Synapse Scaffolding: Intersection of Endocytosis and Growth

Current Biology, Vol. 14, R853–R855, October 5, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.09.042

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Synapse Scaffolding: Intersection of Endocytosis and Growth

Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee 37232, USA. E-mail: [email protected]

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The fusion and recycling of synaptic vesicles must be tightly coupled to sustain high-fidelity neurotransmission. The synapse is compartmentalized into the active zone, which mediates Ca2+-dependent synaptic vesicle fusion, and the surrounding periactive zone, within which resides cell adhesion molecules and endocytosis machinery. Relatively little is known about the architecture and coordinated functions of the periactive zone, but it is thought to be an important site for retrieval of the extra membrane added to the plasma membrane each time a synaptic vesicle undergoes exocytosis. At the heart of membrane retrieval in the periactive zone is the GTPase dynamin, first identified by the Drosophila mutant shibire — from the Japanese word for ‘paralyzed’ — and a host of dynamin-associated proteins (Figure 1). Dynamin-associated protein 160 kDa (Dap160) was isolated by cofractionation with dynamin from Drosophila head extracts, and colocalizes with endocytic proteins in the periactive zone [1]. Dap160 contains two Eps15 homology (EH) domains and four Src homology (SH3) domains, through which it binds dynamin, synaptojanin, a synaptic vesicle-associated phosphoinositide phosphatase, and synapsin, a phosphoprotein synaptic vesicle tether in a reserve pool (Figure 1) [1,2]. Vertebrate Dap160 was named Intersectin to highlight its putative function as a macromolecular complex scaffold in the periactive zone [3]. Like Dap160, Intersectin binds numerous key endocytic proteins, including dynamin, epsin1/2, Stoned B/Stonin 2, Eps15 and synaptojanin (Figure 1) [3–5]. Intersectin localizes to clathrin-coated pits via its EH domains [6], and is enriched at the necks of endocytic intermediates [7]. There are two vertebrate Intersectins. Intersectin 1short (1S) is ubiquitously expressed and most closely resembles Dap160 — it has two EH domains and five SH3 domains, one more than Dap160. Intersectin 1long (1L) is predominantly in neurons and has three additional carboxy-terminal domains not found in Dap160: Dbl homology (DH), pleckstrin homology (PH) and C2 domains [6]. The DH and PH domains act as a

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The Drosophila dynamin-associated protein Dap160, homolog of the vertebrate Intersectins, is thought likely to act as a molecular scaffold in the synaptic periactive zone. New mutant analyses have revealed separable roles for Dap160 in the regulation of vesicular endocytosis and synaptic growth.

guanine nucleotide exchange factor (GEF) for Cdc42 and can thereby modulate the actin cytoskeleton (Figure 1) [8,9]. Intersectin 1L regulates actin cytoskeleton during endocytosis, vesicle mobilization and modulation of synaptic structure [10]. In this capacity, Intersectin binds to WASp, a positive regulator of the Arp2/3 complex which nucleates Factin, and signals via the RasGEF Son-of-sevenless and Cdc42 (Figure 1) [8,9,11]. The absence of comparable domains in Dap160 suggests it may lack cytoskeletal signaling properties. A dominant-negative approach in mammalian cell culture first implicated Intersectin 1S in synaptic vesicle endocytosis. The expression of isolated Intersectin SH3 domains, either alone or in combination, disrupted clathrin-mediated endocytosis [4,8,11,12], presumably by sequestering essential endocytic proteins. The critical genetic tests have now been performed in the Bellen [13] and Davis [14] laboratories through characterization of dap160 lossof-function mutations in Drosophila. Both groups found that Dap160 is essential for viability and transgenic rescue experiments showed the requirement is specifically within neurons. Koh et al. [13] went on to show that viable dap160 mutant flies display temperature-sensitive paralysis

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Figure 1. The endocytic interactome. Dap160 binds multiple proteins in the periactive zone of neuronal synapses, including proteins implicated in endocytosis, actin cytoskeletal organization and cell signaling. The interaction modules EH–NPF, SH3–PXXP and coiled-coil domains mediate protein–protein binding. Protein abbreviations: Dyn, dynamin; Endo, endophilin; Synj, synaptojanin; Syt, synaptotagmin; WASp, Wiskott-Aldrich Syndrome protein; ARP2/3, actin-related protein 2/3 (ARP2/3); and SOS, Son-ofSevenless.

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Model 1: Dynamin allostery Cyto

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Figure 2. Models for Dap160 function during synaptic vesicle endocytosis. Model 1: Dap160 is required to maintain sufficient levels of the GTPase dynamin, which is crucial for synaptic vesicle fission in the periactive zone. Dynamin’s GTPase activity shows allosteric dependence on its concentration during the oligomerization process. Decrease in dynamin levels would lead to a non-linear decrease in dynamin activity. Model 2: Dap160 is required to localize dynamin to the neck of the collared pit during endocytosis. In the absence of Dap160, dynamin fails to tether to the collared pit, dynamin is destabilized/degraded and synaptic vesicle endocytosis occurs with low efficiency. Model 3: Dap160 dimers provide a platform for propulsive force provided by actin polymerization and dynamin action acting coordinately to pinch off vesicles. In all models, synaptic vesicle fission is delayed, causing accumulation of collared pits, enlarged vesicles, and decreased vesicle density.

reminiscent of shibire dynamin mutants. Consistently, but surprisingly, mutant neuromuscular junction synapses show only a mild synaptic vesicle endocytic defect at normal rearing temperature (22°C), although a more severe impairment at elevated temperature (34°C). The complete absence of Dap160 results in phenotypes much less severe than removal of other endocytic proteins, such as dynamin, Stoned or endophilin [15,16]. Physiological nerve stimulation results in near normal neuromuscular junction synaptic potentials in dap160 mutants. Only conditions of severe demand reveal an impairment: for example, 10 minutes of 10 Hz stimulation was found to cause an ~50% decrease in synaptic potential amplitudes [13]. Similarly, assays of synaptic vesicle endocytosis with FM1-43 dye loading revealed no short-term defects in dap160 mutants, although after a 10 minute labeling period dye uptake was found to be reduced by ~30%, with more severe defects at higher temperatures. With higher levels of demand — 30 Hz stimulation for 1 minute — FM4-64 dye loading is reduced by ~50% [14]. The frequency of spontaneous synaptic vesicle fusions is two-fold higher in dap160 mutants, and aberrant large-amplitude events occur quite often, although the majority of events exhibit normal amplitudes. Mutation of other endocytic proteins, such as Stoned, endophilin and AP180, also increases the average quantal amplitude at the Drosophila neuromuscular junction, reflected in the appearance of a class of enlarged synaptic vesicles [15–17]. Electron microscopy of dap160 mutants [13] revealed fewer synaptic vesicles, a population of aberrant enlarged vesicles, and accumulation of endocytic intermediates at both active and periactive zones. Both groups [13,14] found that loss of Dap160 decreases the levels of several endocytic proteins,

including dynamin, endophilin, synaptojanin and AP180. From confocal imaging at the neuromuscular junction, the levels of these proteins are all decreased by an average of about 50%, showing that Dap160 plays a facilitory but non-essential role in maintaining the synaptic endocytic apparatus (Figure 2). All four proteins remain clearly enriched in synaptic boutons in dap160 mutants, which the authors suggest may be due to persisting interactions among endocytic proteins (Figure 1). Whether these proteins are mislocalized or degraded is not clear. The exception is synapsin, the reserve pool synaptic-vesicleassociated protein that also interacts with Dap160 [1], which shows a very interesting redistribution into internal bouton puncta in dap160 mutants [14]. These findings are consistent with the view that DAP160 may scaffold endocytic proteins, but show that it is not essential in this function. They do not make clear precisely how Dap160 may fulfill this putative scaffolding role, and indicate that Dap160 function extends to other synaptic proteins not implicated in endocytosis, such as synapsin. But the integrity of the active zone does appear to be independent of Dap160 [13,14]. Moreover, at least some elements of the periactive zone are also organized independently of Dap160; the cell adhesion protein fasciclin II, for example, appears largely normal in dap160 mutants. Dap160 also plays an important role in the regulation of synaptic architecture. This is rather unexpected, as Dap160 lacks domains found in Intersectin 1L that are implicated in the regulation of actin cytoskeleton and synaptic growth [8,9]. In dap160 mutants, the neuromuscular junction displays an ~10-fold increase in small ‘satellite boutons’ [13,14], which have been suggested to represent a developmentally arrested bouton [18]. Consistently, mutant neuromuscular junctions

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were found not to grow beyond the normal synaptic span, although Koh et al. [13] report a five-fold increase in synaptic branching. These structural defects are not observed in other endocytic mutants, including synaptojanin and endophilin [2,16], suggesting that the regulation of synaptic structure by Dap160 is separable from its role in endocytosis. Koh et al. [13] note aberrant staining of Futsch, a microtubule-associated protein 1B-like protein, and a fragmented synaptic microtubule cytoskeleton in dap160 mutants; both groups [13,14] observed an ~50% decrease in Nervous Wreck, a WASp-binding adaptor protein that controls neuromuscular junction development by regulating actin cytoskeletal dynamics (Figure 1) [19]. These abnormalities may be causative of the observed developmental defects in the dap160 mutants. It should be noted, however, that nervous wreck mutant synapses show only a two-fold increase in satellite bouton formation. Nervous Wreck and Dap160 both interact with the actin-nucleating protein WASp [19], but nervous wreck; wasp double mutants display only a four-fold increase in satellite boutons [19]. Taken together, these data suggest that Dap160 may regulate microtubule and/or actin dynamics, but the mechanism is unclear. New work on Drosophila mutants thus indicates that Dap160 acts as a molecular scaffold for the endocytic machinery at neuromuscular junctions, and separably regulates synaptic architecture. Does this represent convergent or divergent molecular functions? There does appear to be a strong link between endocytosis and cytoskeletal organization. Moreover, loss of dynamin function in shibire mutants impairs growth, and vertebrate dynamin is implicated in synaptic remodeling. But the loss of other endocytic proteins in Drosophila causes only weak defects in neuromuscular junction architecture [2,16], suggesting that disrupted synaptic vesicle endocytosis per se cannot explain developmental growth defects. So there may be multiple, Dap160-dependent scaffolds within the periactive zone, one regulating endocytosis and another developmental growth. Given that Dap160 lacks Intersectin 1L domains implicated in cytoskeletal interactions, the mechanism by which Dap160 might regulate growth remains unclear. Dap160 might localize Nervous Wreck–WASp–Arp2/3 complexes to synaptic boutons, thereby stimulating the production of branched actin filaments, suppressing microtubule stability and driving new bouton formation. As a scaffold for endocytic proteins — dynamin, synaptojanin, endophilin, and AP180 — Dap160 is clearly not essential, although more severe defects at high temperature suggest that it plays an important stabilizing role. Dap160 does not bind all of these proteins, only dynamin and AP180 directly, and so may serve as the foundation for a branching protein tree. As the EH and SH3 domains of Dap160 are involved in multiple other interactions, it is probable that Dap160 functions as a stabilizing scaffold which interacts transiently with different endocytic proteins at different steps of endocytosis, in agreement with models based on biochemical data [20].

References 1. Roos, J., and Kelly, R.B. (1998). Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J. Biol. Chem. 273, 19108-19119. 2. Verstreken, P., Koh, T.W., Schulze, K.L., Zhai, R.G., Hiesinger, P.R., Zhou, Y., Mehta, S.Q., Cao, Y., Roos, J., and Bellen, H.J. (2003). Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating. Neuron 40, 733-748. 3. Yamabhai, M., Hoffman, N.G., Hardison, N.L., McPherson, P.S., Castagnoli, L., Cesareni, G., and Kay, B.K. (1998). Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. J. Biol. Chem. 273, 31401-31407. 4. Sengar, A.S., Wang, W., Bishay, J., Cohen, S., and Egan, S.E. (1999). The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15. EMBO J. 18, 1159-1171. 5. Martina, J.A., Bonangelino, C.J., Aguilar, R.C., and Bonifacino, J.S. (2001). Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery. J. Cell Biol. 153, 1111-1120. 6. Hussain, N.K., Yamabhai, M., Ramjaun, A.R., Guy, A.M., Baranes, D., O'Bryan, J.P., Der, C.J., Kay, B.K., and McPherson, P.S. (1999). Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. J. Biol. Chem. 274, 15671-15677. 7. Predescu, S.A., Predescu, D.N., Timblin, B.K., Stan, R.V., and Malik, A.B. (2003). Intersectin regulates fission and internalization of caveolae in endothelial cells. Mol. Biol. Cell 14, 4997-5010. 8. Hussain, N.K., Jenna, S., Glogauer, M., Quinn, C.C., Wasiak, S., Guipponi, M., Antonarakis, S.E., Kay, B.K., Stossel, T.P., LamarcheVane, N., and McPherson, P.S. (2001). Endocytic protein intersectinl regulates actin assembly via Cdc42 and N-WASP. Nat. Cell Biol. 3, 927-932. 9. Zamanian, J.L., and Kelly, R.B. (2003). Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis. Mol. Biol. Cell 14, 1624-1637. 10. Irie, F., and Yamaguchi, Y. (2002). EphB receptors regulate dendritic spine development via intersectin, Cdc42 and N-WASP. Nat. Neurosci. 5, 1117-1118. 11. Tong, X.K., Hussain, N.K., de Heuvel, E., Kurakin, A., Abi-Jaoude, E., Quinn, C.C., Olson, M.F., Marais, R., Baranes, D., Kay, B.K., and McPherson, P.S. (2000). The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain. EMBO J. 19, 1263-1271. 12. 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. 13. Koh, T.-W., Verstreken, P. and Bellen, H.J. (2004). Dap160/Intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis. Neuron 43, 193-205. 14. Marie, B., Sweeney, S.T., Poskanzer, K.E., Roos, J., Kelly, R.B., and Davis, G.W. (2004). Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth. Neuron 43, 207-219. 15. Fergestad, T., Davis, W.S., and Broadie, K. (1999). The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal. J. Neurosci. 19, 5847-5860. 16. Verstreken, P., Kjaerulff, O., Lloyd, T.E., Atkinson, R., Zhou, Y., Meinertzhagen, I.A., and Bellen, H.J. (2002). Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109, 101-112. 17. Zhang, B., Koh, Y.H., Beckstead, R.B., Budnik, V., Ganetzky, B., and Bellen, H.J. (1998). Synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis. Neuron 21, 1465-1475. 18. Beumer, K.J., Rohrbough, J., Prokop, A., and Broadie, K. (1999). A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila. Development 126, 5833-5846. 19. 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. 20. McPherson, P.S. (2002). The endocytic machinery at an interface with the actin cytoskeleton: a dynamic, hip intersection. Trends Cell Biol. 12, 312-315.