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An Endophilin–Dynamin Complex Promotes Budding of Clathrin-Coated Vesicles During Synaptic Vesicle Recycling

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An Endophilin–Dynamin Complex Promotes Budding of Clathrin-Coated Vesicles During Synaptic Vesicle Recycling Research Article 133 An endophilin–dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling Anna Sundborger1, Cynthia Soderblom1, Olga Vorontsova1, Emma Evergren1,*, Jenny E. Hinshaw2,‡ and Oleg Shupliakov1,‡ 1Department of Neuroscience, DBRM, Karolinska Institutet, 17177 Stockholm, Sweden 2Laboratory of Cell Biochemistry and Biology, NIDDK, NIH, Bethesda, MD 20892, USA *Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK ‡Authors for correspondence ([email protected]; [email protected]) Accepted 20 September 2010 Journal of Cell Science 124, 133-143 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.072686 Summary Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles. Key words: Synapse, Endophilin, Dynamin, Endocytosis Introduction in lamprey giant synapses, one of the effects of acute perturbation Recycling of synaptic vesicles after neurotransmitter release is of the endophilin Src-homology 3 (SH3) domain interaction was crucial for a sustained transmission. This membrane-retrieval an increase in the number of constricted CCPs (Gad et al., 2000). Journal of Cell Science process occurs mainly through clathrin-mediated endocytosis These results indicate that endophilin interactions are not only (Granseth et al., 2007; Jung and Haucke, 2007; Rodal and Littleton, involved in uncoating but also play a role in a stage dealing with 2008). At the final step of this process, clathrin-coated vesicles bud membrane-bound constricted CCPs before the fission step and from the presynaptic membrane and shed their clathrin coat. The might thus involve dynamin. accessory protein endophilin binds to two endocytic molecules There are three dynamin isoforms in neurons. Dynamin-1 is the implicated in the scission and uncoating events in synapses – the neuron-specific isoform required during high levels of neuronal GTPase dynamin and the polyphosphoinositide phosphatase activity (Ferguson et al., 2007; Lou et al., 2008). All dynamin synaptojanin (Liu et al., 2009; Ferguson et al., 2007; Heymann and proteins contain a pleckstrin-homology (PH) domain, which allows Hinshaw, 2009; Mettlen et al., 2009). Dynamin plays the key role the protein to interact with membrane phospholipids, and a proline- in synaptic vesicle budding, as a complete depletion of all dynamin rich domain (PRD), which enables them to bind to SH3 domains isoforms blocks fission of clathrin coated pits (CCPs) (Ferguson et of accessory proteins (Ferguson et al., 2007). The SH3 domain of al., 2007). The interaction of endophilin with synaptojanin serves endophilin 1 has been shown to bind to the PRD of dynamin 1 at mainly to facilitate uncoating of vesicles after fission (Cremona et two sites in vitro (Anggono and Robinson, 2007; Heymann and al., 1999; Gad et al., 2000; Ringstad et al., 1999; Ringstad et al., Hinshaw, 2009). Endophilin also contains an N-terminal BAR 2001; Schuske et al., 2003; Song and Zinsmaier, 2003; Verstreken domain with membrane curvature-generating and curvature-sensing et al., 2003), although recent studies in budding yeast suggest a properties (Gallop et al., 2006; Masuda et al., 2006). Together with possible involvement of synaptojanin in the actual fission reaction. dynamin, endophilin is present on tubules formed by liposomes This property of endophilin implies that it functions as a molecular after addition of brain cytosol (Farsad et al., 2001; Ringstad et al., switch linking fission and uncoating during recycling of synaptic 1999). Taken together, these studies suggest that endophilin might vesicles. The molecular mechanisms underlying this ‘switch’ are regulate fission, although it still remains unclear how the complex unknown, and in particular the role of the endophilin–dynamin of endophilin and dynamin, which is not modified by GTP (Farsad interaction remains to be clarified. et al., 2001), might aid this process. Loss of endophilin in both fly and nematode resulted in a Here, we utilized the lamprey reticulospinal synapse to elucidate slowdown of the endocytic process in neuromuscular junctions the role of endophilin–dynamin interactions in the regulation of the and in an increase in the number of free and membrane-attached fission mechanism during recycling of synaptic vesicles. Using clathrin-coated intermediates (Dickman et al., 2005; Schuske et al., immuno-electron microscopy, we localized endophilin to the sites 2003; Verstreken et al., 2002; Verstreken et al., 2003). Additionally, of synaptic vesicle recycling in relation to dynamin and studied the 134 Journal of Cell Science 124 (1) effects of acute perturbations of endophilin SH3 domain interactions synaptojanin-1-knockout mice. These data led to the conclusion on endocytosis in lamprey synapses. To model the effects observed that endophilin serves as a recruiter of the polyinositolphosphatase in living synapses in vitro, we used recombinant proteins and lipid synaptojanin to CCPs to promote the uncoating reaction (Cremona templates. Our experiments indicate that endophilin accumulates et al., 1999). In the present study, we sought to investigate further at a restricted part of the neck of the CCP. We propose that the origin of another, upstream, effect of PP19 microinjection – the endophilin provides a template for dynamin assembly and forms a accumulation of constricted CCPs – which possibly involves complex with dynamin at the neck. This complex increases dynamin dynamin (Gad et al., 2000). recruitment to the neck and structurally promotes dynamin- We first tested whether PP19 blocks SH3 domain interactions of mediated fission and thus aids sustaining a high rate of synaptic other endocytic proteins interacting with dynamin in lamprey and vesicle recycling during neurotransmitter release. found that PP19 competes efficiently with dynamin for binding to the GST–SH3 domain of lamprey endophilin but does not affect Results the interactions between dynamin and the lamprey amphiphysin Endophilin localizes to the rim of the clathrin coat of CCPs SH3 domain or the whole cassette of five SH3 domains of lamprey and colocalizes with dynamin at a restricted part of the intersectin (supplementary material Fig. S2B). Additionally, coupled neck to beads, PP19 selectively affinity-purifies the lamprey endophilin To study the relative distribution of endophilin and dynamin at orthologue but not amphiphysin or intersectin from lamprey brain sites of synaptic vesicle recycling in intact stimulated synapses, we extracts (supplementary material Fig. S2C). used immunogold labeling (Evergren et al., 2004). Both endophilin We then microinjected PP19 into lamprey giant axons to test and dynamin accumulate in the synaptic vesicle pool at rest whether perturbations of the endophilin SH3-domain interactions (supplementary material Fig. S1A) (Evergren et al., 2007). During affect the localization of dynamin at the necks of constricted CCPs. stimulation, an accumulation of both proteins is observed at CCPs Ultrastructural effects induced by microinjection of PP19 in giant in the periactive zone (Fig. 1A–C,E,F; supplementary material axons (n4) are similar to those described earlier (Gad et al., Fig. S1B,C) (see also Evergren et al., 2007). This is in contrast to 2000). Synaptic vesicle recycling is perturbed and an accumulation synapsin, which has also been shown to reside in the synaptic of constricted CCPs is observed in synaptic periactive zones (Fig. vesicle cluster at rest but does not associate with CCPs in the 2A,G and supplementary material Fig. S2D). The fission of clathrin- periactive zone of stimulated synapses (Evergren et al., 2004). coated vesicles, however, is not completely blocked as free clathrin- Interestingly, endophilin is localized at the edge of the clathrin coat coated vesicles are observed in the axoplasm (Fig. 2A2 and of shallow coated pits (Fig. 1B1,2) and at later stages is concentrated supplementary material Fig. S2D). at the necks of constricted coated pits (Fig. 1B3). Localization of To determine whether the localization of dynamin to constricted endophilin at the rim of the coat was also confirmed by post- CCPs is affected by PP19, we labeled endocytic intermediates with embedding immunogold labeling (supplementary material Fig. antibodies against dynamin. While labeling of the upper part of the S1B,C). Dynamin immunoreactivity is also detected at early coat is unaffected, labeling at the lower areas of CCPs is decreased endocytic stages. Contrary to endophilin, dynamin is found significantly (Fig. 2C vs 2D,H–I). The localization of endophilin
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