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Regulation of Synaptic Vesicle Recycling by Complex Formation Between Intersectin 1 and the Clathrin Adaptor Complex AP2

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Regulation of Synaptic Vesicle Recycling by Complex Formation Between Intersectin 1 and the Clathrin Adaptor Complex AP2 Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2 Arndt Pechsteina,b, Jelena Bacetica,1, Ardeschir Vahedi-Faridia,1, Kira Gromovaa,1, Anna Sundborgerb,1, Nikolay Tomlinb, Georg Krainerc, Olga Vorontsovab, Johannes G. Schäfera, Simen G. Owed, Michael A. Cousine, Wolfram Saengera, Oleg Shupliakovb,2, and Volker Hauckea,c,2 aInstitute of Chemistry and Biochemistry, Freie Universität and Charité Universitätsmedizin Berlin, 14195 Berlin, Germany; cLeibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; bDepartment of Neuroscience, Linné Center in Developmental Biology and Regenerative Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden; dDepartment of Anatomy, University of Oslo, N-0317 Oslo, Norway; and eMembrane Biology Group, Centre for Integrative Physiology, University of Edinburgh, EH8 9XD Edinburgh, Scotland, United Kingdom Edited* by Pietro De Camilli, Yale University and the Howard Hughes Medical Institute, New Haven, CT, and approved January 21, 2010 (received for review September 29, 2009) Clathrin-mediated synaptic vesicle (SV) recycling involves the spa- over, association of the SH3A-B linker region of intersectin with tiotemporally controlled assembly of clathrin coat components at AP2 inhibits binding of the inositol phosphatase synaptojanin 1. phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P2]-enriched mem- These data identify the intersectin-AP2 complex as an important brane sites within the periactive zone. Such spatiotemporal control regulator of clathrin-mediated SV recycling in synapses. is needed to coordinate SV cargo sorting with clathrin/AP2 recruit- ment and to restrain membrane fission and synaptojanin-mediated Results uncoating until membrane deformation and clathrin coat assembly Antibodies Targeting the Linker Region Between SH3 Domains A and are completed. The molecular events underlying these control mech- B of Lamprey Intersectin 1 Disrupt SV Endocytosis at Early Stages. anisms are unknown. Here we show that the endocytic SH3 domain- Acute perurbation of intersectin interactions in living lamprey containing accessory protein intersectin 1 scaffolds the endocytic synapses with antibodies raised against the SH3 domain-containing process by directly associating with the clathrin adaptor AP2. Acute module revealed multiple effects on SV recycling (13). Endocytosis perturbation of the intersectin 1-AP2 interaction in lamprey synapses was inhibited at the stage of constricted CCPs, consistent with a in situ inhibits the onset of SV recycling. Structurally, complex for- defect in dynamin-mediated fission. However, microinjection of mation can be attributed to the direct association of hydrophobic “ antibodies targeting SH3 domains A to C, including the connecting peptides within the intersectin 1 SH3A-B linker region with the side linker regions of intersectin 1, also led to an accumulation of large sites” of the AP2 α-andβ-appendage domains. AP2 appendage membrane expansions, indicative of endocytosis inhibition at early association of the SH3A-B linker region inhibits binding of the inosi- stages (13). To further explore the underlying mechanisms of the tol phosphatase synaptojanin 1 to intersectin 1. These data identify early endocytic phenotype associated with intersectin perturbation the intersectin-AP2 complex as an important regulator of clathrin- mediated SV recycling in synapses. at vertebrate synapses, we raised antibodies against different parts of the SH3 module, including the linker region between SH3 do- fi fi endocytosis | synapse | scaffolding proteins | appendage | synaptojanin mains A and B of lamprey intersectin (LIS-linker). Af nity-puri ed LIS-linker IgG specifically recognized two bands of expected molecular weights of about 200 and 170 kDa (13) (lamprey inter- ynaptic vesicles (SVs), following their activity-dependent exo- sectin 1 short and long isoforms) in detergent-extracted lamprey cytic fusion with the presynaptic plasma membrane, are recy- S brain homogenates, and this reactivity was lost upon preincubation cled by compensatory endocytosis at the periactive zone (1–3), with the antigenic peptide (Fig. S1A). Similar results were seen if largely via clathrin-mediated reinternalization of fully fused SV antibody specificity was assessed by immunoprecipitation of native membrane (4). Clathrin-coated pit (CCP) formation (5) proceeds endogenous intersectin 1 (Fig. S1D). LIS-linker IgG also recog- through the assembly of endocytic proteins at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P ]-enriched membrane sites (6, 7). A nized endogenous intersectin 1 accumulated within SV clusters in 2 reticulospinal axons, as seen previously (13); again, this labeling was key factor in the assembly pathway is the heterotetrameric adaptor B complex AP2, whose α-andβ2-appendage domains act as major completely abolished by preincubation with the antigen (Fig. S1 and C). A similar accumulation of intersectin 1 at SV clusters is also recruitment platforms for accessory proteins (6, 7), regulating dis- fi tinct steps within the pathway. Despite our extensive knowledge observed in mammalian synapses of hippocampal mossy bers from regarding the endocytic interactome, we know comparably little the CA3 region and synapses established by Schaffer recurrent about the structural components within the periactive zone that colaterals from CA1 and at corresponding postsynaptic elements scaffold the endocytic process, thereby allowing the high fidelity of SV recycling. Such spatiotemporal control is needed to coordinate SV cargo protein sorting with coat recruitment (8) and to restrain Author contributions: O.S. and V.H. designed research; A.P., J.B., A.V.-F., K.G., A.S., N.T., fi J.G.S., S.G.O., and O.S. performed research; K.G., A.S., N.T., G.K., O.V., and M.A.C. con- membrane ssion and uncoating until membrane deformation and tributed new reagents/analytic tools; A.P., A.V.-F., K.G., G.K., M.A.C., W.S., and V.H. an- CCP assembly are completed. Moreover, stabilizing scaffolds may alyzed data; and A.P., O.S., and V.H. wrote the paper. aid coupling of SV exo- and endocytosis (1, 3). The Drosophila The authors declare no conflict of interest. multidomain protein Dap160, an ortholog of mammalian inter- *This Direct Submission article had a prearranged editor. sectin, has been postulated to act as an endocytic scaffold of the Data deposition: Protein structures are accessible under PDB ID codes 3HS8 and 3HS9. – periactive zone (9 11), although its precise role in SV recycling in 1J.B., A.V.-F., K.G., and A.S. contributed equally to this work. mammalian nerve terminals remains largely unclear (12). 2To whom correspondence may be addressed. E-mail: [email protected] or oleg. Here we show that intersectin 1 scaffolds the endocytic process [email protected]. by directly associating with AP2. Acute perturbation of intersectin- This article contains supporting information online at www.pnas.org/cgi/content/full/ AP2 complex formation blocks the onset of SV recycling. More- 0911073107/DCSupplemental. 4206–4211 | PNAS | March 2, 2010 | vol. 107 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.0911073107 Downloaded by guest on September 27, 2021 (Fig. 1A and Fig. S2 A–E). Thus, intersectin 1 is accumulated at observed instead in these synapses (Fig. 1 E, F, and I). These presynaptic sites both in lamprey and in mammals. functional data indicate that the linker region between SH3 Having established that LIS-linker IgGs specifically recognize domains A and B of intersectin plays an important role at early intersectin 1 in vitro and in situ, we microinjected these IgGs into stages of clathrin-mediated SV endocytosis, perhaps via direct lamprey reticulospinal axons and stimulated at 5 Hz for 20 min to interactions with one of the major coat components. induce SV recycling at a physiological rate. Analysis of ultrathin sections revealed a profound loss of SVs at active zones in syn- Intersectin 1 via its SH3A-B Linker Region Directly Associates with the α β apses from these axons (Fig. 1 B, C, and H), but not from control - and -Appendage Domains of AP2. To directly address the pos- sibility that intersectin 1 interacts with clathrin/AP2, we per- axons microinjected with nonspecific IgGs (Fig. 1 G and H) (13). formed immunoprecipitations from rat (Fig. 2A) and lamprey No morphological alterations were observed in axons micro- B fi D brain extracts (Fig. 2 ). Antibodies speci c for intersectin 1 (Fig. injected with LIS-linker IgGs kept at rest (Fig. 1 ). This change S1E)efficiently coprecipitated not only the established intersectin was accompanied by an accumulation of large membrane pockets 1 binding partner dynamin 1, but also the plasmalemmal clathrin and invaginations and a concomitant increase in the number of adaptor AP2 (Fig. 2 A and B). Hsc70 or AP1 were absent from the B C CCPs, predominantly at early stages of endocytosis (Fig. 1 , , immunoprecipitates. Conversely, intersectin was found in immu- H–J ), indicative of an inhibition of clathrin-mediated SV recy- noprecipitated material using monoclonal antibodies against AP2 cling. This phenotype was clearly distinct from that observed (see below), indicating that intersectin and AP2 are present in a following microinjection of antibodies against SH3 domains C to tight complex in vivo in both lamprey and in mammals. This E of intersectin, which did not induce large membrane expansions conclusion is supported by the near perfect colocalization of around the active zone (Fig. 1 E,
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