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. 2014 Jan 2;592(Pt 4):621–633. doi: 10.1113/jphysiol.2013.265447

Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

Jeffrey S Dason 1, Alex J Smith 1, Leo Marin 1, Milton P Charlton 1,✉
PMCID: PMC3934705  PMID: 24297851

Abstract

AbstractSynaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

Introduction

The recycling and sorting of synaptic vesicle (SV) proteins are required for sustained synaptic transmission (reviewed in Haucke et al. 2011); however, it is unclear how proteins are sorted into SVs during endocytosis. SV recycling occurs at the periactive zone (PAZ), which surrounds the active zone (AZ) (Roos & Kelly, 1999). Some studies have proposed that SV proteins disperse into the plasma membrane following fusion and subsequently undergo reclustering within the PAZ (Fernández-Alfonso et al. 2006; Wienisch & Klingauf, 2006); however, reclustering SV proteins would probably be rate-limiting during sustained activity (see Haucke et al. 2011). Alternatively, SV proteins may remain clustered (Willig et al. 2006; Opazo et al. 2010). Given that SVs have a high cholesterol content (Takamori et al. 2006), that several SV proteins bind to cholesterol (Thiele et al. 2000) and that SV cholesterol is required during recycling (Dason et al. 2010), vesicular cholesterol could play a role in confining SV proteins during recycling.

The cholesterol content of membranes affects both SV exocytosis (Zamir & Charlton, 2006; Wasser et al. 2007; Smith et al. 2010; Dason et al. 2010; Linetti et al. 2010; Ormerod et al. 2012) and endocytosis (Wasser et al. 2007; Dason et al. 2010; Petrov et al. 2010). Evidence from in vitro studies suggests that cholesterol is required for the spatial segregation of SV and plasma membrane proteins into lipid-raft domains (Thiele et al. 2000; Lang et al. 2001; Mitter et al. 2003; Yoshinaka et al. 2004; Jia et al. 2006; Lv et al. 2008; Geumann et al. 2010). Actin acts as a diffusion barrier to prevent lateral movement of transmembrane proteins (Nakada et al. 2003); phosphatidylinositol (4,5)-bisphosphate (PIP2) and subsequent actin polymerization occur in cholesterol-rich membrane domains (Rozelle et al. 2000; Kwik et al. 2003) and play a role in clustering proteins. The PAZ contains an actin-rich cytomatrix and disruption of actin impairs SV endocytosis (Kuromi & Kidokoro, 1998; Shupliakov et al. 2002; Bloom et al. 2003; Richards et al. 2004). Thus, actin may act as a scaffold to confine SV proteins during recycling (Sankaranarayanan et al. 2003).

SVs and their proteins can be reversibly trapped on the plasma membrane of the Drosophila temperature-sensitive dynamin mutant, shibire-ts1 (shi) (Koenig & Ikeda, 1989; Ramaswami et al. 1994; van de Goor et al. 1995; Estes et al. 1996; Macleod et al. 2004; Dason et al. 2010). We used this mutant to study the mechanisms that regulate clustering of SV proteins during recycling in live presynaptic terminals. We found that extraction of vesicular cholesterol caused SV proteins to disperse, reduced presynaptic PIP2 and altered the distribution of presynaptic actin. Inhibition of actin polymerization while SV proteins were trapped on the plasma membrane caused dispersal of SV proteins, impaired endocytosis and the recovery of synaptic transmission after return to the permissive temperature. These results demonstrate that vesicular cholesterol regulates actin polymerization, which is required to confine SV proteins on the presynaptic plasma membrane following exocytosis.

Methods

Fly stocks

Fly stocks were grown as previously described (Dason et al. 2010). Wandering third-instar larvae were used for all experiments. The temperature-sensitive shi mutant (Grigliatti et al. 1973) was used to reversibly block SV recycling. Male flies from UAS-PLCδ1-PH-EGFP,nsyb-GAL4/TM6B (Verstreken et al. 2009; Khuong et al. 2010) or UAS-Actin-GFP/TS;nsyb-GAL4/TL (Nunes et al. 2006) stocks were crossed to female shi flies and non-tubby male larvae were selected.

Solutions and chemicals

All physiology and imaging experiments were conducted in HL6 saline (Macleod et al. 2002). The osmolality of all solutions was 330 ± 10 mosmol l–l. Methyl-β-cyclodextrin (MβCD) was obtained from Sigma-Aldrich, St. Louis, Missouri Cayman Chemical, Ann Arbor, Michigan. MβCD at a 10 mm concentration was applied for 12 min to extract cholesterol. Latrunculin A (LatA; Cayman Chemical) was prepared as a 10 mm stock solution in DMSO (Sigma-Aldrich). Stock solutions were diluted in HL6 to 10 μm. DMSO (0.1%) was added to HL6 in control experiments for LatA.

Immunohistochemistry and quantification

Immunohistochemistry was performed as previously described (Dason et al. 2009) and imaged on a Leica TCS SL confocal laser-scanning microscope (Heidelberg, Germany) with a ×63 oil-immersion objective (1.32 NA). The following antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-HRP (1:800; Jackson ImmunoResearch, USA), polyclonal rabbit DVGLUT (vglut) antibody (1:5000; Daniels et al. 2004), monoclonal mouse DCSP-2(6D6) (csp) antibody (1:100; Iowa Hybridoma Bank, USA; Zinsmaier et al. 1994), polyclonal rabbit DSYT2 (Syt) antibody (1:800; Littleton et al. 1993), mouse monoclonal NC82 (brp) antibody (1:100 dilution; Iowa Hybridoma Bank; Wagh et al. 2006) and anti-GFP mAB 3E6 (1:500; Molecular Probes, Eugene, Oregon).

Colocalization analysis was performed on each 0.1 μm slice from z-stacks obtained from preparations doubled-stained with SV protein markers and anti-HRP to measure the distribution of SV proteins in the plasma membrane. Pearson's correlation coefficient (PCC) was used as a measure for colocalization and calculated for regions of interest (ROIs), defined as an entire bouton (based on anti-HRP staining), using an intensity correlation analysis plugin in ImageJ (see Li et al. 2004). A PCC close to 1 indicates a high degree of colocalization, whereas a PCC closer to 0 indicates a low degree.

The clustering index (CI) was calculated (Goldstein et al. 2000, 2005; Bouchier-Hayes et al. 2008) to determine if protein distribution was altered. The CI is the standard deviation of the average brightness of all the pixels of projections in ROIs, defined as an entire bouton (based on anti-HRP staining). A high CI indicates a protein is clustered, whereas a low CI indicates it is more diffuse. Two types of axons innervate the NMJs used in this study. These axons give rise to larger tonic-like type 1b and smaller phasic-like type 1s boutons, respectively. We restricted analysis for both CI and PCC to the larger 1b boutons where clustering was more readily observable. All images of fixed preparations shown in figures are z-stacks.

Live imaging

Live green fluorescent protein (GFP) imaging experiments were performed using a Leica TCS SL confocal laser-scanning microscope with a ×63 water dipping objective (0.90 NA). Images of live preparations in figures are single scans.

Electrophysiology

Intracellular recordings were performed as previously described (Romero-Pozuelo et al. 2007). Briefly, sharp glass electrodes filled with 3 m KCl (∼40 MΩ) were used to impale the ventral longitudinal muscle fibre 6 (abdominal segment 3) of dissected larvae to measure stimulus-evoked excitatory junction potentials (EJPs). Cut segmental nerves were stimulated using a suction electrode.

FM1-43 imaging

All FM1-43 (N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide) experiments were performed as previously described (Dason et al. 2010) using a Leica TCS SL confocal laser-scanning microscope with a ×63 water dipping objective (0.9 NA).

Electron microscopy

Electron microscopy was performed as previously described (Dason et al. 2010).

Statistical analysis

Statistical analyses were performed using unpaired t tests. For CI and PCC, n represents the number of boutons analysed from six preparations. n represents the number of preparations analysed in all other experiments. Error bars in all figures represent standard error of the mean.

Results

Vesicular cholesterol extraction causes dispersal of SV proteins

To investigate the trafficking and sorting of SV proteins after exocytosis we studied the shi larval neuromuscular junction (NMJ) where endocytosis can be reversibly blocked. Prolonged high frequency stimulation at non-permissive temperature (30°C) leads to a block in SV recycling and loss of neurotransmitter release due to a complete loss of SVs (Fig. 1A; Delgado et al. 2000; Macleod et al. 2004). This block is reversible and SVs can re-form at permissive temperature. SV proteins can also be trapped on the plasma membrane at non-permissive temperature (Fig. 1B; Ramaswami et al. 1994; Estes et al. 1996). Clusters of synaptotagmin (Syt) and cysteine string protein (csp) colocalize following SV trapping in shi mutants suggesting that SV proteins are clustered together during recycling (van de Goor et al. 1995). Subcellular fractionation of homogenates from shi mutant heads showed that SV proteins are transferred to the plasma membrane following SV trapping (van de Goor et al. 1995). The loss of SVs following SV trapping is not accompanied by an increase in other membranous compartments (Koenig & Ikeda, 1996). Thus, clusters of SV proteins observed by confocal microscopy following SV trapping in shi mutants represent protein clusters on the plasma membrane.

Figure 1.

Figure 1

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A, shi terminals contain numerous SVs at rest. Stimulation (10 Hz for 12 min) at 30°C traps SVs on plasma membrane. B, C, D and E, shi mutants stimulated (10 Hz for 12 min) at 30°C and fixed at 30°C were stained with anti-HRP antibody (green), anti-DSYT2 (red), anti-vglut antibody (red) or anti-csp antibody (red). Arrows indicate clusters of SV proteins. B, Syt is diffusely expressed prior to stimulation, but primarily on the plasma membrane following trapping. C, D, E and F, Syt, vglut and csp appeared more diffuse following vesicular cholesterol extraction. There was a significant increase in colocalization (as indicated by PCC) between Syt and HRP (n = 21–36; P < 0.01) and between vglut and HRP (n = 11–19; P < 0.05). There was also a significant decrease in PCC between csp and HRP (n = 14–18; P < 0.05). This decrease may be due to some csp coming off the plasma membrane since it is not a transmembrane protein like Syt and vglut.

SVs have a high cholesterol content (Takamori et al. 2006), which may regulate spatial organization of recycling SV proteins (Haucke & Gundelfinger, 2009; Opazo & Rizzoli, 2010; Puchkov & Haucke, 2013). This hypothesis has not been previously tested because SV membranes are largely inaccessible to cholesterol extraction in live presynaptic terminals. To test this hypothesis, we examined the effect of vesicular cholesterol extraction on the clustering of SV proteins following exocytosis using our previously established method of SV trapping followed by cholesterol extraction with MβCD, which results in a 30–40% reduction in cholesterol (Dason et al. 2010). While this method results in cholesterol extraction from both SVs and the plasma membrane, the physiological effects that we previously reported were specific to vesicular cholesterol because trapping SVs on plasma membranes depleted of cholesterol (Fig. 3B; MβCD applied at non-permissive temperatures before SV trapping, washed off and followed by SV trapping) did not impair SV recycling. All the effects of MβCD were cholesterol-specific as a 1:10 cholesterol–MβCD complex (made with 10 mm MβCD) that could not extract cholesterol had no effect on synaptic transmission or endocytosis (Dason et al. 2010).

Figure 3.

Figure 3

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A and B, schematic diagrams of cholesterol extraction from exocytosed SVs trapped on plasma membrane, where vesicular cholesterol is more accessible to extraction by MβCD and from the plasma membrane of a presynaptic terminal. C and D, live shi;+;UAS-PLCδ1-PH-EGFP,nsyb-GAL4/+ preparations in HL6 (without or with 10 mm MβCD) were imaged before and after stimulation (10 Hz for 12 min) at 30°C. The increase in PIP2 levels induced by stimulation was significantly impaired following vesicular cholesterol extraction (n = 8; P < 0.01). Fluorescence (F) was reported with background F subtracted. The F response was reported as the change in F relative to the resting F. This change in F was normalized to the resting F, using the equation ΔF/Frest = (F – Frest)/Frest. E, fixed shi;UAS-actin-GFP/+;n-syb-GAL4/+ preparations were stained with anti-GFP (green). E and F, actin clusters (indicated by arrows) were disrupted and the CI was significantly reduced after vesicular cholesterol extraction in preparations stimulated (10 Hz for 12 min) at 30°C and fixed at 30°C (n = 18–21; P < 0.01). E and F, actin clusters (indicated by arrows) were not disrupted following plasma membrane cholesterol extraction (n = 30 for control; n = 30 for MβCD; P > 0.05). Thus, vesicular cholesterol, but not plasma membrane cholesterol, regulates actin polymerization during SV recycling.

Vesicular cholesterol extraction altered the staining pattern of SV proteins (Syt, vesicular glutamate transporter (vglut), and csp) trapped at the plasma membrane (Fig. 1C–E, and Supplemental Fig. S1 available online). We analysed the distribution of SV components in the plasma membrane by determining colocalization with anti-HRP, which recognizes the β subunit of a Na+/K+-ATPase and uniformly stains the plasma membrane of presynaptic terminals (Sun & Salvaterra, 1995; Sun et al. 1998; Xu et al. 1999). Subcellular fractionation of homogenates from Drosophila heads showed that anti-HRP recognizes a protein present in plasma membrane fractions, but not in SV fractions (van de Goor et al. 1995). To determine if anti-HRP distribution changes following SV trapping, we measured the clustering index (CI; see Methods) of anti-HRP and found no difference (rest = 8.33 ± 0.34 (n = 17), trapped = 8.24 ± 0.35 (n = 17); P > 0.05). Vesicular cholesterol extraction also had no effect on anti-HRP distribution (MβCD = 8.29 ± 0.39 (n = 17); P > 0.05), thus anti-HRP is a suitable plasma membrane marker.

SV proteins were clustered in regions of the plasma membrane following SV trapping (see arrows in Fig. 1C–E). Colocalization was measured by calculating Pearson's correlation coefficient (PCC; see Methods). A higher PCC indicates a higher degree of colocalization. We found an increase in the PCC between HRP and Syt or vglut following vesicular cholesterol extraction (Fig. 1F). This demonstrates that Syt and vglut are clustered in the plasma membrane following trapping and that vesicular cholesterol extraction causes them to disperse more uniformly in the plasma membrane.

Interestingly, the PCC between HRP and csp was reduced following vesicular cholesterol extraction (Fig. 1F). This is consistent with the staining pattern that we observe. Following vesicular cholesterol extraction, csp was not observed in the periphery of the bouton, where HRP staining is most prominent (Fig. 1E). The explanation for this may be that Syt and vglut are transmembrane proteins, whereas csp is a palmitoylated protein that may more easily leave the plasma membrane following vesicular cholesterol extraction. Overall, extraction of vesicular cholesterol results in the dispersal of SV proteins.

To determine whether cholesterol extraction affected the structure of presynaptic terminals, we assessed the distribution of AZs by staining for the AZ protein bruchpilot (brp). We did not observe any obvious changes in brp distribution following plasma membrane or vesicular cholesterol extraction (Fig. 2A and B). Thus, cholesterol extraction did not cause complete remodelling of synaptic AZs. In addition, given that the distribution of brp, a plasma membrane protein, was not affected by extracting cholesterol when SVs were trapped on the plasma membrane (Fig. 2B), this supports the conclusion that the altered distribution of SV proteins that we observe (Fig. 1) is due to vesicular cholesterol extraction and not due to a change in plasma membrane structure.

Figure 2.

Figure 2

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A and B, shi terminals contain numerous AZs (as indicated by the AZ marker brp) at rest and following SV trapping on plasma membrane (10 Hz for 12 min at 30°C) in the presence or absence of 10 mm MβCD. Note that brp distribution remains punctate following SV trapping and cholesterol extraction. C, electron micrographs of shi presynaptic terminals, in which SVs were trapped on the plasma membrane (10 Hz for 12 min at 30°C) in the presence or absence of 10 mm MβCD. The structure of presynaptic terminals appeared similar to controls following vesicular cholesterol extraction. C and D, there were no significant differences (n = 21–23; P > 0.05) in the number of collared pits or vesicular structures arrested on the plasma membrane (indicated by arrows) in controls and preparations following vesicular cholesterol extraction. n represents the number of sections analysed from 6 preparations.

We next used electron microscopy to determine whether MβCD caused any obvious changes in plasma membrane structure or an increase in membranous structures when SVs were trapped on the plasma membrane; we detected no differences (Fig. 2C). Note the absence of SVs in the absence or presence of MβCD in comparison to unstimulated preparations (Fig. 1A). In addition, we found a similar number of collared pits or vesicular structures arrested in the process of pinching off the plasma membrane in both conditions (Fig. 2C and D). These structures have been reported in other studies on shi mutants (Koenig & Ikeda, 1989; Macleod et al. 2004). Thus, the changes in SV protein distribution that we observe following vesicular cholesterol extraction are not due to an increase in membranous structures or a change in the number of arrested vesicular structures but instead represent the dispersion of SV proteins on the plasma membrane.

Vesicular cholesterol extraction disrupts PIP2 accumulation and actin clustering

Cholesterol regulates actin by modulating PIP2 in many cell types (Symons et al. 1996; Miki et al. 1996; Rozelle et al. 2000; Kwik et al. 2003). We hypothesized that cholesterol functions to segregate SV proteins spatially during recycling by regulating PIP2 and actin dynamics. To determine whether cholesterol extraction affects PIP2 accumulation and distribution at presynaptic terminals during SV recycling, we used the GAL4/UAS system (Brand & Perrimon, 1993) to express the pleckstrin homology domain of PLCδ1 fused to enhanced green fluorescent protein (EGFP) to visualize PIP2 (Stauffer et al. 1998; Verstreken et al. 2009) in shi presynaptic terminals. This domain is known to bind PIP2 (Varnai & Balla, 1998). PIP2 levels increase during the stimulation of hippocampal neurons (Micheva et al. 2001). We found that PIP2 levels increased following SV trapping in shi mutants (Fig. 3C and D). When we extracted vesicular cholesterol (Fig. 3A), the increase in PIP2 levels (Fig. 3C and D) seen following SV trapping was significantly reduced. We next extracted cholesterol from the plasma membrane at non-permissive temperature (30°C), washed off MβCD, and then trapped SVs on the plasma membrane (Fig. 3B). Plasma membrane cholesterol extraction did not result in any differences in PIP2 levels (control = 0.77 ± 0.29 arbitrary units (a.u.) (n = 8), MβCD = 0.60 ± 0.08 a.u. (n = 6); P > 0.05). Thus, vesicular cholesterol, but not plasma membrane cholesterol, is required for increased PIP2 levels during SV recycling.

Actin colocalizes with SV proteins at the Drosophila larval NMJ (Nunes et al. 2006). To determine whether cholesterol extraction affects actin clustering, we used the GAL4/UAS system (Brand & Perrimon, 1993) to express actin-GFP in shi presynaptic terminals and found that actin became diffuse and its CI significantly reduced following vesicular cholesterol extraction (Fig. 3E and F). We next tested whether plasma membrane cholesterol also regulated actin dynamics. Actin distribution was not altered following plasma membrane cholesterol extraction (Fig. 3E and F). Thus, vesicular cholesterol, but not plasma membrane cholesterol, is required for clustering actin during SV recycling.

Inhibition of actin polymerization causes trapped SV proteins to disperse in the plasma membrane

We hypothesized that inhibition of actin polymerization when SV proteins were trapped on the plasma membrane would cause them to disperse. We found that when shi preparations were treated with 10 μm LatA for 5 min, actin became more diffuse (data not shown), confirming the results of a previous study (Nunes et al. 2006). The effects of inhibition of actin polymerization while SV proteins are trapped on the plasma membrane have not previously been tested. We found that 10 μm LatA treatment for 5 min when SV proteins were trapped on the plasma membrane (Fig. 4A) caused Syt to disperse and resulted in an increase in the PCC between HRP and Syt (Fig. 4B and C, and Supplemental Fig. S2 available online). Thus, actin is required to confine Syt in the plasma membrane following SV trapping. The dispersal of Syt resembled that following vesicular cholesterol extraction (Fig. 1C and F).

Figure 4.

Figure 4

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A, schematic diagram of inhibition of actin polymerization following SV protein trapping. B and C, shi mutants were given a stimulus protocol (10 Hz for 12 min at 30°C, no stimulation for an additional 5 min with or without 10 μm LatA at 30°C and fixed at 30°C) and stained with anti-HRP (green) and anti-DSYT2 (red). Syt dispersed and the PCC between Syt and HRP was significantly increased when actin was disrupted following SV trapping (n = 15–22; P < 0.01).

Inhibition of actin polymerization while SVs are trapped blocks recovery of synaptic transmission

We hypothesized that if actin and cholesterol were acting together to cluster SV proteins, inhibition of actin polymerization would have a similar effect to vesicular cholesterol extraction on synaptic transmission. We found in shi controls that stimulation (10 Hz for 12 min) at 30°C resulted in a progressive impairment of synaptic transmission that recovered after restoration of the permissive temperature (Fig. 5C and D). We next determined the effects of inhibition of actin polymerization by applying the same protocol with 10 μm LatA added for 5 min at 30°C following SV trapping (Fig. 5A). In these preparations, synaptic transmission did not recover (Fig. 5C and D). The inability to recover was reminiscent of the impairment that we previously observed following vesicular cholesterol extraction (Dason et al. 2010).

Figure 5.

Figure 5

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A, schematic diagram of actin disruption in a presynaptic terminal following SV protein trapping. Numbers in diagram indicate the corresponding traces and points in the graph. B, schematic diagram of experimental protocol measuring SV recycling using FM1-43. C, representative traces of EJPs. The segmental nerve was stimulated at 0.05 Hz for 5 min at 22°C, then at 10 Hz for 12 min at 30°C, followed by no stimulation at 30°C for 5 min (with or without 10 μm LatA) and then stimulation at 0.05 Hz for 20 min at 22°C to assess recovery. D, recovery of synaptic transmission was severely impaired following LatA treatment (n = 5; P < 0.01). E, shi mutants were stimulated (90 mm K+ for 10 min) at 30°C, no stimulation with or without 10 μm LatA for 5 min at 30°C, incubated with 10 μm FM1-43 for 20 min at 22°C (load), then washed in 0 mm Ca2+ HL6 (with 75 μm Advasep-7 for first 2 min) for 10 min to remove extracellular FM1-43 and fluorescence (F) was measured. Then 90 mm K+ saline was reapplied for 10 min to cause unloading and F measured again (unload). F, inhibition of actin polymerization after SV trapping significantly reduced FM1 uptake (n = 7–8; P < 0.01). G, a similar fraction of FM1-43 was released in control and LatA-treated shi presynaptic terminals in response to high K+ stimulation for 10 min, demonstrating that some of the endocytosed membrane is capable of exocytosis (P > 0.05). F was reported with background F subtracted.

To determine the cause of the impaired recovery, we used the lipophilic dye FM1-43 (Betz & Bewick, 1992) to monitor SV cycling. SVs were trapped by stimulation (90 mm K+ saline for 10 min) at 30°C followed by a 5 min incubation with no stimulation in HL6 (at 30°C) in the absence or presence of 10 μm LatA. Preparations were then incubated with FM1-43 for 20 min at 22°C, thoroughly washed and then imaged (Fig. 5B). We found a 63% reduction in FM1-43 uptake in shi preparations when actin was disrupted following SV trapping in comparison to controls (Fig. 5E and F), demonstrating that the impaired synaptic transmission (Fig. 5D) was due to deficient SV recycling. Both preparations treated with LatA and their controls released a similar fraction of FM1-43 (Fig. 5E and G) following a second, unloading stimulus with high K+. Therefore, following LatA treatment, the endocytosed membrane is capable of exocytosis in response to high K+ stimulation.

Discussion

Our study is the first to show that extraction of vesicular cholesterol causes SV proteins to disperse in the plasma membrane during SV recycling in presynaptic terminals. We also found that vesicular cholesterol regulates PIP2 levels and actin clustering during SV recycling, while plasma membrane cholesterol extraction had no effect. Similar to vesicular cholesterol extraction, inhibition of actin polymerization while SVs and their proteins were trapped on the plasma membrane led to the dispersal of SV proteins and the inability to recover synaptic transmission due to impaired endocytosis.

Cholesterol and SV protein sorting

Some studies have shown that SV proteins remain clustered in the plasma membrane following exocytosis (Willig et al. 2006; Opazo et al. 2010). We tested the hypothesis that the high cholesterol content of SVs (Takamori et al. 2006) functions to cluster SV proteins in the plasma membrane during recycling. We found that SV proteins appear clustered in the presynaptic plasma membrane during SV recycling and that these clusters were disrupted following vesicular cholesterol extraction (Fig. 1). Our data demonstrate that vesicular cholesterol is important for clustering SV proteins in the presynaptic plasma membrane during recycling. It is tempting to speculate that these vesicular cholesterol-dependent clusters of SV proteins may persist during SV recycling. This could explain how SV proteins may remain clustered with little intermixing during recycling (Willig et al. 2006; Opazo et al. 2010; but also see Fernández-Alfonso et al. 2006; Wienisch & Klingauf, 2006). However, confocal microscopy does not have sufficient resolution for us to image SV proteins from a single SV and determine whether SV proteins from a single SV remain clustered without intermixing with SV proteins from other SVs. The clusters of SV proteins that we imaged are from numerous SVs. Nevertheless, our data show that these clusters are disrupted following extraction of vesicular cholesterol. A recent study on the axons of cultured hippocampal neurons found that clusters of SV proteins were visible with stimulated emission depletion (STED) imaging but not with confocal microscopy (Opazo et al. 2010). However, we were able to see clusters of SV proteins using confocal microscopy due to the significantly larger size of presynaptic terminals (3–5 μm in diameter) at the Drosophila larval NMJ in comparison with those of cultured hippocampal neurons.

The resolution of confocal microscopy is probably the reason why the amplitudes of changes that we observed were relatively small. The staining pattern for Syt that we observed following vesicular cholesterol extraction is similar to that seen using confocal microscopy in Drosophila stoned mutants, which have defects in Syt retrieval from the plasma membrane during SV recycling (Stimson et al. 2001; Estes et al. 2003). Thus, the changes in staining pattern that we observe, while small, are consistent with the dispersal of SV proteins on the plasma membrane.

Vesicular cholesterol regulates PIP2 actin dynamics

Cholesterol regulates actin dynamics in non-neuronal systems by regulating PIP2 (Kwik et al. 2003; Milosevic et al. 2005). The actin-binding protein, Wiscott-Aldrich syndrome protein (WASP), is activated by PIP2 and polymerizes actin (Symons et al. 1996; Miki et al. 1996). WASP-mediated actin polymerization occurs in cholesterol rafts in non-neuronal cells (Rozelle et al. 2000). Here, we show for the first time that vesicular cholesterol extraction in presynaptic terminals reduces PIP2 levels (Fig. 3C and D) and actin clustering (Fig. 3E and F). The effects we observe were specific to vesicular cholesterol, as plasma membrane cholesterol extraction had no effect on PIP2 levels or actin distribution. Thus, PIP2 levels and actin polymerization during SV recycling are regulated by vesicular cholesterol. While cholesterol has been shown to regulate both PIP2 and actin in non-neuronal systems, our study is the first to show that this occurs in neurons. In addition, we show that both PIP2 and actin are regulated by vesicular cholesterol and not plasma membrane cholesterol.

The vesicular cholesterol-dependent increase in PIP2 levels during SV recycling (Fig. 3C and D) suggests that something on the SV is sensitive to cholesterol and regulates PIP2. Phosphatidylinositol 4-kinase (PI4K) type IIα is associated with SVs (Weidemann et al. 1998; Guo et al. 2003; Takamori et al. 2006) and its activity is regulated by cholesterol (Banerji et al. 2010). PI4KIIα accounts for the majority of PI4K activity in brain extracts (Guo et al. 2003) and is responsible for the synthesis of phosphatidylinositol 4-phosphate (PIP) which is subsequently made into PIP2 by phosphatidylinositol 4-phosphate 5-kinase. Thus, we speculate that the extraction of vesicular cholesterol may reduce the activity of PI4KIIα, which may lead to the reduced levels of PIP2.

Actin and SV recycling

Inhibition of actin polymerization while SV proteins were trapped on the presynaptic plasma membrane resulted in the dispersal of SV proteins in the plasma membrane (Fig. 4), similar to what we observed following vesicular cholesterol extraction (Fig. 1). Actin has been implicated in regulating both SV exocytosis and endocytosis (Kuromi & Kidokoro, 1998; Delgado et al. 2000; Shupliakov et al. 2002; Sankaranarayanan et al. 2003; Richards et al. 2004; Bleckert et al. 2012). However, the effects of inhibiting actin polymerization while SV proteins are trapped on the plasma membrane have not been previously tested. We found that inhibiting actin polymerization at this stage prevented the recovery of synaptic transmission when shi presynaptic terminals were returned to permissive temperatures due to impaired SV endocytosis (Fig. 5). These results are similar to our previous findings following vesicular cholesterol extraction (Dason et al. 2010); endocytosis was blocked at permissive temperature following extraction of cholesterol from trapped SV. Given that vesicular cholesterol regulates actin distribution (Fig. 3E and F), our data suggest that actin and vesicular cholesterol function together to confine SV proteins in the plasma membrane during SV recycling. Actin-based membrane skeletons in developing hippocampal neurons form a diffusion barrier to cluster Na+ channels and ankyrin-G (Nakada et al. 2003). Thus, actin may play a scaffolding role in retaining SV proteins during recycling (Shupliakov et al. 2002; Sankaranarayanan et al. 2003). Actin may also play a role in clearing the active zone by conferring directionality to laterally diffusing patches of SV proteins to the periactive zone (see Haucke et al. 2011).

Our study is the first to show that the distribution of trapped SV proteins in the plasma membrane is altered following vesicular cholesterol extraction. We also found that vesicular cholesterol extraction, but not plasma membrane cholesterol extraction, prevents increases in the levels of PIP2 during SV recycling and causes actin to disperse. Similar to vesicular cholesterol extraction, inhibition of actin polymerization while SVs and their proteins are trapped on the plasma membrane leads to dispersal of SV proteins and the inability to recover synaptic transmission due to impaired endocytosis. Thus, our data demonstrate that the high cholesterol content of SVs regulates actin polymerization and functions to cluster SV proteins on the plasma membrane during SV recycling (Fig. 6). Membrane and SV lipids may function to confine recycling SV proteins to endocytic sites and thereby allow synapses to maintain sustained exocytosis and endocytosis. The failure to confine recycling SV proteins to endocytic sites may explain why endocytosis was blocked at permissive temperature after SV cholesterol had been extracted and why we were unable to rescue those effects by adding cholesterol back (Dason et al. 2010). The effects of MβCD on synaptic transmission and endocytosis were cholesterol-specific as a 1:10 cholesterol–MβCD complex that could not extract cholesterol had no effect (Dason et al. 2010). In this regard SV endocytosis in neurons is different from constitutive or receptor-triggered endocytosis in other secretory systems, such as CHO and HeLa cells, where endocytosis was blocked by membrane cholesterol extraction but could be re-established by addition of cholesterol (extraction of vesicular or granular cholesterol has not been reported in these systems; Rodal et al. 1999; Subtil et al. 1999; Urs et al. 2005).

Figure 6.

Figure 6

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SV proteins are clustered by cholesterol and actin on the plasma membrane during SV recycling. Extraction of vesicular cholesterol by MβCD or actin disruption by LatA causes SV proteins to disperse in the plasma membrane.

Key points

  1. Extraction of cholesterol from synaptic vesicles trapped on the presynaptic plasma membrane causes synaptic vesicle proteins to disperse after exocytosis.

  2. Vesicular cholesterol regulates both presynaptic phosphatidylinositol (4,5)-bisphosphate levels and actin distribution during synaptic vesicle recycling.

  3. Inhibition of actin polymerization results in the dispersal of proteins from trapped synaptic vesicles and impairs synaptic vesicle recycling.

  4. Vesicular cholesterol and actin together confine synaptic vesicle proteins on the presynaptic plasma membrane during synaptic vesicle recycling.

  5. Alteration of membrane or synaptic vesicle lipids might therefore affect the ability of synapses to undergo sustained exocytosis and endocytosis by compromising the recycling of synaptic vesicle proteins.

Acknowledgments

We thank Dr Patrik Verstreken, Dr Hugo Bellen, Dr Aaron DiAntonio, Dr Bryan Stewart and the Developmental Studies Hybridoma Bank for antibodies and fly stocks, Dr Harold Atwood for critically reading this manuscript, and Marianne Hegström-Wojtowicz for helping maintain fly stocks.

Glossary

AZ

active zone

brp

bruchpilot

CI

clustering index

csp

cysteine string protein

EJP

excitatory junction potential

LatA

latrunculin A

MβCD

methyl-β-cyclodextrin

NMJ

neuromuscular junction

PAZ

periactive zone

PCC

Pearson's correlation coefficient

PI4K

phosphatidylinositol 4-kinase

PIP

phosphatidylinositol 4-phosphate

PIP2

phosphatidylinositol (4,5)-bisphosphate

ROI

region of interest

shi

shibire-ts1

SV

synaptic vesicle

Syt

synaptotagmin

vglut

vesicular glutamate transporter

WASP

Wiscott-Aldrich syndrome protein

Additional information

Competing interests

The authors have no competing interests.

Author contributions

J.S.D, A.J.S. and M.P.C. designed the experiments and interpreted the data. J.S.D. and L.M. collected the data. J.S.D. analysed the data. J.S.D drafted the manuscript and A.J.S. and M.P.C. critically revised the draft for intellectual content. All the authors approved the final version of the manuscript.

Funding

This work was supported by a grant from the Canadian Institutes of Health Research (MOP-82827 to M.P.C.).

tjp0592-0621-sd1.pdf (248.3KB, pdf)

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