Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation

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Natalia L. Kononenko1,2 and Volker Haucke1,2,* 1Leibniz Institut fu¨ r Molekulare Pharmakologie (FMP) and Freie Universita¨ t Berlin, Robert-Roessle-Straße 10, 13125 Berlin, Germany 2Charite Universita¨ tsmedizin, NeuroCure Cluster of Excellence, Virchowweg 6, 10117 Berlin, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2014.12.016

The function of the nervous system depends on the exocytotic release of neurotransmitter from synaptic vesicles (SVs). To sustain neurotransmission, SV membranes need to be retrieved, and SVs have to be reformed locally within presynaptic nerve terminals. In spite of more than 40 years of research, the mechanisms underlying presynaptic membrane retrieval and SV recycling remain controversial. Here, we review the current state of knowledge in the ﬁeld, focusing on the molecular mechanism involved in presynaptic membrane retrieval and SV reformation. We discuss the challenges associated with studying these pathways and present perspectives for future research.

Introduction biochemical studies that had identified SVs as store for non- Communication within the nervous system relies on the release peptide neurotransmitters (Whittaker, 1969). Evidence for SV of neurotransmitter by the calcium-triggered fusion of synaptic recycling and their endocytic origin was gathered by morpholog- vesicles (SVs) with the presynaptic plasma membrane at the ical studies based on the uptake of extracellular ‘‘tracers’’ such nerve terminal. The number of vesicles available for fusion and as horseradish peroxidase (HRP) into the SV lumen (Figure 1B) their propensity to undergo exocytosis define the efficacy of (Holtzman et al., 1971) and by electron microscopic analyses neurotransmitter release and fine-tune neuronal function. Given of stimulated nerve-muscle preparations (Heuser and Reese, the distance of the presynaptic terminal from the neuronal cell 1973; Heuser et al., 1979; Miller and Heuser, 1984)(Figures body the replacement of fused SVs by de novo synthesis and 1C–1E). Heuser and Reese (1973) used sustained electrical stim- axonal transport would be far too slow (on the timescale of hours ulation of frog neuromuscular junctions, which resulted in the to days) to sustain neuronal activity. In addition, the continuous depletion of SVs and the appearance of cisternal structures fusion of SVs with the plasma membrane in the absence of labeled with HRP (Figures 1C and 1D) as well as HRP-positive compensatory membrane retrieval would cause a detrimental in- clathrin-coated vesicles and coated endocytic intermediates crease in the area of the presynaptic plasma membrane, loss of in a region located away from the active zone (AZ) (Figure 1E). membrane tension, and the misalignment with postsynaptic These transient structures were apparently converted into structures. Hence, neurons have evolved mechanisms to main- functional SVs if synapses were allowed to rest, suggesting tain presynaptic membrane homeostasis by recycling SV mem- that SV recycling largely occurs by clathrin-mediated endocy- branes locally within their nerve terminals. This specific property tosis (CME), possibly involving endosome-like vacuolar interme- of SVs to undergo a local trafficking cycle has drawn the atten- diates (Heuser and Reese, 1973; Miller and Heuser, 1984). tion of neuroscientists for more than 40 years, but in spite of its Using the same preparation and technique, but a different importance for synaptic transmission, the precise mechanisms stimulation paradigm, Ceccarelli et al. (1972, 1973) observed by which exocytosed SV membranes are recycled and SVs are membrane intermediates devoid of clathrin coats directly at reformed remain enigmatic. the AZ (Figure 1D). As the intermediates corresponded in size to single SVs, these observations gave rise to the notion that Early Studies on SV Recycling SVs could fuse and recycle via a transient fusion pore, while Pioneering work in the 1950s and 1960s established the quantal preserving their identity via a mechanism later on referred to by nature of neurotransmitter release (del Castillo and Katz, 1954; Meldolesi and colleagues as ‘‘kiss and run’’ (Fesce et al., 1994). Katz and Miledi, 1967) and its relationship to the presence of small SVs, hypothesized to be the physical correlate of neuro- Monitoring SV Recycling—Tools, Models, and transmitter quanta (Figure 1A) (De Robertis, 1959; De Robertis Discrepancies and Bennett, 1955). Stunning morphological observations by Since these orginal observations by Heuser and Reese (1973) Heuser and Reese (1973) and Heuser et al. (1979) provided direct and Ceccarelli et al. (1972), we have witnessed a revolution in experimental support for this idea. By relating the amplitude and the techniques used to monitor exo-endocytosis as well as in the quantal content of the endplate potential to the number of ve- the molecular analysis of the components involved in SV recy- sicular openings measured by rapid ‘‘freeze-slam’’ electron mi- cling (Dittman and Ryan, 2009; Jahn and Fasshauer, 2012; Riz- croscopy (EM) they concluded that a single SV indeed contains zoli, 2014; Saheki and De Camilli, 2012). Optical probes for a quantum of neurotransmitter (Heuser and Reese, 1973; Heuser the real-time visualization of membrane recycling at synapses et al., 1979; Miller and Heuser, 1984), a postulate corroborating have been developed (Kavalali and Jorgensen, 2014), which

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Figure 1. Electron Microscopy of Recycling SV Membranes (A) Electron micrograph of a large axonal process (PRSN) of the earthworm, containing numerous synaptic vesicles (SVs). PSN, portion of another axon; ER, endoplasmic reticulum; Y, a specialized synaptic contact. Reproduced with permission from De Robertis and Bennett (1955). (B) SV recycling at the lobster neuromuscular junction, by exposing the synapse to HRP during stimulation for three periods of 10 min each, with 16,000 stimuli in total. HRP is present within many small vesicles in an axon ending, indicated by ar- rows. Scale bar represents 0.5 mm. Reproduced with permission from Holtzman et al. (1971). (C) An example of HRP retained within bulk structures (cisternae, C) at the frog neuromuscular junction stimulated constantly for 15 min and ﬁxed immediately after. Reproduced with permission from Heuser and Reese (1973). (D) Original drawings by Heuser and Reese (1973), illustrating a progressive depletion of synaptic vesicles (open circles) and appearance ofcis- ternae (ﬁlled in black) in frog nerve terminal stimulated at 10 Hz for 1 and 15 min. The numbers indicate the area per section of synaptic vesicle membrane + cisternal membrane + surface membrane = total membrane. Reproduced with permission from Heuser and Reese (1973). (E) Clathrin-coated pits and clathrin-coated vesicles observed in the vicinity of the plasma membrane at a frog nerve terminal stimulated at 10 Hz for 1 min. Reproduced with permission from Heuser and Reese (1973). (F) Uncoated U-shaped intermediates at the active zone of frog nerve terminals stimulated at 2 Hz for 2 hr. Reproduced with permission from Ceccarelli et al. (1972).

1998). In contrast, analysis of SV recycling by fluorescent antibodies directed against the lumenal domain of synaptotagmin 1 (Syt1) or by pHluorin re- have allowed unprecedented insights into the mechanisms porters have challenged these findings and instead suggest that of membrane retrieval and SV reformation. These include exog- following fusion SV proteins intermix with plasma membrane enously applied fluorescent probes such as the styryl dye components (Ferna´ ndez-Alfonso et al., 2006; Wienisch and Klin- FM1-43 and its relatives, which fluoresce upon partitioning into gauf, 2006), where they may form a reservoir of readily retriev- membranes (Betz et al., 1992), the internalization of fluorescent able SV proteins internalized during subsequent exo-endocytic antibodies directed against lumenal epitopes of SV proteins cycles. Such a readily retrievable SV pool (Hua et al., 2011a) (Matteoli et al., 1992), the use of pH-sensitive GFP (pHluorin) may correspond to clusters of surface-stranded SV proteins fused to the lumenal domain of a SV protein (Miesenbo¨ ck visualized by super-resolution microscopy (Hoopmann et al., et al., 1998), and finally, channelrhodopsin-mediated optoge- 2010; Willig et al., 2006). netic stimulation paired with fast-freezing EM (Watanabe et al., Discrepancies also exist regarding the time course of SV 2013a, 2013b)(Table 1). In spite of this impressive arsenal of endocytosis. The earliest estimates of the kinetics of SV endocy- weapons, the precise mechanisms by which SV membranes tosis, originating from ultrastructural analysis of HRP uptake, re- are retrieved and SVs are reformed have remained partially vealed a slow time course of SV endocytosis on the order of tens elusive. For instance, monitoring exo-endocytosis of SVs loaded of seconds (Heuser and Reese, 1973). In contrast, capacitance with FM1-43 has revealed a partial retention of fluorescence measurements in retinal bipolar cells (von Gersdorff and Mat- upon action potential (AP)-triggered exocytosis in at least a frac- thews, 1994) and quantum dot imaging of single SVs in hippo- tion of events. As kiss-and-run is thought to involve only transient campal neurons in culture (Zhang et al., 2009) indicated that membrane fusion, such partial FM dye retention within the lumen the newly exocytosed membrane is recovered within 0.5–2 s. of SVs suggests that some SVs may recycle via a kiss-and-run Pulse-chase experiments using FM dyes and measurements mechanism (Aravanis et al., 2003) and thereby preserve their of SV protein internalization and reacidification by pHluorins identity during exo-endocytic cycling (Murthy and Stevens, revealed time constants of 10–40 s (Armbruster and Ryan,

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Neuron Method Example References Measured Parameter Information Obtained Pros Cons Capacitance Neher and Marty, 1982; membrane exo-/endocytosed exquisite temporal blind to the nature of the von Gersdorff and capacitance/area membrane resolution internalized membrane and 85 Matthews, 1994; its content eray4 2015 4, February , Sun et al., 2002 use is limited to large presynaptic terminals (i.e., calyx of Held) Fluorescent styryl dyes Betz et al., 1992; Ryan changes in ﬂuorescent membrane turnover exogenously added inherently ‘‘noisy’’ (FM1-43, FM4-64) et al., 1993; Aravanis intensity upon ﬂuorescent probes poor temporal resolution et al., 2003 binding to membranes recycling monitored live

ª no need for genetic 05Esve Inc. Elsevier 2015 manipulation or transfection Fluorescent antibodies Matteoli et al., 1992; fluorescent intensity of recycling of selected exogenously added monitors localization of a against SV proteins Kraszewski et al., antibodies SV protein fluorescent probes single SV protein lumenal epitopes 1995; Wienisch and recycling can be poor time resolution Klingauf, 2006 monitored live error prone as internalized, no need for genetic and surface bound fractions manipulation or may be difficult to discern transfection pH reporters (pHluorin, Miesenbo¨ ck et al., 1998; pH dependence of pH environment of recycling can be monitored requires genetic CypHer, Quantum dots) Ferna´ ndez-Alfonso et al., fluorescence exo-/endocytosed in live preparation manipulation/transfection 2006; Zhang et al., 2009; SV proteins (for pHluorin) Hua et al., 2011a indirect monitoring of SV recycling as prerequisite for acidification Super-resolution Willig et al., 2006; localization of fluorescent spatial organization exquisite spatial resolution bleaching imaging Hoopmann et al., 2010; molecules of SV proteins limited temporal resolution Wilhelm et al., localization precision and 2014 spatial resolution rely on fluorophore property EM Ceccarelli et al., 1972; membrane morphology size, number, and exquisite spatial resolution poor temporal resolution Heuser and Reese, 1973; morphology of time consuming Koenig and Ikeda, 1996 endocytic intermediates requires careful tissue preservation to avoid artifacts Fluid phase markers Clayton and Cousin, 2009 morphology of plasma- size, number, and exquisite spatial resolution poor temporal resolution (HRP, dextrans) membrane- morphology of time consuming

and EM derived intermediates endocytic intermediates requires careful tissue Review preservation to avoid artifacts Optogenetics and Watanabe et al., 2013a, membrane morphology size, number, and exquisite temporal time consuming high-pressure 2013b, 2014) morphology of endocytic and spatial resolution requires careful tissue freeze EM intermediates preservation to avoid Neuron artifacts EM, electron microscopy; HRP, horseradish peroxidase; SV, synaptic vesicles. Neuron Review

2011; Granseth et al., 2006; Ryan et al., 1993; Ryan and Smith, been inferred from electron microscopic observations of ribbon 1995; Wu and Betz, 1996). Finally, recent data in Caenorhabditis synapses from temperature-sensitive dynamin shibire mutants elegans and mammalian hippocampal neurons in culture using in Drosophila (Koenig and Ikeda, 1996) and from pHluorin-meas- single optogenetic stimuli paired with fast high-pressure freezing urments of single-vesicle fusion and endocytosis events in EM suggest an ultrafast pathway of endocytosis with a time con- cultured neurons (Gandhi and Stevens, 2003; Zhu et al., 2009) stant of 20–50 ms (Watanabe et al., 2013a, 2013b). (but see Balaji and Ryan, 2007 for a different view). Thus, SV re- What are possible reasons for these differences? An important cycling likely involves more than one pathway. point to consider is the fact that many of the approaches described above monitor distinct parameters that often serve SV Recycling Involves Membrane Retrieval and SV as surrogate measures of SV recycling (see Table 1). Capaci- Reformation tance recordings are a fast and precise means to monitor exo- If, indeed, several pathways of SV recycling at the synapse exist, endocytic membrane turnover (Lindau and Neher, 1988; Neher what are the mechanisms underlying them and what are the and Marty, 1982), but are blind to the nature of the internalized components involved? As synapses differ greatly with respect membrane and its properties (i.e., whether or not SVs are to their firing pattern and the size of the SV reserve pool, it is reformed). Moreover, capacitance measurements are limited to conceivable that synapse-specific adaptations exist that may the study of large synapses, which arguably may differ from be tailored to their needs with respect to the retrieval of newly small central synapses in the mammalian brain. Similar concerns exocytosed membrane and the reformation of functional SVs with respect to the nature of the cycling membrane hold true for to replenish their releasable vesicle pool. For example, high-fre- the use of FM dyes as reporters for presynaptic membrane turn- quency firing synapses such as the calyx of Held (firing at hun- over. However, when combined with photoconversion and EM dreds of Hz and capitalizing on a large reserve pool of SVs) (or super-resolution microscopy in case of the newly developed may depend on efficient modes to retrieve newly exocytosed membrane-binding fluorophore mCLING) (Revelo et al., 2014) SV membrane from the active zone (AZ) area to clear release these probes allow the visualization and tracing of the internal- sites and make them available for the next round of fusion ized membrane on a timescale of seconds. PHluorins enable (Haucke et al., 2011; Neher, 2010), while the reserve pool of the monitoring of the activity-induced recycling of a specific SV SVs may be refilled at a much slower rate. Recent data indicate protein, but only do so indirectly as their fluorescence reports that such release site clearance depends on components of the on the pH environment, not on exo- or endocytosis per se. endocytic machinery such as intersectin 1 (Hosoi et al., 2009; Hence, kinetic analyses of pHluorin measurements to assay SV Sakaba et al., 2013). On the other hand, all synapses need to endocytosis may have to be interpreted with caution as fast replenish their SV pool by regenerating new SVs of proper size modes of endocytosis could well be missed if they operate on and shape, a complex process that requires high-fidelity sorting a timescale faster than the time course of acidification (1–4 s) of SV protein components that is unlikely to be accomplished on (Atluri and Ryan, 2006; Budzinski et al., 2011). a timescale of milliseconds. Another potential source of the observed variability in mem- Thus, SV recycling involves both (1) retrieval of SV compo- brane retrieval kinetics is the stimulus intensity used to trigger nents from the plasma membrane, and (2) the regeneration of SV exocytosis. Data from the calyx of Held suggest that rapid functional SVs of proper composition (Takamori et al., 2006). endocytosis is activated by calcium influx and is the dominant mechanism of SV recycling during intense nerve activity (Wu Retrieval of SV Membranes et al., 2005). Fast endocytosis during excessive stimulation has At least four different pathways for the retrieval of SV membranes also been observed in other model systems (de Lange et al., have been put forward: (1) ‘‘kiss-and-run’’ endocytosis, (2) ultra- 2003; Klingauf et al., 1998; Richards et al., 2000). On the other fast endocytosis, (3) CME, and (4) bulk endocytosis, which differ hand, studies in cultured hippocampal neurons have suggested with respect to speed, maintenance of SV identity, and likely that the time course of endocytosis slows down in parallel with molecular components, although our knowledge about the latter the stimulation intensity and the amount of exocytosis (Armbrus- remains limited for some of these routes. ter et al., 2013; Ferna´ ndez-Alfonso and Ryan, 2004). One possi- Kiss and Run bility to explain these findings is that several mechanisms for Several studies have reported a fast form of membrane retrieval endocytic membrane retrieval with different kinetics operate at the synapse (Klingauf et al., 1998; Murthy and Stevens, 1998; within the same nerve terminal and their use may depend on cal- Pyle et al., 2000; Stevens and Williams, 2000), which has been as- cium levels, stimulation frequency, exocytotic load, and/or even signed to a kiss-and-run mechanism first suggested by Ceccar- temperature (Watanabe et al., 2014). Evidence for the coexis- elli et al. (1972, 1973) based on electron micrographs of uncoated tence of several SV recycling pathways within a single synaptic vesicular openings. Kiss-and-run exo-endocytosis is thought to terminal has been gathered from electrophysiological measure- occur directly at the AZ and involves the transient opening and ments at retinal bipolar neuron synapses (Neves and Lagnado, rapid closure of a fusion pore without collapse of the vesicle, 1999; von Gersdorff and Matthews, 1994). These studies have which therefore maintains its molecular identity bypassing the reported on the coexistence of two kinetically distinguishable need for endocytic sorting of SV proteins or passage through en- mechanisms of endocytosis that are regulated by different cal- dosomal intermediates (Alabi and Tsien, 2013)(Figure 2A). Exper- cium levels (Neves et al., 2001) and involve distinct sets of com- imental data supporting kiss-and-run include the rapid time ponents (Jockusch et al., 2005). The presence of fast and slow course of membrane retrieval in capacitance measurements recycling pathways within a single synaptic terminal has also (He et al., 2006; Xu et al., 2008), high-speed imaging of quantum

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Figure 2. Current Models of SV Membrane Retrieval (A) Kiss-and-run mode. Opening of a fusion pore is sufficient for discharge of SV content. After closure of the pore vesicles detach from the plasma membrane and are directly reused through a fast mechanism. (B) ‘‘Classical’’ CME. Opening of a fusion pore is followed by complete SV fusion with the plasma membrane. SV components are subsequently retrieved directly from the cell surface through the formation of a clathrin-coated vesicle, which following uncoating, reenters the SV reserve pool. This mechanism operates on a timescale of tens of seconds. (C) Ultrafast mode. Fast retrieval of SV membranes after single or more APs occurs by membrane invaginations corresponding to approximately 4 SVs formed laterally to the active zone. SVs may be regenerated primarily from the resulting primary endocytic vesicles or from endosome-like vacuoles (ELVs) through a slow, presumably clathrin- dependent mechanism. (D) Strong tetanic or chemical stimulations cause the appearance of large bulk endocytic structures, which are subsequently resolved into SVs via clathrin-dependent and/or clathrin-independent mechanisms. dots as reporters for single SV collapse and reuptake (Zhang such as dynamins, endophilins, amphiphysin, synaptojanin, or et al., 2009), or the lack of effect of molecular perturbations that auxilin perturbs SV recycling measured by pHluorins or FM dyes affect CME (Jockusch et al., 2005; Neef et al., 2014). and causes the accumulation of various intermediates of CME While the existence of kiss-and-run in neurons remains hotly (Dickman et al., 2005; Ferguson et al., 2007; Gad et al., 2000; debated, compelling evidence in favor of such a pathway has Kasprowicz et al., 2014; Milosevic et al., 2011; Shupliakov been obtained in endocrine cells (Albillos et al., 1997; Ale´ s et al., 1997; Yim et al., 2010). However, as mutation and/or ge- et al., 1999), where secretory granules undergo transient, appar- netic deletion of such proteins failed to result in a complete block ently reversible fusion pore openings and closures (Chiang et al., of SV membrane retrieval, it is conceivable if not likely (as further 2014; Wu et al., 2014a). discussed below) that SV recycling does not exclusively occur by A key unresolved question with respect to kiss-and-run exo- CME. Furthermore, some CME components such as dynamin or endocytosis is how full collapse of the fusing vesicle membrane endophilin are possibly shared with clathrin-independent path- is prevented. In chromaffin cells, such a role may be executed by ways of endocytosis (Fuchs et al., 2014; Jockusch et al., 2005; the GTPase dynamin (Holroyd et al., 2002), which facilitates Llobet et al., 2011; Sandvig et al., 2011; Boucrot et al., 2015). fission of endocytic structure in both CME as well as in cla- Clathrin-coated intermediates have been observed both on the thrin-independent routes of endocytosis. Consistent with a role presynaptic membranes, often lateral to AZs (Gad et al., 2000), for dynamin, infusion of GTPgS or dynamin inhibitors, treatments and on endocytic cisternae (Ferguson et al., 2007; Heuser and that inactivate dynamin among other targets, inhibit fast capac- Reese, 1973; Takei et al., 1996), some of which may be con- itive membrane retrieval at the calyx of Held (Xu et al., 2008) and nected with the plasma membrane (Ferguson et al., 2007; Heuser in rod photoreceptors of salamander retina (Van Hook and Thor- and Reese, 1973; Takei et al., 1996). Clathrin coats are formed by eson, 2012). the assembly of clathrin triskelia comprising three 190 kDa cla- CME thrin heavy chains and 25 kDa clathrin light chains into a poly- CME, defined as the uptake of material into the cell from the hedral lattice (Brodsky et al., 2001). The assembly of the clathrin membrane surface by clathrin-coated vesicles, arguably repre- coat is initiated by early acting endocytic adaptors such as the sents the best characterized pathway for the endocytic entry of assembly protein (AP) complex 2 (AP-2), a heterotetramer of proteins and lipids into eukaryotic cells (McMahon and Boucrot, two large (a, b2) and two small subunits (m2, s2), the Bin-amphi- 2011). A plethora of morphological, biochemical, ultrastructural, physin-rvs (BAR) domain protein FCHo, epsins, and intersectin and genetic studies indicate a key role for CME in SV recycling. among other factors. These serve to link clathrin to the underlying As discussed above, sustained or repetetive stimulation of syn- membrane via their association with phosphoinositides, in partic- apses is seen to correlate with a transient rise in the number of ular with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and clathrin-coated endocytic intermediates. SV proteins are the ma- to coordinate the structural assembly of the coat with the selec- jor cargo of clathrin-coated vesicles (CCVs) isolated from nerve tion of transmembrane cargo proteins (i.e., SV proteins) (Owen terminals (Blondeau et al., 2004; Maycox et al., 1992), et al., 2004). AP-2 targets transmembrane proteins for internali- suggesting that SV proteins at least during some stage of their zation carrying tyrosine- (YxxØ, Ø bulky hydrophobic residue) life cycle pass through CME (Figure 2B). Furthermore, genetic or dileucine-based ([DE]XXXL[LI]) motifs by concentrating them or acute biochemical perturbation of CME-associated factors in the emerging clathrin-coated pit (CCP). Among the known

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SV proteins recognized by AP-2 only VGLUT1 has been shown to PI(4,5)P2-phosphatase synaptojanin 1 (Mani et al., 2007) and contain a dileucine-based ([DE]XXXL[LI]) motif (Voglmaier et al., the clathrin disassembling chaperone Hsc70 and its J-domain 2006). In addition to AP-2, neurons express a variety of other cofactor auxilin (Ungewickell et al., 1995). Recruitment of synap- CME-associated adaptors for SV cargo. These include stonin tojanin 1 to endocytic intermediates requires endophilin (Gad 2, the mammalian ortholog of Drosophila stoned B and et al., 2000; Milosevic et al., 2011; Verstreken et al., 2002), which C. elegans unc41, which binds to and sorts the SV calcium directly binds to the proline-rich domain of synaptojanin 1 and sensor synaptotagmin 1 (Diril et al., 2006; Fergestad and Broadie, activates its enzymatic activity in a curvature-dependent manner 2001; Kononenko et al., 2013; Maritzen et al., 2010; Mullen et al., (Chang-Ileto et al., 2011). Inactivation of the synaptojanin 1 gene

2012), AP180 and its ubiquitous paralog CALM (clathrin assem- in mice leads to increased PI(4,5)P2 levels, accumulation of bly lymphoid myeloid protein), specific sorting adaptors for syn- CCVs, and to severe neurological abnormalities such as ataxia aptobrevin/VAMP 2 (Koo et al., 2011; Zhang et al., 1998), an (Cremona et al., 1999). Strikingly similar phenotypes are seen essential component of neuronal soluble NSF attachment protein in KO mice lacking auxilin (Yim et al., 2010), or endophilin A1-3 receptor (SNARE) complexes that mediate SV exocytosis. Ge- (Milosevic et al., 2011), in line with the close functional interaction netic inactivation of stonins in mice, worms, or fruit flies results between these proteins. in missorting of synaptotagmin 1 to the neuronal surface and/or Live-imaging experiments in nonneuronal cells expressing its targeting for degradation, suggesting that SV proteins indeed GFP-tagged clathrin coat components revealed that the entire have to pass through CME for correct sorting to SVs (Diril et al., process of CME (coating, fission, and uncoating) operates on a 2006; Fergestad and Broadie, 2001; Kononenko et al., 2013; timescale of 20–60 s (McMahon and Boucrot, 2011). While it is Maritzen et al., 2010; Mullen et al., 2012). Surprisingly, while the conceivable that CME is faster in neurons due to the high local fidelity of synaptotagmin 1 sorting is compromised in stonin 2 concentration of CME components at synapses, it seems un- knockout (KO) mice, synaptotagmin 1-pHluorin retrieval kinetics likely that CME is able to retrieve SV membranes on a timescale are accelerated (Kononenko et al., 2013). Thus, at least in the of tens of milliseconds as observed in some systems (Sun et al., case of synaptotagmin endocytic membrane retrieval and sorting 2002; Watanabe et al., 2013a, 2013b). to SVs appear to be partially independent processes. While the above data support a key role for clathrin and CME Early shallow CCPs undergo maturation during which they in SV recycling, recent genetic data in a variety of models progressively invaginate in a poorly understood process that argue that clathrin may be largely dispensable for SV likely involves BAR domain proteins such as SNX9/18, amphi- membrane retrieval. While acute inactivation of clathrin by fluo- physin, and endophilin and possibly others (Daumke et al., rophore-assisted light inactivation (FALI) in Drosophila 2014; Frost et al., 2009). BAR domain proteins often also harbor melanogaster caused dramatic defects in neurotransmission SH3 domains that bind to and recruit the mechanochemical and resulted in the accumulation of large internal vacuoles, en- GTPase dynamin, which catalyzes membrane scission of late docytic membrane uptake following evoked SV exocytosis pro- stage U-shaped CCPs. Data from nonneuronal cells (Meinecke ceeded unperturbed (Heerssen et al., 2008; Kasprowicz et al., et al., 2013), mouse neurons (Di Paolo et al., 2002; Milosevic 2008). Likewise, RNAi-mediated ablation of clathrin expression et al., 2011), and neurons in C. elegans (Bai et al., 2010) suggest in C. elegans had surprisingly mild effects on neurotransmission that endophilin, amphiphysin, and SNX9/18 serve partially over- and resulted in SVs that were reduced in size but normal in num- lapping roles in the recruitment of dynamin to CCPs. Oligomeric ber (Sato et al., 2009), while CME of other cargo was inhibited. In assembly of dynamin into helical rings around the narrow neck primary hippocampal neurons, depletion of clathrin by lentiviral of U-shaped CCPs facilitates GTP hydrolysis and membrane RNAi led to moderately reduced rates of pHluorin retrieval in scission. The actual mechanism by which dynamin drives this re- response to sustained low-frequency stimulation, but had no action is debated and may involve membrane constriction, as effect on membrane retrieval induced by higher frequency stimuli well as insertion of dynamin’s pleckstrin homology domain (i.e., 20 or 40 Hz). Similar results were obtained in neurons lack- into the underlying membrane, resulting in the generation of ing AP-2(m), an essential factor in CME and major recruiter of free clathrin-coated vesicles. Mammals express three dynamin clathrin, due to conditional genetic inactivation (Kononenko isoforms (Daumke et al., 2014; Ferguson and De Camilli, 2012; et al., 2014), consistent with earlier data using RNAi (Kim and McMahon and Boucrot, 2011) of which dynamin 1 and 3 are ex- Ryan, 2009). These data indicate that clathrin/AP-2, and there- pressed highest in brain, while dynamin 2 is found ubiquitously. fore CME, may be partly dispensable for SV membrane retrieval Ablation of dynamin 1, the major brain isoform, results in the pre- and further suggest that synapses in addition to CME capitalize synaptic accumulation of branched, tubular plasma membrane on clathrin-independent mechanisms of membrane retrieval invaginations capped by CCPs and in severely impaired SV such as ultrafast or bulk endocytosis. endocytosis during high-frequency stimulation (Ferguson et al., Ultrafast Endocytosis 2007), a phenotype further aggravated in dynamin 1/3 double Elegant recent experiments in C. elegans and mouse hippocam- knockout (KO) mice (Raimondi et al., 2011). However, it appears pal neurons capitalizing on single optogenetic stimuli paired with that even the extremely low levels of dynamin 2 are sufficient to rapid high pressure fast-freezing EM have suggested the exis- support basic neuronal function (Raimondi et al., 2011), low-level tence of an ultrafast form of endocytosis (Figure 2C) (Watanabe synaptic transmission and SV reformation in the absence of dy- et al., 2013a, 2013b). During ultrafast endocytosis, docked SVs namins 1 and 3 (Wu et al., 2014c). apparently fully collapse into the plasma membrane and are Concomitant with or shortly after fission, the released endo- then retrieved via endocytic invaginations of 80 nm diameter cytic vesicles shed their coat by the coordinated action of the that appear within 50 to 100 ms at sites flanking the AZ. As the

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surface area of these endocytic invaginations, observed at sites Elegant electron microscopic studies of stimulated synapto- distinct from those of fusion, was equivalent to roughly four SVs somes have shown that budding events on internalized endo- this pathway does not correspond to kiss-and-run endocytosis. some-like vacuoles might involve clathrin (Takei et al., 1996). Moreover, its rapid timescale and the lack of apparent clathrin This could imply a role of CME in resolving bulk endosomes coat structures at the EM level distinguish it from CME. Consis- into SVs. Indeed, several lines of evidence suggest a role for tent with these morphological observations recent data show CME in SV regeneration from bulk endosomes (Heerssen et al., that ultrafast membrane internalization proceeds unaffected in 2008; Kasprowicz et al., 2014; Kononenko et al., 2014; Wata- the near absence of clathrin (Watanabe et al., 2014). Hence, ul- nabe et al., 2014), although this may not be the only pathway trafast endocytosis may be a specialized mechanism for neurons (Wu et al., 2014b) (further discussed below). to rapidly restore the surface area of the presynaptic membrane. Several other aspects of bulk endocytosis, such as the role of Ultrafast endocytosis can explain previous reports of rapid dynamin, remain also under debate. Mutation of the two key endocytic membrane retrieval in hippocampal neurons (Klingauf phosphorylation sites on dynamin 1 was reported to block bulk et al., 1998; Murthy and Stevens, 1998; Pyle et al., 2000; Stevens endocytosis (Clayton and Cousin, 2009), while in another study, and Williams, 2000) and in several other model systems. For bulk endocytosis proceeded in nerve terminals from dynamin 1/3 example, endocytic invaginations similar to those reported by double knockout mice (Wu et al., 2014c), possibly due to the Watanabe et al. (2013a, 2013b) have been observed at frog presence of low levels of dynamin 2. Moreover, acute inactiva- neuromuscular junctions (Miller and Heuser, 1984) and capaci- tion of dynamin in Drosophila (Kasprowicz et al., 2014) led tance measurements have revealed rapid SV membrane retrieval to the formation of large cisternae that remained connected in goldfish retinal bipolar cells (Neves et al., 2001; von Gersdorff with the plasma membrane, suggesting that dynamin activity is and Matthews, 1994) and at the calyx of Held (Sun et al., 2002). required for fission but not formation of bulk endosomes. A The molecular mechanisms underlying ultrafast endocytosis role for dynamin in bulk endocytosis is also supported by genetic at this point remain largely unexplored. Pharmacological pertur- studies where inactivation of the dynamin-associated BAR bations using inhibitors of actin polymerization and of dynamin domain protein syndapin leads to defects in bulk endocytosis GTPase activity suggest a role for both actin and dynamin in due to impaired dynamin recruitment resulting in excitatory/ the formation and resolution of ultrafast endocytic intermediates inhibitory imbalance (Koch et al., 2011). Additional studies are (Watanabe et al., 2013b), although other, more specific ap- required to elucidate the precise mechanism of bulk endocytosis proaches are required to confirm this. One of the most puzzling and its relationship to ultrafast endocytosis described above. aspects of this pathway is the extremely fast kinetics of membrane fission. In vitro studies on dynamin have shown that SV Reformation membrane fission requires seconds or even tens of seconds to Important features of SVs are their morphological uniformity and complete, depending on membrane elastic properties and the precise protein composition, which are efficiently maintained concentration of GTP (Morlot et al., 2012). While these time over repetitive rounds of exo-/endocytosis to maintain their scales are grossly consistent with the kinetics of CME in non- fusion competence. A single SV contains 600 transmembrane neuronal cells (McMahon and Boucrot, 2011), they are much domains, roughly representing a quarter of the vesicle surface slower than the time course of ultrafast endocytosis (Watanabe (Takamori et al., 2006). Hence, sorting of SV proteins, many of et al., 2013b). It thus seems likely that ultrafast membrane which (e.g., the vATPase) are present in just a few copies (Taka- retrieval besides dynamin may require additional factors, most mori et al., 2006), is a major task for the presynaptic endocytic notably actin (Watanabe et al., 2013b), BAR domain proteins machinery (Wilhelm et al., 2014). Moreover, if SVs lose their iden- such as endophilin (Kononenko et al., 2014; Llobet et al., tity over multiple rounds of exo-endocytosis, as current data 2011), or membrane lipids. Furthermore, it appears that ultrafast suggest (Ferna´ ndez-Alfonso et al., 2006; Wienisch and Klingauf, membrane retrieval at the lateral edges of the AZ is coupled 2006), specific mechanisms must exist to orchestrate the fidelity to changes in membrane tension induced by vesicle fusion. of SV protein sorting and to ensure SV uniformity. Although the How such relaxation of membrane tension then couples to the precise pathway(s) responsible for SV re-generation remain(s) endocytic machinery remains one of the challenges for future to be discovered, there is a general consensus that clathrin studies. and CME-associated adaptors play a central role in this (Heers- Bulk Endocytosis sen et al., 2008; Heuser and Reese, 1973; Maycox et al., 1992; Finally, SV membranes can also be retrieved by bulk endocytosis Shupliakov et al., 1997; Verstreken et al., 2002), in addition to involving large infoldings of the plasma membrane distal to the clathrin and AP-2, which may sort VGLUT1 via recognition of AZ in response to very strong stimulation, i.e., chemical stimula- its [DE]XXXL[LI] motif (Voglmaier et al., 2006) (see CME). tion or exocytotic bursts of hundreds of APs (Figure 2D) (Cheung Several studies have indicated that disruption of clathrin or and Cousin, 2013; Clayton and Cousin, 2009; Koenig and Ikeda, CME-associated adaptors leads to defects in sorting SV cargoes 1996; Richards et al., 2000). Bulk endocytosis leads to the gen- and to alterations in SV size (Kononenko et al., 2013; Koo et al., eration of large endocytic vacuoles (Holt et al., 2003; Miller and 2011; Petralia et al., 2013; Zhang et al., 1998). Furthermore, Heuser, 1984; Paillart et al., 2003) that are subsequently con- CCVs isolated from nerve terminals are highly enriched in SV verted into SVs via mechanisms, which remain elusive (see SV proteins (Blondeau et al., 2004; Maycox et al., 1992). Recent regeneration). How bulk endocytosis and the generation of data showed that depletion of clathrin by lentiviral short hairpin endosome-like vacuoles are related to ultrafast endocytosis is RNA (shRNA) in primary neurons (Watanabe et al., 2014)orKO unclear. of AP-2(m) in neurons or in cortical neurons in vivo (Kononenko

490 Neuron 85, February 4, 2015 ª2015 Elsevier Inc. Neuron Review

et al., 2014) resulted in the accumulation of postfission endo- SV proteins to distinct vesicle populations within a single syn- some-like vacuoles and a corresponding depletion of properly apse (Raingo et al., 2012; Ramirez et al., 2012). shaped SV, while in contrast, SV membrane retrieval was largely unaffected (see CME) (Kononenko et al., 2014). These data are An Integrative Hypothetical Model of SV Recycling most consistent with a model whereby SVs are reformed primar- It appears that synapses capitalize on much of the endocytic and ily via clathrin/AP-2-mediated vesicle budding either directly endosomal trafficking machineries known from other cell types from the plasma membrane or via endosome-like vacuolar to maintain presynaptic membrane homeostasis and to re- intermediates generated by clathrin-independent membrane plenish SV pools (Wilhelm et al., 2014). But what determines retrieval (i.e., ultrafast or bulk endocytosis). In line with this the pathway of choice at a particular synapse or following a model, studies of synaptosomal preparations have shown that certain stimulus? We hypothesize that at least to some degree the generation of clathrin coats on endosome-like vacuoles this ‘‘choice’’ may be an inherent property of the presynaptic ma- might occur via mechanisms and components similar to those chinery for SV recycling and the availability of factors that are mediating CME-budding from the plasma membrane (Takei rate-limiting for membrane retrieval and SV reformation. et al., 1996). As PI(4,5)P2 plays an obligatory role of in the assem- SV recycling fulfills at least two crucially important tasks. First, bly of clathrin/AP-2 coats (Di Paolo and De Camilli, 2006), it is compensatory endocytosis removes fused SV membrane from likely that endosome-like vacuoles derived from the plasma the AZ to restore membrane tension and to clear release sites membrane preserve their plasma membrane identity to allow that need to become available for subsequent rounds of SV for clathrin/AP-2 to assemble (McMahon and Boucrot, 2011). exocytosis (Hosoi et al., 2009). Based on theoretical consider- However, in spite of the prominent phenotypes resulting from ations, the rate of sustained neuroexocytosis may be limited by interference with clathrin function, recent data using chemical the availability of specific sites to which vesicles can dock, rather stimulation indicate that synapses, in addition to CME, are than the availability of SVs (Neher, 2010). As exocytosis is rapid, equipped with other clathrin-independent pathways of SV occurring on a millisecond timescale, compensatory membrane reformation that may operate preferentially under conditions retrieval may well be the primary task for the endocytic machin- of high sustained activity (Wu et al., 2014c). Evidence from ery at many types of synapses. Second, to sustain exocytosis neuroendocrine cells (Blumstein et al., 2001), cultured neurons over long periods of activity synapses need to replenish their (Cheung and Cousin, 2012), and genetic studies in mice (Na- releasable SV pool by reforming SVs from internalized mem- katsu et al., 2004) suggests that one such compensatory branes. This task is inherently more complex as SVs are homog- pathway for SV reformation involves SV budding from endo- enous in size and display a defined protein and lipid composition somes via assembly protein 3 (AP-3) coats. AP-3 is an d, that can only be maintained by high fidelity adaptor-mediated b3A/B, m3A/B, s3 heterotetramer similar to AP-2, that may sorting processes that serve to ‘‘proofread’’ SV composition. function independently of clathrin and associates with the late This task may be further complicated by the existence of func- endosomal BLOC-1 complex (Nakatsu et al., 2004; Newell- tionally distinct pools of vesicles that may display compositional Litwa et al., 2010) and is found in all cells, although its b3B heterogeneity (Hua et al., 2011b; Raingo et al., 2012; Ramirez and m3B isoforms are exclusively expressed in neurons (Na- et al., 2012). However, CME as well as endosomal pathways of katsu et al., 2004; Newell-Litwa et al., 2010). Mutations in AP- vesicle budding employed to reform functional SVs operate on 3d in Drosophila garnet mutants (Ooi et al., 1997) and in mocha a timescale of tens of seconds and, thus, provide a potential mice cause pigmentation defects due to impaired biogenesis of kinetic bottleneck when used for compensatory membrane lysosome-related pigment granules (Kantheti et al., 1998), in retrieval at synapses undergoing high rates of firing. We hypoth- line with the notion that AP-3 sorts transmembrane cargo to ly- esize, therefore, that neurons are equipped with mechanisms sosomes. AP-3 has also been implicated in SV recycling that allow them to plastically segregate SV membrane retrieval (Cheung and Cousin, 2012; Voglmaier et al., 2006) and the from SV reformation depending on their activity level (Figure 3). neuronal form of AP3 is able to produce SVs from endosomes In this model, clathrin/AP-2-mediated vesicle budding serves in vitro (Blumstein et al., 2001). Mocha mice display subtle as the major, although perhaps not the only pathway to regen- neurological deficits, including balance and hearing problems, erate SVs of proper size and composition (Wu et al., 2014c). Cla- but show a normal SV pool size, suggesting that the AP-3 thrin/AP-2-coated vesicles according to this model either bud pathway is not essential for SV reformation in vivo, although it from the plasma membrane proper (i.e., local recycling via ‘‘con- may be of importance in subsets of neurons (i.e., inhibitory neu- ventional CME’’) or from plasma membrane-derived endosome- rons) (Nakatsu et al., 2004) and/or under conditions of sus- like vacuoles (Hoopmann et al., 2010; Kononenko et al., 2014; tained high level activity when bulk endocytosis is activated Takei et al., 1996; Watanabe et al., 2014), internalized by cla- (Cheung and Cousin, 2012). The role of AP-3 in SV reformation thrin-independent dynamin-mediated retrieval of previously from endosomes may partially overlap with that of AP-1, exocytosed SV membranes. This hypothesis is consistent with another adaptor complex that operates together with clathrin observations that depletion of clathrin as well as downregulation in vesicular traffic to and from endosomes (Glyvuk et al., or KO of AP-2(m)(Kim and Ryan, 2009; Kononenko et al., 2014) 2010). A regulatory role for endosomes in SV reformation is only moderately affect the kinetics of membrane retrieval in supported by the observation that mutations in Rab5 impair response to low-frequency but not high-frequency stimulation, neurotransmission at Drosophila neuromuscular junctions (Wu- yet, dramatically impair SV replenishment. Whether or not the cherpfennig et al., 2003). Moreover, endosomal processing clathrin/AP-2 machinery operates directly at the plasma mem- (i.e., via AP-3; Hua et al., 2011b) may contribute to sorting of brane as in conventional CME in nonneuronal cells or mediates

Neuron 85, February 4, 2015 ª2015 Elsevier Inc. 491 Neuron Review

Figure 3. Hypothetical Model for SV Membrane Retrieval and Reformation Left: high activity firing induces a steep rise in presynaptic calcium levels and triggers fast membrane retrieval via endosome-like vacuoles (ELVs) upstream of clathrin coat assembly. ELVs are subsequently converted into SVs via clathrin/ AP-2-mediated budding and possibly additional pathways (i.e., via AP-3). Right: under conditions of low-frequency activity presynaptic calcium levels remain comparably low and membrane fission is rate limiting. This allows clathrin/AP-2 coats to assemble prior to membrane fission, resulting in SV reformation directly from the plasma membrane.

and fast pathways of SV recycling may represent the extreme ends of a spec- trum that are distinguished by their time constant(s) of membrane fission and their regulation by synaptic activity. Finally, this hypothetical model can explain why select SV proteins accumulate on the neuronal plasma membrane and/or are subject to degradation (Koo et al., 2011; SV reformation from endosome-like vacuoles depends on the ef- Zhang et al., 1998), while the kinetics of their retrieval from the ficiency of membrane fission. In line with the important regulatory plasma membrane proceeds nearly unaltered (Kononenko role of Ca2+/calcineurin (Wu et al., 2014b) and its function in dy- et al., 2013). namin 1 dephosphorylation (Armbruster et al., 2013), we hypoth- It seems likely that additional factors other than calcium esize that membrane fission is highly regulated by Ca2+ and contribute to the regulation of fast and slow modes of SV recy- neuronal activity (Figure 3). Such a model is compatible with cling: these include membrane tension, the number and concen- the existence of slow (Armbruster and Ryan, 2011; Ryan et al., tration of SV proteins accumulating on the cell surface, and 1993; Ryan and Smith, 1995; Wu and Betz, 1996) and fast modes possibly their clustering into a readily retrievable pool that could (Armbruster et al., 2013; von Gersdorff and Matthews, 1994; Wa- form a substrate for fast membrane retrieval (Hua et al., 2011a). tanabe et al., 2013b) of SV membrane retrieval observed in a Further experiments are required to support either of these variety of models and can explain how specialized types of neu- possibilities. rons, such as globular bushy cells in the auditory brain stem that synapse via the calyx of Held to MNTB principal neurons can Perspectives transmit signals with rates of up to 800 Hz (Sun et al., 2002). Clearly, in spite of the tremendous progress made in the past High-frequency firing-induced rise in presynaptic calcium levels decades, much is to be learned about the mechanisms of SV may serve as the trigger to stimulate calcineurin-mediated recycling and their modulation by synaptic activity. Progress dephosphorylation of dynamin and its partners, thereby facili- will depend on the further development of techniques to control tating fast membrane fission. Under these conditions membrane synaptic activity and to monitor SV recycling in real-time. These fission may occur before clathrin coats can assemble, resulting techniques include combining optogenetic stimulation with ge- in ultrafast retrieval of SV membranes. The resulting endo- netic manipulations and optical imaging or rapid high pressure some-like vacuoles are subsequently converted to SVs via freezing EM as well as novel approaches to tag SV membranes CME-like budding and possibly additional pathways (i.e., via during their exo-endocytotic itinerary independent of associated AP-3). Conversely, calcium levels may remain comparably low pH changes (Revelo et al., 2014). While the long half-life of SV during sustained low-frequency stimulation, conditions under proteins (days) (Cohen et al., 2013) is compatible with their recy- which few dynamin 1 molecules are active and fission is com- cling through multiple rounds of exo-endocytosis, it is likely that parably slow, allowing clathrin/AP-2 coats to assemble prior to quality control mechanisms exist to ensure that SV proteins vesicle budding leading to SV reformation directly from the incorporated into reformed SVs are fully functional, whereas plasma membrane. Consistent with this model, EM observa- dysfunctional proteins are targeted for degradation. The mecha- tions, made more than 40 years ago, show that prolonged nisms underlying such quality control are currently unknown, stimulation at elevated frequency (15 min at 10 Hz) at the frog but seem to involve endosomal sorting of SV proteins via neuromuscular junction triggers the formation of HRP-filled en- Rab35 and its GTPase-activating protein skywalker/TBC1D10 dosome-like vacuoles (Heuser and Reese, 1973), while HRP- (Uytterhoeven et al., 2011). Another remaining open question filled SVs predominate in the same preparation stimulated at pertains to the mechanisms that ensure presynaptic membrane low frequency (4 hr at 2 Hz) (Ceccarelli et al., 1973). Thus, slow homeostasis. While calcium may serve critical roles in regulating

492 Neuron 85, February 4, 2015 ª2015 Elsevier Inc. Neuron Review

the pathway of SV recycling, it is unlikely to have a direct role Armbruster, M., Messa, M., Ferguson, S.M., De Camilli, P., and Ryan, T.A. in controlling presynaptic membrane homeostasis. How do (2013). Dynamin phosphorylation controls optimization of endocytosis for brief action potential bursts. eLife 2, e00845. synapses ‘‘know’’ how many SVs have undergone exocytosis? One option is that SV proteins such as synaptotagmin 1, whose Atluri, P.P., and Ryan, T.A. (2006). The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals. J. Neurosci. 26, 2313–2320. plasma membrane levels might increase upon exocytosis, serve as coupling devices that trigger endocytic membrane retrieval Bai, J., Hu, Z., Dittman, J.S., Pym, E.C., and Kaplan, J.M. (2010). Endophilin functions as a membrane-bending molecule and is delivered to endocytic (Poskanzer et al., 2006; Yao et al., 2012), i.e., by recruiting endo- zones by exocytosis. Cell 143, 430–441. cytic factors or by modulating phospholipid content. Alterna- tively, synapses could be equipped with tension sensors that Betz, W.J., Mao, F., and Bewick, G.S. (1992). Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. trigger membrane retrieval as soon as membrane tension drops J. Neurosci. 12, 363–375. below a threshold following calcium-triggered neuroexocytosis. Balaji, J., and Ryan, T.A. (2007). Single-vesicle imaging reveals that synaptic The molecular mechanisms of endocytosis and SV regenera- vesicle exocytosis and endocytosis are coupled by a single stochastic tion are remarkably complex. Not surprisingly, the machinery mode. Proc. Natl. Acad. Sci. USA 104, 20576–20581. for SV recycling is implicated in brain dysfunction and disease. Blondeau, F., Ritter, B., Allaire, P.D., Wasiak, S., Girard, M., Hussain, N.K., An- For example, the PI(4,5)P2-phosphatase synaptojanin 1, is over- gers, A., Legendre-Guillemin, V., Roy, L., Boismenu, D., et al. (2004). Tandem expressed in Down syndrome resulting in phosphoinositide MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc. Natl. Acad. Sci. USA 101, 3833–3838. dyshomeostasis and cognitive disabilities, while reduced synaptojanin 1 levels ameliorate synaptic and behavioral impairments Blumstein, J., Faundez, V., Nakatsu, F., Saito, T., Ohno, H., and Kelly, R.B. (2001). The neuronal form of adaptor protein-3 is required for synaptic vesicle caused by Ab-mediated disruption of PI(4,5)P2 metabolism in formation from endosomes. J. Neurosci. 21, 8034–8042. Alzheimer’s disease models (McIntire et al., 2012). Moreover, genome-wide association studies have identified single nucleo- Boucrot, E., Ferreira, A.P., Almeida-Souza, L., Debard, S., Vallis, Y., Howard, G., Bertot, L., Sauvonnet, N., and McMahon, H.T. (2015). Endophilin marks tide polymorphisms (SNPs) in the genes encoding for the AP180 and controls a clathrin-independent endocytic pathway. Nature 517, 460–465. paralog CALM, dynamin 2, and amphiphysin 1 in familial and Brodsky, F.M., Chen, C.-Y., Knuehl, C., Towler, M.C., and Wakeham, D.E. sporadic cases of Alzheimer’s disease (Aidaralieva et al., 2008; (2001). Biological basket weaving: formation and function of clathrin-coated Harold et al., 2009; Seshadri et al., 2010). Finally, GIT1 (termed vesicles. Annu. Rev. Cell Dev. Biol. 17, 517–568. dGIT in Drosophila), an AZ-associated scaffold with GTPase Budzinski, K.L., Zeigler, M., Fujimoto, B.S., Bajjalieh, S.M., and Chiu, D.T. activating protein activity toward Arf6, associates with stonin (2011). Measurements of the acidification kinetics of single SynaptopHluorin 2/stoned B and regulates SV recycling (Podufall et al., 2014). Hu- vesicles. Biophys. J. 101, 1580–1589. man mutations in GIT1, which is also found postsynaptically, are Ceccarelli, B., Hurlbut, W.P., and Mauro, A. (1972). Depletion of vesicles from associated with attention deficit hyperactivity disorder (ADHD) frog neuromuscular junctions by prolonged tetanic stimulation. J. Cell Biol. 54, 30–38. and cause ADHD-like phenotypes in GIT1 mutant mice (Won et al., 2011). How precisely mutations in the machinery for SV Ceccarelli, B., Hurlbut, W.P., and Mauro, A. (1973). Turnover of transmitter and recycling lead to these diseases also remains an important synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57, 499–524. area for future studies. Chang-Ileto, B., Frere, S.G., Chan, R.B., Voronov, S.V., Roux, A., and Di Paolo, G. (2011). Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission. Dev. Cell 20, 206–218. ACKNOWLEDGMENTS Cheung, G., and Cousin, M.A. (2012). Adaptor protein complexes 1 and 3 are Work in the N.L.K. and V.H. laboratory is supported by a grant from the Deut- essential for generation of synaptic vesicles from activity-dependent bulk endosomes. J. Neurosci. 32, 6014–6023. sche Forschungsgemeinschaft (SFB 958/A01 to V.H.). Cheung, G., and Cousin, M.A. (2013). Synaptic vesicle generation from activ- REFERENCES ity-dependent bulk endosomes requires calcium and calcineurin. J. Neurosci. 33, 3370–3379.

Aidaralieva, N.J., Kamino, K., Kimura, R., Yamamoto, M., Morihara, T., Kazui, Chiang, H.C., Shin, W., Zhao, W.D., Hamid, E., Sheng, J., Baydyuk, M., Wen, H., Hashimoto, R., Tanaka, T., Kudo, T., Kida, T., et al. (2008). Dynamin 2 gene P.J., Jin, A., Momboisse, F., and Wu, L.G. (2014). Post-fusion structural is a novel susceptibility gene for late-onset Alzheimer disease in non-APOE- changes and their roles in exocytosis and endocytosis of dense-core vesicles. epsilon4 carriers. J. Hum. Genet. 53, 296–302. Nat. Commun. 5, 3356.

Alabi, A.A., and Tsien, R.W. (2013). Perspectives on kiss-and-run: role in Clayton, E.L., and Cousin, M.A. (2009). The molecular physiology of activity- exocytosis, endocytosis, and neurotransmission. Annu. Rev. Physiol. 75, dependent bulk endocytosis of synaptic vesicles. J. Neurochem. 111, 393–422. 901–914.

Albillos, A., Dernick, G., Horstmann, H., Almers, W., Alvarez de Toledo, G., and Cohen, L.D., Zuchman, R., Sorokina, O., Mu¨ ller, A., Dieterich, D.C., Armstrong, Lindau, M. (1997). The exocytotic event in chromafﬁn cells revealed by patch J.D., Ziv, T., and Ziv, N.E. (2013). Metabolic turnover of synaptic proteins: ki- amperometry. Nature 389, 509–512. netics, interdependencies and implications for synaptic maintenance. PLoS ONE 8, e63191. Ale´ s, E., Tabares, L., Poyato, J.M., Valero, V., Lindau, M., and Alvarez de Toledo, G. (1999). High calcium concentrations shift the mode of exocytosis Cremona, O., Di Paolo, G., Wenk, M.R., Lu¨ thi, A., Kim, W.T., Takei, K., Daniell, to the kiss-and-run mechanism. Nat. Cell Biol. 1, 40–44. L., Nemoto, Y., Shears, S.B., Flavell, R.A., et al. (1999). Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, 179–188. Aravanis, A.M., Pyle, J.L., and Tsien, R.W. (2003). Single synaptic vesicles fusing transiently and successively without loss of identity. Nature 423, Daumke, O., Roux, A., and Haucke, V. (2014). BAR domain scaffolds in dyna- 643–647. min-mediated membrane ﬁssion. Cell 156, 882–892.

Armbruster, M., and Ryan, T.A. (2011). Synaptic vesicle retrieval time is a cell- de Lange, R.P.J., de Roos, A.D.G., and Borst, J.G.G. (2003). Two modes of wide rather than individual-synapse property. Nat. Neurosci. 14, 824–826. vesicle recycling in the rat calyx of Held. J. Neurosci. 23, 10164–10173.

Neuron 85, February 4, 2015 ª2015 Elsevier Inc. 493 Neuron Review

De Robertis, E. (1959). Submicroscopic morphology of the synapse. In Interna- He, L., Wu, X.-S., Mohan, R., and Wu, L.-G. (2006). Two modes of fusion pore tional Review of Cytology, G.H. Bourne and J.F. Danielli, eds. (Academic opening revealed by cell-attached recordings at a synapse. Nature 444, Press), pp. 61–96. 102–105.

De Robertis, E.D.P., and Bennett, H.S. (1955). Some features of the submicro- Heerssen, H., Fetter, R.D., and Davis, G.W. (2008). Clathrin dependence of scopic morphology of synapses in frog and earthworm. J. Biophys. Biochem. synaptic-vesicle formation at the Drosophila neuromuscular junction. Curr. Cytol. 1, 47–58. Biol. 18, 401–409. del Castillo, J., and Katz, B. (1954). Quantal components of the end-plate po- Heuser, J.E., and Reese, T.S. (1973). Evidence for recycling of synaptic vesicle tential. J. Physiol. 124, 560–573. membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344. Di Paolo, G., and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657. Heuser, J.E., Reese, T.S., Dennis, M.J., Jan, Y., Jan, L., and Evans, L. (1979). Synaptic vesicle exocytosis captured by quick freezing and correlated with Di Paolo, G., Sankaranarayanan, S., Wenk, M.R., Daniell, L., Perucco, E., Cal- quantal transmitter release. J. Cell Biol. 81, 275–300. darone, B.J., Flavell, R., Picciotto, M.R., Ryan, T.A., Cremona, O., and De Ca- milli, P. (2002). Decreased synaptic vesicle recycling efﬁciency and cognitive Holroyd, P., Lang, T., Wenzel, D., De Camilli, P., and Jahn, R. (2002). Imaging deﬁcits in amphiphysin 1 knockout mice. Neuron 33, 789–804. direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl. Acad. Sci. USA 99, 16806– Dickman, D.K., Horne, J.A., Meinertzhagen, I.A., and Schwarz, T.L. (2005). A 16811. slowed classical pathway rather than kiss-and-run mediates endocytosis at synapses lacking synaptojanin and endophilin. Cell 123, 521–533. Holt, M., Cooke, A., Wu, M.M., and Lagnado, L. (2003). Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells. J. Neurosci. 23, Diril, M.K., Wienisch, M., Jung, N., Klingauf, J., and Haucke, V. (2006). Stonin 2 1329–1339. is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling. Dev. Cell 10, 233–244. Holtzman, E., Freeman, A.R., and Kashner, L.A. (1971). Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions. Science Dittman, J., and Ryan, T.A. (2009). Molecular circuitry of endocytosis at nerve 173, 733–736. terminals. Annu. Rev. Cell Dev. Biol. 25, 133–160. Hoopmann, P., Punge, A., Barysch, S.V., Westphal, V., Bu¨ ckers, J., Opazo, F., Fergestad, T., and Broadie, K. (2001). Interaction of stoned and synaptotagmin Bethani, I., Lauterbach, M.A., Hell, S.W., and Rizzoli, S.O. (2010). Endosomal in synaptic vesicle endocytosis. J. Neurosci. 21, 1218–1227. sorting of readily releasable synaptic vesicles. Proc. Natl. Acad. Sci. USA 107, 19055–19060. Ferguson, S.M., and De Camilli, P. (2012). Dynamin, a membrane-remodelling GTPase. Nat. Rev. Mol. Cell Biol. 13, 75–88. Hosoi, N., Holt, M., and Sakaba, T. (2009). Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 63, 216–229. Ferguson, S.M., Brasnjo, G., Hayashi, M., Wo¨ lfel, M., Collesi, C., Giovedi, S., Raimondi, A., Gong, L.W., Ariel, P., Paradise, S., et al. (2007). A selective ac- Hua, Y., Sinha, R., Thiel, C.S., Schmidt, R., Hu¨ ve, J., Martens, H., Hell, S.W., tivity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Egner, A., and Klingauf, J. (2011a). A readily retrievable pool of synaptic vesi- Science 316, 570–574. cles. Nat. Neurosci. 14, 833–839.

Ferna´ ndez-Alfonso, T., and Ryan, T.A. (2004). The kinetics of synaptic vesicle Hua, Z., Leal-Ortiz, S., Foss, S.M., Waites, C.L., Garner, C.C., Voglmaier, S.M., pool depletion at CNS synaptic terminals. Neuron 41, 943–953. and Edwards, R.H. (2011b). v-SNARE composition distinguishes synaptic vesicle pools. Neuron 71, 474–487. Ferna´ ndez-Alfonso, T., Kwan, R., and Ryan, T.A. (2006). Synaptic vesicles interchange their membrane proteins with a large surface reservoir during re- Jahn, R., and Fasshauer, D. (2012). Molecular machines governing exocytosis cycling. Neuron 51, 179–186. of synaptic vesicles. Nature 490, 201–207.

Fesce, R., Grohovaz, F., Valtorta, F., and Meldolesi, J. (1994). Neurotransmitter Jockusch, W.J., Praefcke, G.J., McMahon, H.T., and Lagnado, L. (2005). Cla- release: fusion or ‘kiss-and-run’? Trends Cell Biol. 4, 1–4. thrin-dependent and clathrin-independent retrieval of synaptic vesicles in retinal bipolar cells. Neuron 46, 869–878. Frost, A., Unger, V.M., and De Camilli, P. (2009). The BAR domain superfamily: membrane-molding macromolecules. Cell 137, 191–196. Kantheti, P., Qiao, X., Diaz, M.E., Peden, A.A., Meyer, G.E., Carskadon, S.L., Kapfhamer, D., Sufalko, D., Robinson, M.S., Noebels, J.L., and Burmeister, Fuchs, M., Brandsta¨ tter, J.H., and Regus-Leidig, H. (2014). Evidence for a M. (1998). Mutation in AP-3 delta in the mocha mouse links endosomal trans- Clathrin-independent mode of endocytosis at a continuously active sensory port to storage deﬁciency in platelets, melanosomes, and synaptic vesicles. synapse. Front. Cell. Neurosci. 8, 60. Neuron 21, 111–122.

Gad, H., Ringstad, N., Lo¨ w, P., Kjaerulff, O., Gustafsson, J., Wenk, M., Di Kasprowicz, J., Kuenen, S., Miskiewicz, K., Habets, R.L.P., Smitz, L., and Ver- Paolo, G., Nemoto, Y., Crun, J., Ellisman, M.H., et al. (2000). Fission and streken, P. (2008). Inactivation of clathrin heavy chain inhibits synaptic recy- uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of cling but allows bulk membrane uptake. J. Cell Biol. 182, 1007–1016. interactions with the SH3 domain of endophilin. Neuron 27, 301–312. Kasprowicz, J., Kuenen, S., Swerts, J., Miskiewicz, K., and Verstreken, P. Gandhi, S.P., and Stevens, C.F. (2003). Three modes of synaptic vesicular re- (2014). Dynamin photoinactivation blocks Clathrin and a-adaptin recruitment cycling revealed by single-vesicle imaging. Nature 423, 607–613. and induces bulk membrane retrieval. J. Cell Biol. 204, 1141–1156.

Glyvuk, N., Tsytsyura, Y., Geumann, C., D’Hooge, R., Hu¨ ve, J., Kratzke, M., Katz, B., and Miledi, R. (1967). A study of synaptic transmission in the absence Baltes, J., Boening, D., Klingauf, J., and Schu, P. (2010). AP-1/sigma1B-adap- of nerve impulses. J. Physiol. 192, 407–436. tin mediates endosomal synaptic vesicle recycling, learning and memory. EMBO J. 29, 1318–1330. Kavalali, E.T., and Jorgensen, E.M. (2014). Visualizing presynaptic function. Nat. Neurosci. 17, 10–16. Granseth, B., Odermatt, B., Royle, S.J., and Lagnado, L. (2006). Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocam- Kim, S.H., and Ryan, T.A. (2009). Synaptic vesicle recycling at CNS snapses pal synapses. Neuron 51, 773–786. without AP-2. J. Neurosci. 29, 3865–3874.

Harold, D., Abraham, R., Hollingworth, P., Sims, R., Gerrish, A., Hamshere, Klingauf, J., Kavalali, E.T., and Tsien, R.W. (1998). Kinetics and regulation of M.L., Pahwa, J.S., Moskvina, V., Dowzell, K., Williams, A., et al. (2009). fast endocytosis at hippocampal synapses. Nature 394, 581–585. Genome-wide association study identiﬁes variants at CLU and PICALM associated with Alzheimer’s disease. Nat. Genet. 41, 1088–1093. Koch, D., Spiwoks-Becker, I., Sabanov, V., Sinning, A., Dugladze, T., Stell- macher, A., Ahuja, R., Grimm, J., Schu¨ ler, S., Mu¨ ller, A., et al. (2011). Proper Haucke, V., Neher, E., and Sigrist, S.J. (2011). Protein scaffolds in the coupling synaptic vesicle formation and neuronal network activity critically rely on syn- of synaptic exocytosis and endocytosis. Nat. Rev. Neurosci. 12, 127–138. dapin I. EMBO J. 30, 4955–4969.

494 Neuron 85, February 4, 2015 ª2015 Elsevier Inc. Neuron Review

Koenig, J.H., and Ikeda, K. (1996). Synaptic vesicles have two distinct recy- (2012). UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Cae- cling pathways. J. Cell Biol. 135, 797–808. norhabditis elegans. PLoS ONE 7, e40095.

Kononenko, N.L., Diril, M.K., Puchkov, D., Kintscher, M., Koo, S.J., Pfuhl, G., Murthy, V.N., and Stevens, C.F. (1998). Synaptic vesicles retain their identity Winter, Y., Wienisch, M., Klingauf, J., Breustedt, J., et al. (2013). Compromised through the endocytic cycle. Nature 392, 497–501. ﬁdelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2. Proc. Natl. Acad. Sci. USA 110, E526–E535. Nakatsu, F., Okada, M., Mori, F., Kumazawa, N., Iwasa, H., Zhu, G., Kasagi, Y., Kamiya, H., Harada, A., Nishimura, K., et al. (2004). Defective function of Kononenko, N.L., Puchkov, D., Classen, G.A., Walter, A.M., Pechstein, A., Sa- GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor. wade, L., Kaempf, N., Trimbuch, T., Lorenz, D., Rosenmund, C., et al. (2014). J. Cell Biol. 167, 293–302. Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron Neef, J., Jung, S., Wong, A.B., Reuter, K., Pangrsic, T., Chakrabarti, R., Ku¨ gler, 82, 981–988. S., Lenz, C., Nouvian, R., Boumil, R.M., et al. (2014). Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells. J. Neurosci. 34, Koo, S.J., Markovic, S., Puchkov, D., Mahrenholz, C.C., Beceren-Braun, F., 705–716. Maritzen, T., Dernedde, J., Volkmer, R., Oschkinat, H., and Haucke, V. (2011). SNARE motif-mediated sorting of synaptobrevin by the endocytic Neher, E. (2010). What is rate-limiting during sustained synaptic activity: adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 vesicle supply or the availability of release sites. Front. Synaptic Neurosci. at synapses. Proc. Natl. Acad. Sci. USA 108, 13540–13545. 2, 144.

Kraszewski, K., Mundigl, O., Daniell, L., Verderio, C., Matteoli, M., and De Ca- Neher, E., and Marty, A. (1982). Discrete changes of cell membrane capaci- milli, P. (1995). Synaptic vesicle dynamics in living cultured hippocampal neu- tance observed under conditions of enhanced secretion in bovine adrenal rons visualized with CY3-conjugated antibodies directed against the lumenal chromaffin cells. Proc. Natl. Acad. Sci. USA 79, 6712–6716. domain of synaptotagmin. J. Neurosci. 15, 4328–4342. Neves, G., and Lagnado, L. (1999). The kinetics of exocytosis and endocytosis Lindau, M., and Neher, E. (1988). Patch-clamp techniques for time-resolved in the synaptic terminal of goldfish retinal bipolar cells. J. Physiol. 515, capacitance measurements in single cells. Pflugers Arch. 411, 137–146. 181–202.

Llobet, A., Gallop, J.L., Burden, J.J., Camdere, G., Chandra, P., Vallis, Y., Hop- Neves, G., Gomis, A., and Lagnado, L. (2001). Calcium inﬂux selects the fast kins, C.R., Lagnado, L., and McMahon, H.T. (2011). Endophilin drives the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells. Proc. mode of vesicle retrieval in a ribbon synapse. J. Neurosci. 31, 8512–8519. Natl. Acad. Sci. USA 98, 15282–15287.

Mani, M., Lee, S.Y., Lucast, L., Cremona, O., Di Paolo, G., De Camilli, P., and Newell-Litwa, K., Chintala, S., Jenkins, S., Pare, J.-F., McGaha, L., Smith, Y., Ryan, T.A. (2007). The dual phosphatase activity of synaptojanin1 is required and Faundez, V. (2010). Hermansky-Pudlak protein complexes, AP-3 and for both efﬁcient synaptic vesicle endocytosis and reavailability at nerve termi- BLOC-1, differentially regulate presynaptic composition in the striatum and nals. Neuron 56, 1004–1018. hippocampus. J. Neurosci. 30, 820–831.

Maritzen, T., Podufall, J., and Haucke, V. (2010). Stonins—specialized adap- Ooi, C.E., Moreira, J.E., Dell’Angelica, E.C., Poy, G., Wassarman, D.A., and tors for synaptic vesicle recycling and beyond? Trafﬁc 11, 8–15. Bonifacino, J.S. (1997). Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet. Matteoli, M., Takei, K., Perin, M.S., Su¨ dhof, T.C., and De Camilli, P. (1992). EMBO J. 16, 4508–4518. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J. Cell Biol. 117, 849–861. Owen, D.J., Collins, B.M., and Evans, P.R. (2004). Adaptors for clathrin coats: Maycox, P.R., Link, E., Reetz, A., Morris, S.A., and Jahn, R. (1992). Clathrin- structure and function. Annu. Rev. Cell Dev. Biol. 20, 153–191. coated vesicles in nervous tissue are involved primarily in synaptic vesicle re- Paillart, C., Li, J., Matthews, G., and Sterling, P. (2003). Endocytosis and cycling. J. Cell Biol. 118, 1379–1388. vesicle recycling at a ribbon synapse. J. Neurosci. 23, 4092–4099. McIntire, L.B., Berman, D.E., Myaeng, J., Staniszewski, A., Arancio, O., Di Paolo, G., and Kim, T.W. (2012). Reduction of synaptojanin 1 ameliorates syn- Petralia, R.S., Wang, Y.-X., Indig, F.E., Bushlin, I., Wu, F., Mattson, M.P., and aptic and behavioral impairments in a mouse model of Alzheimer’s disease. Yao, P.J. (2013). Reduction of AP180 and CALM produces defects in synaptic J. Neurosci. 32, 15271–15276. vesicle size and density. Neuromolecular Med. 15, 49–60.

McMahon, H.T., and Boucrot, E. (2011). Molecular mechanism and physiolog- Podufall, J., Tian, R., Knoche, E., Puchkov, D., Walter, A.M., Rosa, S., Quentin, ical functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, C., Vukoja, A., Jung, N., Lampe, A., et al. (2014). A presynaptic role for the cy- 517–533. tomatrix protein GIT in synaptic vesicle recycling. Cell Rep. 7, 1417–1425.

Meinecke, M., Boucrot, E., Camdere, G., Hon, W.C., Mittal, R., and McMahon, Poskanzer, K.E., Fetter, R.D., and Davis, G.W. (2006). Discrete residues in the H.T. (2013). Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs c(2)b domain of synaptotagmin I independently specify endocytic rate and (BAR) domain-containing proteins leads to GTP-dependent membrane scis- synaptic vesicle size. Neuron 50, 49–62. sion. J. Biol. Chem. 288, 6651–6661. Pyle, J.L., Kavalali, E.T., Piedras-Renterıá, E.S., and Tsien, R.W. (2000). Rapid Miesenbo¨ ck, G., De Angelis, D.A., and Rothman, J.E. (1998). Visualizing secre- reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, tion and synaptic transmission with pH-sensitive green fluorescent proteins. 221–231. Nature 394, 192–195. Raimondi, A., Ferguson, S.M., Lou, X., Armbruster, M., Paradise, S., Giovedi, Miller, T.M., and Heuser, J.E. (1984). Endocytosis of synaptic vesicle mem- S., Messa, M., Kono, N., Takasaki, J., Cappello, V., et al. (2011). Overlapping brane at the frog neuromuscular junction. J. Cell Biol. 98, 685–698. role of dynamin isoforms in synaptic vesicle endocytosis. Neuron 70, 1100– 1114. Milosevic, I., Giovedi, S., Lou, X., Raimondi, A., Collesi, C., Shen, H., Paradise, S., O’Toole, E., Ferguson, S., Cremona, O., and De Camilli, P. (2011). Recruit- Raingo, J., Khvotchev, M., Liu, P., Darios, F., Li, Y.C., Ramirez, D.M.O., ment of endophilin to clathrin-coated pit necks is required for efficient vesicle Adachi, M., Lemieux, P., Toth, K., Davletov, B., et al. (2012). VAMP4 directs uncoating after fission. Neuron 72, 587–601. synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat. Neurosci. 15, 738–745. Morlot, S., Galli, V., Klein, M., Chiaruttini, N., Manzi, J., Humbert, F., Dinis, L., Lenz, M., Cappello, G., and Roux, A. (2012). Membrane shape at the edge of Ramirez, D.M., Khvotchev, M., Trauterman, B., and Kavalali, E.T. (2012). Vti1a the dynamin helix sets location and duration of the fission reaction. Cell 151, identifies a vesicle pool that preferentially recycles at rest and maintains spon- 619–629. taneous neurotransmission. Neuron 73, 121–134.

Mullen, G.P., Grundahl, K.M., Gu, M., Watanabe, S., Hobson, R.J., Crowell, Revelo, N.H., Kamin, D., Truckenbrodt, S., Wong, A.B., Reuter-Jessen, K., J.A., McManus, J.R., Mathews, E.A., Jorgensen, E.M., and Rand, J.B. Reisinger, E., Moser, T., and Rizzoli, S.O. (2014). A new probe for super-

Neuron 85, February 4, 2015 ª2015 Elsevier Inc. 495 Neuron Review

resolution imaging of membranes elucidates trafficking pathways. J. Cell Biol. Watanabe, S., Liu, Q., Davis, M.W., Hollopeter, G., Thomas, N., Jorgensen, 205, 591–606. N.B., and Jorgensen, E.M. (2013a). Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. eLife 2, e00723. Richards, D.A., Guatimosim, C., and Betz, W.J. (2000). Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Watanabe, S., Rost, B.R., Camacho-Pe´ rez, M., Davis, M.W., So¨ hl-Kielczynski, Neuron 27, 551–559. B., Rosenmund, C., and Jorgensen, E.M. (2013b). Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247. Rizzoli, S.O. (2014). Synaptic vesicle recycling: steps and principles. EMBO J. 33, 788–822. Watanabe, S., Trimbuch, T., Camacho-Pe´ rez, M., Rost, B.R., Brokowski, B., So¨ hl-Kielczynski, B., Felies, A., Davis, M.W., Rosenmund, C., and Jorgensen, Ryan, T.A., and Smith, S.J. (1995). Vesicle pool mobilization during action po- E.M. (2014). Clathrin regenerates synaptic vesicles from endosomes. Nature tential firing at hippocampal synapses. Neuron 14, 983–989. 515, 228–233.

Ryan, T.A., Reuter, H., Wendland, B., Schweizer, F.E., Tsien, R.W., and Smith, Whittaker, V.P. (1969). The nature of the acetylcholine pools in brain tissue. S.J. (1993). The kinetics of synaptic vesicle recycling measured at single pre- Prog. Brain Res. 31, 211–222. synaptic boutons. Neuron 11, 713–724. Wienisch, M., and Klingauf, J. (2006). Vesicular proteins exocytosed and sub- Saheki, Y., and De Camilli, P. (2012). Synaptic vesicle endocytosis. Cold sequently retrieved by compensatory endocytosis are nonidentical. Nat. Neu- Spring Harb. Perspect. Biol. 4, a005645. rosci. 9, 1019–1027. Sakaba, T., Kononenko, N.L., Bacetic, J., Pechstein, A., Schmoranzer, J., Yao, L., Barth, H., Shupliakov, O., Kobler, O., Aktories, K., and Haucke, V. (2013). Wilhelm, B.G., Mandad, S., Truckenbrodt, S., Kro¨ hnert, K., Scha¨ fer, C., Fast neurotransmitter release regulated by the endocytic scaffold intersectin. Rammner, B., Koo, S.J., Claßen, G.A., Krauss, M., Haucke, V., et al. (2014). Proc. Natl. Acad. Sci. USA 110, 8266–8271. Composition of isolated synaptic boutons reveals the amounts of vesicle traf- ﬁcking proteins. Science 344, 1023–1028. Sandvig, K., Pust, S., Skotland, T., and van Deurs, B. (2011). Clathrin-independent endocytosis: mechanisms and function. Curr. Opin. Cell Biol. 23, Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R., and Hell, S.W. (2006). STED 413–420. microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939. Sato, K., Ernstrom, G.G., Watanabe, S., Weimer, R.M., Chen, C.-H., Sato, M., Siddiqui, A., Jorgensen, E.M., and Grant, B.D. (2009). Differential requirements Won, H., Mah, W., Kim, E., Kim, J.W., Hahm, E.K., Kim, M.H., Cho, S., Kim, J., for clathrin in receptor-mediated endocytosis and maintenance of synaptic Jang, H., Cho, S.C., et al. (2011). GIT1 is associated with ADHD in humans and vesicle pools. Proc. Natl. Acad. Sci. USA 106, 1139–1144. ADHD-like behaviors in mice. Nat. Med. 17, 566–572.

Seshadri, S., Fitzpatrick, A.L., Ikram, M.A., DeStefano, A.L., Gudnason, V., Wu, L.G., and Betz, W.J. (1996). Nerve activity but not intracellular calcium de- Boada, M., Bis, J.C., Smith, A.V., Carassquillo, M.M., Lambert, J.C., et al.; termines the time course of endocytosis at the frog neuromuscular junction. CHARGE Consortium; GERAD1 Consortium; EADI1 Consortium (2010). Neuron 17, 769–779. Genome-wide analysis of genetic loci associated with Alzheimer disease. JAMA 303, 1832–1840. Wu, W., Xu, J., Wu, X.-S., and Wu, L.-G. (2005). Activity-dependent accelera- tion of endocytosis at a central synapse. J. Neurosci. 25, 11676–11683. Shupliakov, O., Lo¨ w, P., Grabs, D., Gad, H., Chen, H., David, C., Takei, K., De Camilli, P., and Brodin, L. (1997). Synaptic vesicle endocytosis impaired by Wu, L.G., Hamid, E., Shin, W., and Chiang, H.C. (2014a). Exocytosis and endo- disruption of dynamin-SH3 domain interactions. Science 276, 259–263. cytosis: modes, functions, and coupling mechanisms. Annu. Rev. Physiol. 76, 301–331. Stevens, C.F., and Williams, J.H. (2000). ‘‘Kiss and run’’ exocytosis at hippocampal synapses. Proc. Natl. Acad. Sci. USA 97, 12828–12833. Wu, X.S., Zhang, Z., Zhao, W.D., Wang, D., Luo, F., and Wu, L.G. (2014b). Cal- cineurin is universally involved in vesicle endocytosis at neuronal and non- Sun, J.-Y., Wu, X.-S., and Wu, L.-G. (2002). Single and multiple vesicle fusion neuronal secretory cells. Cell Rep. 7, 982–988. induce different rates of endocytosis at a central synapse. Nature 417, 555–559. Wu, Y., O’Toole, E.T., Girard, M., Ritter, B., Messa, M., Liu, X., McPherson, P.S., Ferguson, S.M., and De Camilli, P. (2014c). A dynamin 1-, dynamin 3- Takamori, S., Holt, M., Stenius, K., Lemke, E.A., Grønborg, M., Riedel, D., Ur- and clathrin-independent pathway of synaptic vesicle recycling mediated by laub, H., Schenck, S., Bru¨ gger, B., Ringler, P., et al. (2006). Molecular anatomy bulk endocytosis. eLife 3, e01621. of a trafﬁcking organelle. Cell 127, 831–846. Wucherpfennig, T., Wilsch-Bra¨ uninger, M., and Gonza´ lez-Gaita´ n, M. (2003). Takei, K., Mundigl, O., Daniell, L., and De Camilli, P. (1996). The synaptic Role of Drosophila Rab5 during endosomal trafﬁcking at the synapse and vesicle cycle: a single vesicle budding step involving clathrin and dynamin. evoked neurotransmitter release. J. Cell Biol. 161, 609–624. J. Cell Biol. 133, 1237–1250. Xu, J., McNeil, B., Wu, W., Nees, D., Bai, L., and Wu, L.-G. (2008). GTP-inde- Ungewickell, E., Ungewickell, H., Holstein, S.E.H., Lindner, R., Prasad, K., Bar- pendent rapid and slow endocytosis at a central synapse. Nat. Neurosci. 11, ouch, W., Martin, B., Greene, L.E., and Eisenberg, E. (1995). Role of auxilin in 45–53. uncoating clathrin-coated vesicles. Nature 378, 632–635.

Uytterhoeven, V., Kuenen, S., Kasprowicz, J., Miskiewicz, K., and Verstreken, Yao, J., Kwon, S.E., Gaffaney, J.D., Dunning, F.M., and Chapman, E.R. (2012). P. (2011). Loss of skywalker reveals synaptic endosomes as sorting stations Uncoupling the roles of synaptotagmin I during endo- and exocytosis of syn- for synaptic vesicle proteins. Cell 145, 117–132. aptic vesicles. Nat. Neurosci. 15, 243–249.

Van Hook, M.J., and Thoreson, W.B. (2012). Rapid synaptic vesicle endocy- Yim, Y.-I., Sun, T., Wu, L.-G., Raimondi, A., De Camilli, P., Eisenberg, E., and tosis in cone photoreceptors of salamander retina. J. Neurosci. 32, 18112– Greene, L.E. (2010). Endocytosis and clathrin-uncoating defects at synapses 18123. of auxilin knockout mice. Proc. Natl. Acad. Sci. USA 107, 4412–4417.

Verstreken, P., Kjaerulff, O., Lloyd, T.E., Atkinson, R., Zhou, Y., Meinertzhagen, Zhang, B., Koh, Y.H., Beckstead, R.B., Budnik, V., Ganetzky, B., and Bellen, I.A., and Bellen, H.J. (2002). Endophilin mutations block clathrin-mediated H.J. (1998). Synaptic vesicle size and number are regulated by a clathrin endocytosis but not neurotransmitter release. Cell 109, 101–112. adaptor protein required for endocytosis. Neuron 21, 1465–1475.

Voglmaier, S.M., Kam, K., Yang, H., Fortin, D.L., Hua, Z., Nicoll, R.A., and Ed- Zhang, Q., Li, Y., and Tsien, R.W. (2009). The dynamic control of kiss-and-run wards, R.H. (2006). Distinct endocytic pathways control the rate and extent of and vesicular reuse probed with single nanoparticles. Science 323, 1448– synaptic vesicle protein recycling. Neuron 51, 71–84. 1453. von Gersdorff, H., and Matthews, G. (1994). Dynamics of synaptic vesicle Zhu, Y., Xu, J., and Heinemann, S.F. (2009). Two pathways of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367, 735–739. retrieval revealed by single-vesicle imaging. Neuron 61, 397–411.

496 Neuron 85, February 4, 2015 ª2015 Elsevier Inc.