular fractionation. In binding assays, the 31-amino acid sequence was also found to facilitate amphiphysin dimerization mediated through the N terminus. Taken together, these data support a role for the N terminus of amphiphysin II in membrane targeting during endocytosis.     

The calcineurin-binding protein cain is a negative regulator of synaptic vesicle endocytosis

During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.     

An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.     

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.