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 associated with the entire coat at early endocytic stages (Fig. to lower parts of CCPs remains unchanged (Fig. 2E vs Fig. 2F,H),
Journal of Cell Science 1C1,2), and, at late stages, dynamin is detected on both the coat and indicating that the interaction between endophilin and dynamin is the neck of CCPs (Fig. 1C3). Gold particles signaling for another important for the localization of dynamin at the neck of the coated endophilin binding partner, synaptojanin, were also associated with pit. the entire coat at early endocytic stages. At late stages, however, gold particles were found preferentially concentrated at the lower Endophilin and dynamin form a complex on the necks of half of CCPs (upper:lower CCP area labeling ratio0.33, n35; constricted CCPs in living synapses supplementary material Fig. S1D–F). Only 40% of CCPs were To investigate further the organization of dynamin and endophilin labeled, which was in agreement with earlier observations (Haffner on the necks of CCPs just before fission, we microinjected GTPS et al., 1997). By contrast, all CCPs investigated were labeled with into living reticulospinal axons (Evergren et al., 2007). Endocytic antibodies against dynamin and endophilin. These experiments intermediates formed in the presence of GTPS have elongated show that, during formation of coated vesicles in the periactive necks decorated with a striation pattern. Tilting of sections in an zone, endophilin and dynamin are in position to interact at the electron microscope allowed us to define these striations as protein necks of constricted CCPs. spirals (Fig. 3B and Fig. 4B,C). We labeled these structures with antibodies against endophilin, dynamin, intersectin and Perturbation of endophilin–SH3 domain interactions amphiphysin and compared the distributions of gold particles (Fig. prevents dynamin localization at the neck of constricted 3A,C–G and supplementary material Fig. S1G,H). Interestingly, CCPs and inhibits fission in a living synapse endophilin localizes predominately to the area of the neck proximal PP19, a peptide derived from the PRD of synaptojanin binds to rat to the coat [Fig. 3A (region I), Fig. 3C,D,G]. Dynamin and endophilin 1 and blocks interactions of the endophilin SH3 domain amphiphysin, however, are distributed evenly along the spiral (Fig. selectively (Ringstad et al., 2001). Previous studies showed that 3E–G and supplementary material Fig. S1G), and intersectin microinjection of PP19 into living lamprey synapses blocks immunolabeling is confined to the coat region (supplementary interactions of endophilin with its endocytic binding partners – material Fig. S1H). The distribution of gold particles signaling for synaptojanin and dynamin – which results in an inhibition of synaptojanin on protein spirals formed in synapses in the presence clathrin-mediated endocytosis at late stages (Gad et al., 2000) (see of GTPS using the same technique has been reported previously also supplementary material Fig. S2A). One of the striking effects (Haffner et al., 1997). Clusters of synaptojanin 1 immunogold, was an accumulation of free clathrin-coated vesicles. An which have been observed, are clearly different from the heavy, accumulation of clathrin-coated vesicles was also observed in continuous labeling produced by antibodies against dynamin and Pre-fission complex in synaptic vesicle recycling 135
Fig. 1. Endophilin accumulates at the periactive zone after stimulation and colocalizes with dynamin at the necks of CCPs. (A)TEM image of the periactive zone of a reticulospinal synapse in lamprey stimulated at 5 Hz for 20 minutes and labeled with antibodies against endophilin. Gold particles are accumulated at CCPs and vesicles (sv) in the periactive zone. (B,C)Clathrin-coated intermediates labeled with antibodies against endophilin (B) and dynamin (C) at higher magnification (1 – shallow, 2 – non-constricted, and 3 – constricted states). (D)Schematic illustration of a constricted CCP divided into upper and lower areas used for the quantifications of gold particle labeling. (E)Bar graph showing the densities of gold particle labeling for endophilin and dynamin in the region 15 nm from the presynaptic membrane in the periactive zone (500 nm from the active zone) and outside synaptic regions. (F)Bar graph showing gold particle labeling for endophilin and dynamin in the axoplasmic matrix 500m2 lateral to the active zone and outside the synaptic region. (G)Bar graph showing the ratios of gold particle densities in the ‘upper’ versus ‘lower’ areas of the constricted CCPs labeled for endophilin and dynamin. (D,G)Quantification of immunogold labeling on constricted CCPs shows that endophilin predominantly resides Journal of Cell Science at the lower half of CCPs (upper:lower ratio0.37±0.17), whereas dynamin is found more frequently in the upper half of constricted CCPs (upper:lower ratio2.3±0.35). Bars represent means ± s.e.m. (***P<0.001, Student’s t-test; n35 CCPs). Scale bars: 100 nm (A); 50 nm (B,C).
the restricted signal produced by immunogold labeling for into spirals (Farsad et al., 2001). When adding dynamin and endophilin observed in our experiments. Thus, before fission, endophilin to 1,2-dioleoyl-sn-glycero-3-(phospho-l-serine) endophilin is concentrated at the base of the coat of the CCPs, (hereafter referred to as PS) liposomes, we find that lipid tubules where it colocalizes with dynamin. When the dynamin spiral are decorated by protein complexes similar to those observed on continues to form in the presence of GTPS, other SH3-domain- the necks of constricted CCPs in vivo, as shown in previous figures containing proteins come into interaction with the PRD of dynamin. (Fig. 3B). Importantly, these endophilin–dynamin complexes are One such protein appears to be amphiphysin. As dynamin and structurally different from those formed by either dynamin or endophilin interact directly, this suggests that, in synapses, they endophilin alone (Fig. 4A,D vs 4F,G). This is further visualized by assemble into a complex in a restricted part of the spiral proximal cryoelectron microscopy (Fig. 4H,I vs 4J,K). Dynamin-decorated to the coat. tubes have an average diameter and pitch of 43.8±0.42 nm and 13.3±0.26 nm, respectively (mean ± s.e.m.; n100 tubes; Fig. Endophilin and dynamin form a structurally distinct 4F,J,L,M). Together, endophilin and dynamin assemble into distinct complex, which promotes dynamin lipid binding in vitro spirals with significantly smaller diameters, 32.1±0.26 nm, as To investigate further the assembly and structure of an endophilin– compared with the spirals formed by dynamin alone, and the pitch dynamin complex, we employed an in vitro model. Previous of the endophilin–dynamin complex spirals is larger, 25.7±0.44 negative-stain electron microscopy studies have shown that nm (mean ± s.e.m.; n100 tubes; Fig. 4D,I,L,M). Also, the average endophilin and dynamin together tubulate liposomes and assemble inner diameter of endophilin–dynamin-decorated tubes is 8.1 nm 136 Journal of Cell Science 124 (1)
(Fig. 4L,M), similar to that of the tubes decorated by endophilin concentration of 150 mM, indicating that the mixed proteins form alone (Fig. 5G,K). Immunogold labeling of the tubes confirms that distinct complexes even in the absence of lipids (supplementary both proteins are components of these structures (Fig. 4N,O). material Fig. S3A). By measuring the protein–lipid tube diameter, and the pitch of To investigate whether endophilin aids in the recruitment of the protein spirals, we further confirmed that the endophilin– dynamin to lipid membranes, we changed the composition of the dynamin complex assembles on liposomes of different lipid liposome templates by decreasing the amount of PS, which compositions, including PS, PS:PC (PC, 1-acyl-2-acyl-sn-glycero- dramatically reduces the lipid binding and tubulation properties of 3-phosphocholine), brain total lipid extract plus PIP2 (hereafter dynamin (Table 1). After testing different PS:PC ratios, we found referred to as BTLEPIP2) and PS:PI4 [PI4, 1,2-dioctanoyl-sn- that neither dynamin nor endophilin binds or tubulates PS:PC glycero-3-phosphate (1Ј-myo-inositol-4Ј-phosphate)] (Fig. 5B,D, liposomes mixed at a 30:70% ratio as efficiently as PS liposomes and data not shown). Also, after addition of endophilin to dynamin, (Fig. 5A and Table 1). Mixing the two proteins together, however, many ring-like structures are observed in solution at a salt increases the amount of dynamin bound to PS:PC lipids by an average of 1.8-fold under these conditions (Fig. 5H; n8, P<0.05; supplementary material Fig. S4A). The number of tubulated liposomes decorated with endophilin–dynamin complexes is also increased (Fig. 5A vs 5B; supplementary material Fig. S4B). The structural organization of the spirals assembled on this template is similar to that observed in Fig. 4. To test whether a similar improvement of dynamin recruitment might occur under more physiological conditions, we used brain total lipids plus 10% PIP2 (BTLEPIP2) (Fig. 5C vs 5D). A 6.6-fold average increase in dynamin recruitment to liposomes is observed after addition of endophilin (Fig. 5H; n2; P<0.05). To determine whether the interaction between the SH3 domain and PRD is important for endophilin–dynamin complex formation on lipid templates, we added PP19 to the endophilin–dynamin mixture. When endophilin and dynamin are pre-incubated with PP19 and then added to PS:PC (30:70% ratio) or BTLEPIP2 liposomes, a significant reduction in dynamin liposome binding is observed. The presence of PP19 results in an average 2.5-fold reduction in dynamin binding to PS:PC liposomes (Fig. 5I; n4; P<0.05) and an average 4.7-fold reduction in dynamin binding to total brain lipid extract liposomes (Fig. 5I; n2; P<0.05). Furthermore, electron microscopy (EM) analysis shows that
Journal of Cell Science endophilin–dynamin complexes are disrupted in the presence of PP19 and large gaps in the protein decoration of lipid tubes are visible, whereas addition of 1 mM GTPS has no effect (Fig. 5E vs 5F; supplementary material Fig. S5D). Furthermore, addition of PP19 has no effect on lipid binding and tubulation properties of the
Fig. 2. Acute perturbation of endophilin SH3-domain interactions perturbs localization of dynamin at necks of CCPs and inhibits fission in living synapses. (A1)Electron micrograph showing a periactive zone of a lamprey synapse stimulated at 5 Hz for 20 minutes after microinjection of PP19 and labeling with antibodies against dynamin. (A2)Free clathrin-coated vesicle found in the injected synapse shown in A1, but here at high magnification. (B)TEM image of a periactive zone in a control, non-injected synapse stimulated at 5 Hz and labeled with antibodies against dynamin. (C–F)TEM images of CCPs from stimulated synapses in axons microinjected with PP19 and labeled with antibodies against dynamin (C) and endophilin (E) and in stimulated control axons labeled with the same antibodies (D and F, respectively). (G)Bar graph showing an accumulation of CCPs in synapses injected with PP19 as compared with control synapses (P<0.001, Student’s t-test; n6–8 synapses). (H)Bar graph showing dynamin and endophilin labeling in the lower area of CCPs in synapses injected with PP19 and stimulated at 5 Hz for 20 minutes compared with that of stimulated control synapses from the same preparation. (I)Bar graph showing dynamin labeling in the upper area of CCPs in synapses injected with PP19 and stimulated at 5 Hz for 20 minutes compared with that of stimulated control synapses from the same preparation (*P<0.05, Student’s t-test; n35 CCPs). Bars in (G,H,I) represent means ± s.e.m. Scale bars: 50 nm (A2,C–F); 200 nm (A1,B). Pre-fission complex in synaptic vesicle recycling 137
Fig. 3. Endophilin and dynamin colocalize in the region proximal to the coat of elongated constricted necks induced by GTPS. (A)Schematic illustration of areas 1 (‘I’) and 2 (‘II’) of the CCP neck defined for quantifications of antibody labeling. (B)TEM image of a constricted CCP in the periactive zone of a synapse injected with GTPS and stimulated at 5 Hz for 20 minutes. Note the presence of dense rings (arrowheads). (C,D)and (E,F) Electron micrographs of the CCPs labeled with antibodies against endophilin and dynamin, respectively. Areas ‘I’ and ‘II’ are marked by dashed lines. (G)Bar graph showing the number of gold particles labeling endophilin, dynamin and amphiphysin in the two regions defined in (A). Bars represent means ± s.e.m. (***P<0.001, Student’s t-test; n35 CCPs). Scale bar: 50 nm (B–F).
Journal of Cell Science individual proteins (supplementary material Fig. S5A–C) and does injection sites was adjusted to allow analysis of each compound at not have any effect on endophilin dimerization in solution, as separate sites (which served as a control) and to achieve mixing of revealed by size-exclusion chromatography and crosslinking of the two compounds to investigate the effect of the double-injection endophilin (supplementary material Fig. S5E). Recombinant in the same axon. To monitor the diffusion of the reagents within the ⌬PRD-dynamin fails to form the distinct dynamin–endophilin axon, Texas Red was added as an injection marker. Extracellular spirals seen on lipids incubated with wild-type dynamin. stimulation at 5 Hz was initiated to induce cycling of synaptic Additionally, endophilin does not facilitate the lipid binding of vesicles. PP19 was injected first, to block selectively interactions of ⌬PRD-dynamin (Fig. 5G,I). Thus, the interaction between the the endophilin SH3 domain, followed by stimulation at 5 Hz to endophilin SH3 domain and dynamin PRD mediates assembly of increase the number of accumulated constricted coated pits at the proteins into a structurally distinct complex on lipids that leads periactive zones before introduction of GTPS (Farsad et al., 2001), to the formation of protein–lipid tubes of a smaller diameter and which was injected 10 minutes after the onset of stimulation. The facilitates further recruitment of dynamin to lipids. stimulation was maintained for an additional 20 minutes before fixation. For controls, neighboring axons were microinjected with Blocking endophilin SH3 domain interactions in a living Texas Red alone or Texas Red and GTPS to allow comparison of synapse perturbs the assembly of an endophilin–dynamin the morphology of endocytic intermediates at the same distance complex from the site of injection in the same spinal cord preparation (Fig. To investigate in vivo how blocking of endophilin SH3 domain 6A; supplementary material Fig. S2F). Ultrastructural analysis of interactions affects the assembly of the endophilin–dynamin clathrin-coated intermediates was performed at the site of injection complex, which promotes fission in intact synapses, we of each compound (Fig. 6C, ‘area 1’ and ‘area 2’) and at several microinjected GTPS into lamprey giant axons in the presence of levels (up to eight) where PP19 and GTPS mixed (Fig. 6C, ‘area PP19. GTPS, microinjected alone (n10), strongly blocks 3’). Fig. 6B1–4 shows the effects of double-injection of PP19 and endocytosis at the fission step. Numerous constricted coated pits GTPS. A massive accumulation of constricted CCPs with short with long necks decorated with protein spirals are observed at the necks is observed (Fig. 6D,E). This is in clear contrast to adjacent periactive zone of these synapses (Fig. 6A1,2,D). axons microinjected with GTPS alone at the same level (‘area 3’), In three isolated spinal cord preparations, PP19 and GTPS were where only long necks decorated by dynamin spirals were found microinjected into the same axon at distances of 200 m to 2 mm (Fig. 6A). Thus, the average neck length of the CCPs is significantly from each other, as shown in Fig. 6C. The distance between the two lower in PP19–GTPS double-injected axons as compared with the 138 Journal of Cell Science 124 (1)
Fig. 4. Ultrastructural characterization of the endophilin–dynamin complex in vitro. (A,H)TEM images of PS liposomes tubulated by endophilin and dynamin and visualized by negative staining (A) and cryo- EM (H). (B,C)Stereo pair of TEM images of CCPs taken at ± 4° tilts from synapses injected with GTPS and stimulated at 5 Hz. (D,I)High magnifications of endophilin–dynamin- decorated PS tubes in negative stain (D) and vitreous ice (I). Note the regular arrangement of the protein complex and its structural similarity to the protein complex assembled on the neck of a constricted CCP (E, arrows). (F,J)TEM images of dynamin-decorated PS tubes in negative stain (F)
Journal of Cell Science and vitreous ice (J). Note that the diameter of the protein– lipid tube in F and J is larger than the one shown in D and I. (G,K)TEM images of tubulated PS liposomes decorated by endophilin visualized by negative stain (G) and cryo-EM (K). (L)Measurements of protein tube diameter (d), tube lumen diameter (l) and protein spiral pitch (p), measured in protein–lipid tubes embedded in amorphous ice quantified in the bar graph shown in (M). Note the statistically significant difference in each case between dynamin- decorated tubes and tubes decorated with dynamin and endophilin (***P<0.001, Student’s t-test; n100 tubes). (N,O)Electron micrographs of PS liposomes decorated by endophilin–dynamin spirals and labeled with antibodies against endophilin and dynamin, respectively. Scale bars: 100 nm (A,H); 50 nm (B,C); 50 nm (D–G,I–K,N,O).
ones found in axons injected with GTPS alone, as well as in control synaptic vesicle recycling at the fission step. Injection of Texas Red non-injected axons (Fig. 6E). Thin spirals, as compared with those alone has no effect on the ultrastructure of stimulated synapses observed in control non-injected axons, are occasionally detected at (supplementary material Fig. S2E vs Fig. S2F). high magnification decorating the short necks (Fig. 6B4, arrow). These experiments further indicate that endophilin recruits These spirals are also thinner than the ones induced by GTPS alone dynamin to the CCP neck and that the assembly of an endophilin– (Fig. 3B and Fig. 6A2). We also found that the amount of dynamin dynamin complex is a crucial step in the fission reaction in a living detectable at these necks is reduced by 84% (1.9±0.24 vs 0.3±0.12 synapse. gold particles per lower CCP area, in control synapses compared with synapses in axons injected with PP19 and GTPS; mean ± Discussion s.e.m., n35 CCPs, P<0.001). Moreover, free clathrin-coated vesicles, Our experiments show that, in living synapses, endophilin 1 is one observed in PP19-only microinjected synapses, are no longer present, of the proteins selectively recruited to the neck of clathrin-coated suggesting that PP19 and GTPS together completely blocked vesicles during recycling of synaptic vesicles. The inhibition of Pre-fission complex in synaptic vesicle recycling 139
Fig. 5. Endophilin–dynamin complex formation facilitates recruitment of dynamin to liposomes. (A,B)TEM images of PS:PC liposomes decorated by dynamin alone (A) or decorated with endophilin and dynamin in negative stain (B). (C,D)TEM images of total- brain-lipid plus 10% PIP2 liposomes (BTLEPIP2) decorated with dynamin (C) or endophilin–dynamin complexes (D). Note an increase in the number of tubules in (B) and (D) compared with A, and C. (E,F)Electron micrographs of PS:PC liposomes in the presence of endophilin and dynamin (E) and following addition of 300M PP19 (F). Note the gaps in the decoration exposing naked liposomes (arrows). (G)TEM image of PS liposomes in the presence of endophilin and PRD- dynamin. Note the absence of well-ordered complexes seen in (E). (H)Bar graph shows the increase in the amount of dynamin (expressed as a percentage) in the pellet fraction after mixing with PS:PC or BTLEPIP2 in the absence (white bars) and presence of endophilin (black bars). (I)Bar graph shows reduction in the amount of dynamin (expressed as a percentage) in the pellet fraction after mixing with PS:PC or BTLEPIP2 and endophilin Journal of Cell Science (black bars) in the absence and presence of PP19 (grey bars) and following deletion of its PRD (hatched bar). Bars show means ± s.e.m. (*P<0.05, **P<0.01, Student’s t-test; n8 experiments for PS:PC and 2 for BTLEPIP2). Scale bars: 500 nm (A–D), 50 nm (E–G).
endophilin SH3-domain interactions by PP19 reduces the amount Interestingly, a number of endophilin-interacting proteins – for of dynamin at the neck and inhibits budding of synaptic vesicles. example, CD2AP or CIN85 (Ferguson et al., 2009) and This further implies that the dynamin–endophilin interaction is of oligophrenin-1 (Nakano-Kobayashi et al., 2009) – interact with the functional importance at this location. PP19 also inhibits the actin cytoskeleton, suggesting that endophilin might be guided to formation of CCPs with elongated necks in the presence of GTPS. the periactive zone by one of these proteins as the actin matrix Instead, an accumulation of constricted CCPs with short necks is assembles. The role of the endophilin BAR domain seems to be observed in these synapses. Also, no free clathrin-coated vesicles unique. Supporting this, Drosophila endophilin-null mutants cannot were found, which is indicative of a complete block of fission. Taken together, these data indicate that endophilin and dynamin are important players in the membrane budding-reaction in Table 1. Endophilin and dynamin lipid binding and synapses. tubulation The organization of the endophilin–dynamin interaction at the Dynamin Endophilin Dynamin plus endophilin neck of the CCPs seems to be dictated by endophilin. Our PS:PC (70:30) ++ ++ +++ experiments show that endophilin localizes in the synaptic vesicle PS:PC (30:70) ++ ++ +++ pool at rest and accumulates in the periactive zone during recycling PS:PC (20:80) + + ++ of synaptic vesicles (Shupliakov, 2009). Endophilin is probably BTLE – – – BTLEPIP – – ++ recruited to CCPs by means of its BAR domain (Andersson et al., 2 2010; Ringstad et al., 1999). Recent studies in non-neuronal cells +: Some tubes and <50% of the protein in the pellet after sedimentation. have shown that formation of an actin matrix around CCPs is ++: Tubes and ≥50% of the protein in the pellet after sedimentation. required for efficient endophilin recruitment (Ferguson et al., 2009). +++: Many tubes and >90% of the protein in the pellet after sedimentation. 140 Journal of Cell Science 124 (1) Journal of Cell Science
Fig. 6. PP19 prevents the formation of elongated necks decorated with dynamin-containing spirals induced by GTPS. (A)Control TEM image of the periactive zone of a reticulospinal synapse from an axon microinjected with GTPS and Texas Red (see panel C, ‘axon 3’, ‘area 3’). Coated pits with elongated necks decorated by spirals are seen accumulated in the periactive zone. (A2)High-magnification image of a constricted coated pit with an elongated neck from the synapse shown in A1. Note the thick spiral. (B)Electron micrograph of the periactive zone of a reticulospinal synapse injected with both PP19 and GTPS (see panel C, ‘axon 1’, ‘area 3’). Note the accumulation of CCPs with short necks in the periactive zone (B1,2). (B3)TEM image illustrating dynamin immunogold labeling of constricted CCPs with short necks in a synapse injected with PP19 and GTPS. (B4,5) High-magnification images of constricted coated pits with short necks from the synapse shown in (B1) (arrow indicates a thin spiral). (C)Schematics of the microinjection experiment. The zone ‘area 1’ (blue) delineates the PP19 microinjection site, and ‘area 2’ (yellow) delineates the GTPS microinjection site. The region ‘Area 3’ (green) marks where the two compounds mixed in the axon that was microinjected with both compounds. Control axons were injected with either PP19 (‘axon 2’) or GTPS (‘axon 3’). (D)Bar graph showing the number of CCPs in the periactive zones of synapses microinjected with PP19, GTPS, PP19 plus GTPS or non-injected control axons stimulated at 5 Hz. Values are normalized to the length of the active zone. (E)Bar graph showing the length of necks of CCPs from the synapses described in (D). Statistical significance of differences between means ± s.e.m. was determined by a Student’s t-test (**P<0.01, ***P<0.001; n35 CCPs). Scale bars: 200 nm (A1,B1,B3), 50 nm (A2,B4,B5), 500 nm (B2).
be rescued with endophilin constructs containing F-BAR domains bottom of the coat after microinjection of GTPS. The organization of other proteins or the N-BAR domain of amphiphysin (Jung et of this protein spiral has strong similarities with the complex al., 2010). between endophilin 1 and dynamin 1 observed in vitro. To form The interaction between endophilin and dynamin results in the this complex, the interaction between the endophilin SH3 domain formation of a complex, which can be visualized as a spiral at the and the dynamin PRD is required, as perturbations of this interaction Pre-fission complex in synaptic vesicle recycling 141
including total brain lipids, which allows us to propose a model for the sequence of events leading to fission of the neck of CCPs in vertebrate synapses (Fig. 7): endophilin accumulates at the base of the coat, where it serves as a template for dynamin. Together, they assemble into a ‘pre-fission complex’. This complex shapes the lipid neck to an average inner diameter of 8.1 nm (3.8–14.8 nm) and brings the neck to the hemi-fission state (Lenz et al., 2009). More dynamin is recruited to the neck and oligomerizes into a ‘constrictable’ spiral. The smaller diameter of the pre-fission complex might accelerate membrane fission by conformational changes in the constrictable dynamin spiral below the pre-fission complex. The pre-fission complex might also function as a template for rapid spiral disassembly. Synaptojanin, recruited after, or Fig. 7. Schematic illustrating the proposed function of the endophilin– possibly during, the fission reaction might compete with dynamin dynamin interaction at the neck of CCPs during synaptic vesicle for binding to endophilin and thus aid the disassembly of the pre- recycling. Dynamin and endophilin are recruited to different regions of the fission complex. coated pit. Endophilin induces curvature of the neck and serves as a template for dynamin. An endophilin–dynamin fission complex forms at the base of the Materials and Methods coat. This complex promotes recruitment of dynamin to the neck, which Proteins results in GTP-mediated fission. The presence of PP19 blocks the dynamin Full-length cDNA encoding human neuronal dynamin (dynamin-1) (obtained from binding sites on endophilin, preventing formation of the complex and efficient A. van der Bliek, UCLA, CA) was subcloned into the pBlueBac baculovirus recruitment of dynamin, resulting in perturbation of fission (dashed arrow). expression vector (Invitrogen) and expressed in TN5-JE cells [modified from Warnock et al. (Warnock et al., 1995)]. Cells were harvested 48 hours after infection, pelleted and frozen in liquid nitrogen before purification. Cell pellets were thawed in 25 ml of HCB100 (20 mM HEPES (pH 7.2), 2 mM EGTA, 1 mM MgCl2, 100 mM NaCl) both in vitro and in situ reduce dynamin recruitment to lipid with protease inhibitor cocktail (Roche), calpain inhibitor (85 l) and 25 l of templates and to presynaptic membranes in living synapses, pefabloc (4-2-amioethyl benzenesulfonyl fluoride hydrochloride, Sigma, 100 mg/ml). Cells were broken open by nitrogen cavitation at ~3400 kPa for 25 minutes in a cold respectively. room, and centrifuged at 20,000 g in a 70.1 Ti rotor for 1 hour at 4°C. Supernatant In vitro studies have shown that the endophilin–dynamin was collected and saturated by adding ammonium sulfate (in HCB100) drop-wise complex is stable after addition of GTP (Farsad et al., 2001). This while stirring until a concentration of 35% (ammonium sulfate) was reached. The stable complex might form a non-constrictable spiral, similar to solution was incubated for 1 hour at 4°C before centrifugation at 10,000 g for 15 minutes at 4°C. The ammonium sulfate pellet was resuspended in 30 ml HCB50 and the one formed by Rvs proteins in budding yeast, which precedes centrifuged at 8000 g for 10 minutes to remove aggregates. The supernatant was the fission reaction (Liu et al., 2009). Imaging and molecular applied to a High Q ion exchange column, washed with HCB50 followed by modeling experiments suggest that, in budding yeast, fission is HCB100 and eluted with HCB250. The peak fractions were pooled, loaded onto a HAP column, washed with HCB250 until flow-through reached baseline, washed triggered by lipid phase separation achieved by hydrolysis of PIP 2 with 200 mM KH2PO4 (pH 7.2), and protein was eluted in 400 mM KH2PO4. Peak driven by the phosphatase sjl2 (synaptojanin-like protein 2), which fractions were pooled, pefabloc was added and aliquots were frozen in liquid nitrogen and stored at –80°C. ⌬PRD-dynamin construct, obtained from Sandy Schmid (The Journal of Cell Science enhances curvature, and the assembly of the actin cytoskeleton Scripps Research Institute, CA), was produced by introducing a stop codon at (Liu et al., 2009). Although it cannot be excluded that hydrolysis residue 751 into the wild-type dynamin sequence. The construct was cloned into the of PIP2 might promote fission during recycling of synaptic vesicles, pBlueBac baculovirus expression vector (Invitrogen), expressed and purified from there is strong evidence that dynamin plays the major role (Ferguson TN5-JE cells as described above for full-length dynamin. et al., 2009). Dynamin depletion in non-neuronal cells results in pGEX-2 constructs of rat full-length endophilin or its SH3 domain were received from Volker Haucke. Full-length endophilin was subcloned into the pGEX-6P-2 the formation of coated pits with elongated necks decorated with vector before both constructs were transformed into competent Escherichia coli spirals containing endophilin and actin (Ferguson et al., 2009). In Bl21(DE3) cells (Invitrogen). The cells were plated onto 100 g/l ampicillin plates these cells, fission cannot occur in the presence of the phosphatase and incubated at 37°C overnight. Colonies were resuspended in 5 ml LB media with 100 g/l ampicillin and grown at 37°C overnight on a shaker. The bacteria synaptojanin, thus supporting the idea that regulation of fission in suspension was diluted 1:100 in YTG media with 10% glucose and 100 g/ml eukaryotic cells is more complex than in yeasts (Conibear, 2010). ampicillin and grown at 37°C with O2 until the OD590 was 0.6–0.8. Isopropyl -D- Endophilin is not the only protein that might recruit synaptojanin 1-thiogalactopyranoside (IPTG; 0.1–0.3 mM) was added and the cells were grown in nerve terminals. It has been shown recently that one mechanism for another 1.5 hours at 30°C. The suspension was centrifuged at 5500 g for 10 minutes. Cell pellets were frozen in liquid nitrogen and stored at –80°C. Cell pellets of synaptojanin recruitment to the clathrin coat in neurons involves were thawed and resuspended in 40 ml PBS with a protease inhibitor cocktail intersectin 1, and the binding of synaptojanin to intersectin is (Sigma). The suspension was frozen in liquid nitrogen and thawed twice before regulated by the adaptor protein complex AP2 (Pechstein et al., sonication at 4°C. 1% Tween-20 was added, and the suspension was incubated at 4°C for 30 minutes on a shaker following centrifugation at 18,000 g for 30 minutes. 2010). This allows for a possible synaptojanin–endophilin Supernatant was filtered and applied to a 1 ml Fastflow GST column (GE Healthcare). interaction, which is crucial for efficient uncoating of synaptic The column was washed with PBS until baseline OD280 level was regained. GST- vesicles, to occur directly after fission, following disassembly of endophilin or GST-SH3 domain of endophilin was eluted in 10 mM glutathione in 50 mM Tris, pH 8. The GST-tag was cleaved with Prescission protease enzyme (GE the endophilin–dynamin complex. Further studies are needed, Healthcare) according to the manufacturer’s instructions. Protein was aliquoted, however, to verify this hypothesis. frozen in liquid nitrogen and stored at –80°C. How might the formation of a non-constrictable complex of endophilin–dynamin promote fission in synapses? Spontaneous Antibodies and reagents All lamprey antibodies were protein A and affinity purified and stored in stocks at polymerization of dynamin can be initiated on tubes with radii concentrations of 1–2 mg/ml. Antibodies against lamprey dynamin GTPase domain ranging between 10 and 30 nm, but not on larger tubes (Roux et (Evergren et al., 2007; Evergren et al., 2004) were diluted 1:100 in blocking solution al., 2009). We show that the diameter of the endophilin–dynamin- containing 1% BSA and 1% HSA in Tris–PBS (pH 7.2) for electron microscopy and 1:10,000 for western blotting. Antibodies against the lamprey endophilin SH3 decorated lipid tube is within this range. In the presence of domain, rat endophilin and synaptojanin have been characterized previously and endophilin, dynamin is efficiently recruited to lipid templates, were diluted 1:100 for electron microscopy and 1:1000 for western blotting 142 Journal of Cell Science 124 (1)
(Andersson et al., 2010; Gad et al., 2000; Ringstad et al., 1999). Antibodies against total lipid extract (BTLE; Avanti Polar Lipids cat. # 131101), 1,2-dioctanoyl-sn- lamprey intersectin SH3 A–C and C–E domains (Evergren et al., 2007) were diluted glycero-3-phosphate (PI4; Avanti Polar Lipids cat. # 850182) and 2-(4,4-difluoro- 1:100 in blocking solution for electron microscopy and 1:1700 for western blotting. 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero- Antibodies against the E-domain of lamprey synapsin and monoclonal antibodies 3-phosphocholine (-BODIPY; Invitrogen cat. # 3793) were mixed (w/v), dried against the D-domain of rat synapsin (Synaptic Systems cat. # 106-001) were diluted using argon or nitrogen gases and incubated in a vacuum desiccator overnight before 1:3000 and 1:2000 in 0.3% Tween–TBS, respectively. Goat anti-rabbit secondary rehydration in HCB150 (with gentle shaking for 20 minutes at room temperature). antibodies conjugated to 5 nm colloidal gold particles (GE Healthcare) were diluted All rehydrated lipids were stored at 4°C and all stocks solutions at –20°C. The 1:50 in TPBS. HRP-conjugated goat anti-rabbit or -mouse secondary antibodies following liposome preparations were made: (1) 100% PS, (2) 10% PIP2 and 90% (Invitrogen) were diluted 1:10,000 in 0.1–0.3% Tween–TBS. PS, (3) 70% PC and 30% PS, (4) 70% PC and 30% PS with -BODIPY added at a PP19 (VAPPARPAPPQRPPPPSGA), C-PP19 (CVAPPARPAPPQRPPPPSGA) and final concentration of 0.06 mg/ml, (5) 10% PIP2 and 90% BTLE. Liposomes were PP15 (CPPPQVPRPNRAPPG) peptides were purchased from Sigma Genosys. used directly, or after extrusion through 0.4 m polycarbonate filters using a mini Powder was dissolved in 1% TFA in H2O, lyophilized for 2 hours at room temperature lipid extruder (Avanti Polar Lipids). and aliquots stored at 4°C. For injections, aliquots were dissolved in injection buffer (250 mM potassium acetate, 100 mM HEPES, pH 7.4) to a final concentration of 30 Light microscopy and electron microscopy analysis of protein–lipid tubes mM in the electrode. GTPS (Roche) was dissolved in 10 mM Texas Red (Sigma) Proteins were added at a concentration range of 0.1–0.2 mg/ml (mixed together or in injection buffer to a final concentration of 30 mM in the electrode. separately) to liposomes, and the protein–lipid solution was incubated for 1–120 minutes at room temperature. For fluorescence microscopic analysis of protein Microinjection experiments mixtures, liposome preparation #4 (see above) was used. Protein–lipid tubes were Lampreys (Lampetra fluviatilis) were kept in aquaria at 4°C. All animals were prepared as described above, transferred onto glass slides and analyzed in a treated according to the Swedish Animal Welfare Act (SFS 1988: 534), as approved fluorescence microscope (Zeiss Axioplan 2). For electron microscopy analysis, by the Local Animal Research Committee of Stockholm. All efforts were made to carbon-coated copper mesh grids (200 mesh, EMS), with or without a nitrocellulose minimize animal suffering and to reduce the number of animals used. supporting film, were put on droplets of protein–lipid solution for 1 minute before Microinjections into axons of isolated lamprey spinal cords were performed as negative staining in 1% uranyl acetate. Protein–lipid tube structures were observed described previously (Evergren et al., 2007). For double-injections of PP19 and and photographed at 80 kV in a Tecnai 12 or at 100 kV in a CM120 electron GTPS, four giant reticulospinal axons were injected in the same preparation (n4) microscope. NIH Image J software was used to quantify the number of protein–lipid with PP19 followed by stimulation at 5 Hz for 10 minutes, during which time GTPS tubes, their diameter and the pitch of the protein helix. One hundred randomly was injected into the same four axons (injected by PP19). GTPS was injected at a selected tube segments were measured in each case. A two-sided Student’s t-test was site isolated from the site of PP19 injection, more rostral in the spinal cord preparation used to compare means. (Fig. 6C). For controls, neighboring axons were injected with PP19, GTPS or Texas Red, or double injected with GTPS–PP19 and Texas Red. For ultrastructural analysis Immunogold labeling of endophlin–dynamin lipid tubes of the effect of the injected compounds on the synapses, spinal cord preparations Protein–lipid tubes made as previously described were absorbed onto grids and were fixed during stimulation and embedded in Durcupan ACM resin (Fluka), as incubated on droplets of blocking solution (1% BSA in HCB100) for 30 minutes at described previously (Evergren et al., 2007; Evergren et al., 2004). For pre-embedding 4°C. Grids were transferred to droplets of primary antibody against dynamin or immunogold labeling (see below), injected preparations were fixed during stimulation endophilin and incubated for 2 hours at 4°C. Grids were washed in HCB100 and in 0.5% glutaraldehyde, 3% paraformaldehyde, in 0.1 M cacodylate buffer for 4 incubated on droplets of secondary antibody for another 2 hours at 4°C before hours at 4°C. washing and staining with 1% in uranyl acetate.
Immunogold labeling Cryo-EM For pre-embedding immunogold labeling, after fixation, spinal cord preparations Vitrified samples were prepared in a Vitrobot (FEI) at a relative humidity of 85%. were washed in 0.1 M cacodylate buffer, and axons were cut longitudinally using a Samples were applied to plasma-cleaned (Fishione) holey-carbon grid (Quantifoil). vibratome (Leica) to expose synaptic periactive zones to antibodies (Evergren et al., Samples were blotted, immediately plunged into liquid ethane and stored in liquid 2007). Blocking solution (1% BSA–1% HSA in Tris–PBS) was added for 30 minutes nitrogen before imaging in the electron microscope. Images were acquired on a to block nonspecific antibody binding. Specimens were incubated with primary Philips CM12 (FEI), operated at 100 kV, using a Gatan 791 Multiscan CCD camera antibodies: anti-dynamin, anti-endophilin, anti-amphiphysin and anti-intersectin (A- with the Digital Micrograph software package.
Journal of Cell Science E), anti-synaptojanin and secondary antibody overnight at 4°C with agitation. Specimens were washed in Tris–PBS, post-fixed in 3% glutaraldehyde in 0.1 M Lipid binding assays cacodylate buffer for 1 hour at 4°C, washed in 0.1 M cacodylate buffer for 1 hour Proteins were spun down separately before mixing with liposomes to remove at 4°C, stained in 1% osmium tetroxide for 1 hour at 4°C, stained en bloc in 2% aggregates before mixing with lipids. Protein–lipid samples (prepared as described uranyl acetate, dehydrated in increasing concentrations of alcohol and embedded in above) were centrifuged at 200,000 g for 1 hour at room temperature in an Durcupan. For post-embedding immunogold labeling, specimens were embedded in ultracentrifuge (Beckman) or in an Airfuge ultracentrifuge (Beckman) at top speed LR Gold resin (Fluka), sectioned onto mesh grids and stained with primary anti- for 60 minutes at room temperature. Supernatant was removed and mixed 1:1 in endophilin antibodies and secondary antibodies conjugated to 5 nm gold particles sample buffer (200 mM Tris–HCl, 4% SDS, 16% glycerol, 0.01% bromophenol (Amersham Bioscience), as described previously (Evergren et al., 2004). blue, pH 6.8). Pellets were resuspended in 2ϫ sample buffer. Samples were boiled, analyzed by SDS-PAGE and stained with Coomassie. Protein band intensities were EM analysis of lamprey synapses quantified using Adobe Photoshop CS3 software. The amount of dynamin in the Serial ultrathin (70–120 nm) sections were put onto Formvar-coated copper–rubidium pellet was calculated as a percentage of the total protein content in the sample. slot grids (EMS) and stained with uranyl acetate and lead citrate. Sections were Dynamin and endophilin were mixed together in the absence of lipids, and less than analyzed and photographed in a Technai 12 electron microscope (FEI) at 80 kV. The 10% of dynamin and less than 7% of endophilin were found in the pellet fraction effects of microinjections were analyzed in 25–60 synapses in each injected axon at (n3). Thus, dynamin–endophilin complex formation in solution could not contribute various distances from the site of injection. The number of synaptic vesicles and significantly to the increase of dynamin in the pellet fraction observed when lipids CCPs was quantified from three middle sections of five synapses at the level of were added. maximum effect in each case. The numbers were normalized to the length of the active zone (Gustafsson et al., 2002). Stereo pairs were assembled from images Crosslinking and gel filtration collected at ±4°. Endophilin was incubated with 1 mM crosslinker DSP [dithiobis(succinimidyl NIH Image J software was used for measurements. Quantifications of gold propionate), Thermo Scientific/Pierce Biotechnology) dissolved in dimethyl sulfoxide particles were performed in the axoplasm in the region 15 nm from the presynaptic (DMSO), or equal volume of DMSO in the presence and absence of increasing membrane in the periactive zone (in a stretch of 500 nm from the active zone) and concentrations of PP19. The reaction was quenched after 40 minutes at room outside synaptic regions, and in the axoplasmic matrix 500 m2 lateral to the active temperature with sample buffer, and the samples were analyzed by SDS-PAGE. zone and outside the synaptic region. Densities of gold particles were calculated as FPLC analysis was carried out using a Superdex 200 HR 10/30 column (GE gold particles/m2. At least 35 randomly selected clathrin-coated intermediates were Healthcare) in HBS (150 mM NaCl, 10 mM HEPES HBS, pH 7.5) buffer. used for statistical analysis in each experiment. Lower and upper areas of constricted CCPs were defined as shown in Fig. 1. Ratios between these values were used to Competition experiment compare changes in the distribution of gold particles in different labeling experiments. GST–fusion protein constructs of the lamprey SH3 A-E domain of intersectin [amino A two-sided Student’s t-test was used to compare means. acids 740–1064; amino acid numbering of lamprey intersectin here corresponds to that of human intersectin 1L; GenBank accession number NP003015], lamprey Protein-lipid tube formation amphiphysin [GenBank accession number AAT45899] and lamprey endophilin Stock solutions 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (PS; Avanti Polar Lipids [GenBank accession number AF304353] were generated, using the c vector (GE cat. # 840035), 1-acyl-2-acyl-sn-glycero-3-phosphocholine (PC; Avanti Polar Lipids Healthcare). GST–fusion proteins were coupled to glutathione sepharose (GE cat. # 850375), PtdIns(4,5)P2 (PIP2; Avanti Polar Lipids cat. # 840046) and brain Healthcare) and incubated with a 10% lamprey brain detergent extract in buffer A Pre-fission complex in synaptic vesicle recycling 143
(10 mM HEPES buffer, pH 7.4, 100 mM KCl, 2 mM MgCl2, and 1% Triton X-100) clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain plus Pefablock (Sigma), E-64, bestatin, leupeptin and aprotinin (Sigma) or 1 mM of endophilin. Neuron 27, 301-312. PMSF and 10–300 M PP19 for 1–2 hours at 4°C on a rotating wheel. Samples were Gallop, J. L., Jao, C. C., Kent, H. M., Butler, P. J., Evans, P. R., Langen, R. and washed three times with buffer A and three times with buffer A without Triton X- McMahon, H. T. (2006). Mechanism of endophilin N-BAR domain-mediated membrane 100, eluted with sample buffer and analyzed by SDS-PAGE, followed by detection curvature. EMBO J. 25, 2898-2910. with antibodies against dynamin, synaptojanin and synapsin. Granseth, B., Odermatt, B., Royle, S. J. and Lagnado, L. (2007). Clathrin-mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses. Pull-down experiment J. Physiol. 585, 681-686. 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