Synaptic Vesicle Endocytosis - Department of Biochemistry

NeuroMolecular Medicine Copyright © 2002 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN1535-1084/02/02:101–114/$20.00

Synaptic Vesicle Endocytosis The Races, Places, and Molecular Faces

Jennifer R. Morgan,1 George J. Augustine,2 and Eileen M. Lafer * ,3 1

Yale University School of Medicine—HHMI; Department of Cell Biology, New Haven, CT 06510; 2Duke University School of Medicine; Department of Neurobiology, Durham, NC 27710; and 3University of Texas Health Science Center at San Antonio; Department of Biochemistry, San Antonio, TX 78229 Received May 28, 2002; Accepted May 29, 2002

Abstract The classical experiments on synaptic vesicle recycling in the 1970s by Heuser and Reese, Ceccarelli, and their colleagues raised opposing theories regarding the speed, mechanisms, and locations of membrane retrieval at the synapse. The Heuser and Reese experiments supported a model in which synaptic vesicle recycling is mediated by the formation of coated vesicles, is relatively slow, and occurs distally from active zones, the sites of neurotransmitter release. Because heavy levels of stimulation were needed to visualize the coated vesicles, Ceccarelli’s experiments argued that synaptic vesicle recycling does not require the formation of coated vesicles, is relatively fast, and occurs directly at the active zone in a “kiss-and-run” reversal of exocytosis under more physiological conditions. For the next thirty years, these models have provided the foundation for studies of the rates, locations, and molecular elements involved in synaptic vesicle endocytosis. Here, we describe the evidence supporting each model and argue that the coated vesicle pathway is the most predominant physiological mechanism for recycling synaptic vesicles. Index Entries: Clathrin; synaptic vesicle trafficking; membrane recycling.

Introduction Communication between neurons relies critically upon the release of neurotransmitter molecules from the presynaptic nerve terminal onto the neighbor-

ing postsynaptic neuron. This is achieved when neurotransmitter-filled synaptic vesicles in a readily releasable pool fuse with the presynaptic plasma membrane, releasing transmitter in response to activity-dependent increases in the concentration of

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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102 intracellular calcium. With each round of synaptic vesicle exocytosis, vesicular membrane is added to the plasma membrane and synaptic vesicles are lost from the readily releasable pool. Thus, in order for neurotransmitter release to be maintained, the readily releasable pool of vesicles must be replaced for the next round of fusion and neurotransmitter release. Because the total number of synaptic vesicles in a nerve terminal is limited, a recycling mechanism must exist to maintain the vesicle pool. Further, because vesicular constituents are synthesized some distance away from the nerve terminal, at the neuronal cell body, rapid replenishment of vesicles requires that synaptic vesicle recycling must occur locally within the nerve terminal. However, the mechanisms involved in such local endocytotic recycling are still under debate.

Classical Studies Suggest Two Models for Synaptic Vesicle Recycling The first demonstrations of local synapticvesicle recycling were made at the frog neuromuscular junction (NMJ) in the 1970s. Classical experiments done in parallel by Heuser and Reese, as well as by Ceccarelli and their colleagues, examined the effects of synaptic activity on the ultrastructure of this synapse (Ceccarelli et al., 1972; Ceccarelli et al., 1973; Heuser and Reese, 1973; Ceccarelli et al., 1979; Miller and Heuser, 1984). In these studies, the NMJ was stimulated vigorously to cause the fusion of many synaptic vesicles with the plasma membrane. Although the number of exocytotic events produced by such stimuli was much greater than the number of synaptic vesicles initially present in the nerve terminal, there was only a partial depletion of the synaptic vesicle pool. Thus, vesicles must have been replenished in response to the exocytotic activity (Ceccarelli et al., 1972; Ceccarelli et al., 1973; Heuser and Reese, 1973). Both groups of investigators demonstrated that a tracer (horseradish peroxidase, or HRP) present in the extracellular space surrounding active terminals eventually appeared within newly formed synaptic vesicles indicating that vesicles are recycled from the plasma membrane following exocytotic release of transmitter (Ceccarelli et al., 1972; Ceccarelli et al., 1973; Heuser and Reese, 1973). However, although both groups used identical synapses and similar experimental methods, there was sub-


Morgan et al. stantial disagreement about the speed, location, and molecular identity of synaptic vesicle recycling mechanisms. In the Heuser and Reese experiments, 10-Hz stimulus was used to label recycling synaptic vesicles with HRP, and the nerve terminals were fixed at varying times after stimulation to examine the localization of the tracer. This approach revealed that HRP first appeared within vesicles surrounded by an electron dense coat, then later within larger endosomal-like membrane compartments known as “cisternae,” and finally within synaptic vesicles (Heuser and Reese, 1973). This study supports a model in which synaptic vesicles are recycled via the formation of coated vesicles, which then pass through an endosomal compartment before being refilled with neurotransmitter for use in another bout of exocytosis (Fig. 1). The (endocytic) coated vesicles were always observed peripherally to active zones, the sites of synaptic vesicle exocytosis (Heuser and Reese, 1973; Heuser et al., 1974; Miller and Heuser, 1984). Further, in order to examine the time-course of synaptic vesicle recycling, endocytosing vesicles were captured by immediately freezing terminals at varying times following stimulation. Following freeze-fracture of the nerve terminals, the membrane dimples or openings representing the endocytotic events on the cytosolic face were counted at each time-point (Heuser et al., 1979; Miller and Heuser, 1984). This approach indicated that synaptic vesicle recycling proceeds for 30–60 s after a bout of exocytosis. In summary, the experiments by Heuser, Reese, and colleagues indicated that synaptic vesicle recycling requires coated vesicles, occurs away from active zones, and proceeds over a time-course of a minute (Heuser and Reese, 1973; Miller and Heuser, 1984). In contrast, Ceccarelli and colleagues argued that the 10-Hz stimulus employed in Heuser and Reese’s experiments was unphysiological, and suggested that a mechanism not requiring coated vesicles is used for synaptic vesicle recycling under more physiological conditions (Ceccarelli et al., 1972; Ceccarelli et al., 1973; Ceccarelli et al., 1979; Torri-Tarelli et al., 1987). In their experiments, HRP-labeled recycling synaptic vesicles were observed directly at the active zones following 2-Hz stimulation (Ceccarelli et al., 1972). Similarly, freeze-fracture electron micrographs revealed transient openings of synaptic vesicles at the active zones during times when endocytosis should predominate over exocytosis (Ceccarelli et al., 1979). Critics of these experiments

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Fig. 1. Two models for synaptic vesicle recycling in nerve terminals suggested by classical studies. The earliest demonstrations of local synaptic vesicle recycling by Heuser and Reese, Ceccarelli, and their colleagues suggested that there may be two pathways—the coated vesicle pathway and the kiss-and-run pathway. These pathways could be distinguished on the basis of their speed, molecular mechanisms, and location. Heuser and Reese’s experiments suggested that vesicle recycling occurs through the formation of coated vesicles, which then pass through an endosomal compartment before re-emerging as synaptic vesicles ready for refilling and reuse. Further, they believed that vesicle recycling is relatively slow and occurs distally from active zones (AZs). In contrast, Ceccarelli’s experiments suggested that vesicle recycling is simply a “kiss-and-run” reversal of exocytosis, such that it occurs at AZs and does not require the formation of coated vesicles.

argue that such structures probably represent exocytotic events. However, because these openings eventually disappeared, Ceccarelli and colleagues argued that local retrieval of vesicular membrane from the plasma membrane was occurring. The fact that this membrane retrieval occurred at active zones indicates that it did not arise from coated vesicles (Heuser and Reese, 1973; Ceccarelli et al., 1979). Similarly, the vesicular openings were the size of synaptic vesicles, and not the larger size expected of coated vesicles. In these experiments there seemed to be no correlation between the number of coated vesicles and either the amount of transmitter released or the estimated number of recycled vesicles, leading Ceccarelli and colleagues to argue that synaptic vesicle recycling does not require the formation of coated vesicles (Torri-Tarelli et al., 1987). Instead, these experiments led to the proposal that synaptic vesicles transiently fuse with the plasma membrane, release their neurotransmitter contents, and then


pinch back off without collapsing fully into the presynaptic plasma membrane in a reversal of exocytosis—a process now colloquially known as “kissand-run” endocytosis (Fig. 1) (Burgoyne et al., 2001; Valtorta et al., 2001). In summary, these classical experiments revealed two potential models for synaptic vesicle recycling in nerve terminals: the coated vesicle pathway and the kiss-and-run pathway (Fig. 1). These models can be distinguished on the basis of their speed, molecular mechanisms, and location in the nerve terminal. Currently, the vast majority of data supports a critical role for the coated vesicle pathway in recycling synaptic vesicles (Brodin et al., 2000; Slepnev and De Camilli, 2000). However, more recent evidence indicates that a fast mode of membrane retrieval exists, which might be consistent with the kiss-and-run model (Burgoyne et al., 2001; Valtorta et al., 2001). Therefore, it is possible that nerve terminals may use both mechanisms for synaptic

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104 vesicle recycling. If these two modes of recycling exist in nerve terminals that correlate to the coated vesicle and kiss-and-run pathways, then several predictions can be made. First, nerve terminals should exhibit both fast and slow rates of endocytosis. Second, the endocytotic pathways should employ different molecular mechanisms. Finally, synaptic vesicle recycling should occur both at and away from active zones. We will discuss these possibilities by addressing what is currently known about the speed, molecular mechanisms, and location of synaptic vesicle recycling in nerve terminals.

Rates of Synaptic Vesicle Endocytosis The best evidence that two pathways for recycling synaptic vesicles may exist in nerve terminals comes from recent demonstrations of two distinct rates of vesicle recycling. Many measurements of the speed of synaptic vesicle recycling have been made using FM1-43, a fluorescent lipophilic dye that partitions into membranes and labels recycling synaptic vesicles in an activity-dependent manner (Betz et al., 1992; Ryan et al., 1993; Ryan et al., 1996; Wu and Betz, 1996). In such experiments, the progression of synaptic vesicle endocytosis can be monitored by stimulating nerve terminals and then applying FM1-43 with variable time delays, with the amount of dye uptake indicative of the kinetics of endocytosis. In this paradigm, synaptic vesicle retrieval is reported to occur over a time-course of 30–60 s (Betz and Bewick, 1992; Ryan et al., 1993; Ryan et al., 1996; Wu and Betz, 1996), a time frame consistent with the coated vesicle pathway from earlier measurements that employed electron microscopy (Miller and Heuser, 1984). Thus, slow synaptic vesicle recycling is generally believed to represent the life cycle of an endocytotic vesicle retrieved by the coated vesicle pathway. Recent membrane capacitance measurements indicate that a much more rapid mode of vesicular recycling also exists (Chow et al., 1992; Alvarez de Toledo et al., 1993; von Gersdorff and Matthews, 1994; Artalejo et al., 1995; Hsu and Jackson, 1996; Albillos et al., 1997; Smith and Neher, 1997). Because capacitance is proportional to the surface area of the membrane, a decrease in capacitance can be used as a high-resolution method to monitor the rate of endocytotic retrieval of membrane. In several types of cells—such as adrenal chromaffin cells, mast cells, retinal bipolar nerve terminals, and pituitary nerve


Morgan et al. terminals—capacitance measurements indicate a form of endocytosis that occurs on a time scale of 1–5 s (von Gersdorff and Matthews, 1994; Artalejo et al., 1995; Hsu and Jackson, 1996; Smith and Neher, 1997; Cousin and Robinson, 2000). A comparably rapid component of synapticvesicle endocytosis at both mammalian central synapses and frog NMJs has been deduced from the kinetics of FM1-43 fluorescence destaining (Klingauf et al., 1998; Kavalali et al., 1999; Pyle et al., 2000; Richards et al., 2000; Sankaranarayanan and Ryan, 2000; Stevens and Williams, 2000). In such experiments, synaptic vesicles are prelabeled with FM1-43 and then destained when fusing vesicles discharge their cargo of FM1-43. It has been found that FM1-43 destaining is incomplete and occurs in bursts, suggesting that some vesicles can be retrieved faster than the FM1-43 can escape. Since the departitioning time constant of FM1-43 is 3 s, these measurements are consistent with the idea that fast endocytosis can retrieve vesicles within a few seconds. Also consistent with this hypothesis are observations that FM2-10, an FM1-43 derivative that departitions from fused vesicles with a time constant of less than a second, destained more completely following exocytosis (Klingauf et al., 1998; Kavalali et al., 1999; Pyle et al., 2000; Richards et al., 2000). Because fast endocytosis takes such a short time, it is generally assumed to be mediated by a kissand-run mechanism, which should be coupled to the millisecond time scale of exocytosis. However, limitations in the time resolution of FM1-43 measurements to 1 or 2 seconds make it impossible to determine whether fast endocytosis is mediated by kiss-and-run. Clear evidence for such rapid kissand-run endocytosis comes from measurements of vesicle fusion from neurosecretory cells. The large size of the secretory vesicles in these cells permits detection of the fusion of individual vesicles by capacitance measurements (Chow et al., 1992; Alvarez de Toledo et al., 1993; Albillos et al., 1997). The increases in capacitance associated with such events are often observed to reverse within milliseconds, indicating a very rapid endocytosis event. However, although these events are brief, they are sufficient in duration to allow the transmitter contents of vesicles to be released. These data provide unequivocal evidence that kiss-and-run endocytosis can occur. Recent capacitance measurements at a mammalian central synapse, the calyx of Held,

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Synaptic Vesicle Endocytosis also revealed rapid, reversible synaptic vesicle fusions that resemble kiss-and-run (Sun et al., 2002). Taken together, the fast and slow rates of synaptic vesicle recycling measured with capacitance and FM1-43 support the notion of two modes of endocytosis in nerve terminals, consistent with both the Heuser and Ceccarelli models. However, whether fast and slow endocytosis represents two separate mechanisms has been challenged by recent real-time endocytosis measurements employing synaptoPHlorin, a pH-sensitive fluorescent protein tethered to the lumen of synaptic vesicles (Sankaranarayanan and Ryan, 2000; Sankaranarayanan and Ryan, 2001). Before fusion, the acidic pH of the lumen of the synaptic vesicle keeps the fluorescence of synaptoPHlorin low. Upon fusion, exposure to the higher extracellular pH causes the fluorescence of synaptoPHlorin to increase dramatically. This permits the rate of synaptic vesicle recycling to be determined in real time by monitoring the time-course of decay of the synaptoPHlorin fluorescence signal. Using this technique, Ryan and colleagues showed that the rate of synaptic vesicle recycling depends upon the amount of exocytosis, occurring on a continuum with time constants that range from 4–90 s (Sankaranarayanan and Ryan, 2000). This suggests a single mode of membrane retrieval that proceeds at a speed which depends upon the load of membrane to be retrieved. The variable rate of endocytosis, in turn, arises from a maximum capacity of one synaptic vesicle endocytosed per seconds (Sankaranarayanan and Ryan, 2001). Thus, synaptic vesicle recycling may be mediated by a single retrieval process with variable speed, which argues that rapid endocytosis is not mediated by a simple kiss-and-run reversal of synaptic vesicle fusion. Although the coated vesicle pathway is presumed to be slow, recent experiments suggest that it may proceed faster than originally imagined by eliminating an endosomal-sorting step. Electron micrographs of stimulated nerve terminals indicates that many endosome-like cisternae observed, such as those observed in the Heuser and Reese experiments, are in fact long extensions of the plasma membrane (Koenig and Ikeda, 1996; Takei et al., 1996; Teng and Wilkinson, 2000; Wilkinson and Cole, 2001). These long membrane extensions often end in coated buds and stain positively for synaptic vesicle proteins, indicating that synaptic vesicles can be formed directly from these structures


105 (Maycox et al., 1992; Takei et al., 1996). Thus, synaptic vesicles can be reformed without going through an endosomal compartment, providing a means for more rapid vesicle recycling via coated vesicles.

Molecular Mechanisms of Synaptic Vesicle Endocytosis If both the coated vesicle and kiss-and-run recycling pathways exist, then they should utilize separate molecular mechanisms. An overwhelming amount of evidence has defined the molecular basis for the coated vesicle pathway in synaptic vesicle endocytosis, whereas a molecular mechanism has not yet been identified for kiss-and-run endocytosis. It is now known that the coated vesicles observed in the Heuser and Reese experiments are utilized for vesicle budding from the plasma membrane of all eukaryotic cell types, and that one major constituent of the coat is a protein known as clathrin (Pearse, 1976; Pearse and Bretscher, 1981; Heuser, 1989). Therefore the coated vesicles are referred to as clathrin-coated vesicles (CCVs) (Pearse, 1976). In addition to clathrin, a number of other coat and accessory proteins have been identified that are required for assembling and/or uncoating CCVs in vitro. Based on perturbations of these various CCVassociated proteins in vivo at the synapse, it is now clear that clathrin-mediated synaptic vesicle endocytosis can be divided into several morphologically and functionally distinct steps, known as adaptor recruitment, clathrin assembly, fission, and uncoating (Fig. 2) and that the CCV pathway represents a predominant molecular mechanism for recycling synaptic vesicles (Brodin et al., 2000; Slepnev and De Camilli, 2000). The initial stage of clathrin-mediated synaptic vesicle endocytosis probably requires the recruitment of a subset of coat proteins, the clathrin adaptor/assembly proteins (APs), to the plasma membrane via interactions with plasma membrane phospholipids and integral membrane proteins (Fig. 2) (Ohno et al., 1995; Kirchhausen et al., 1997; Jost et al., 1998; Gaidarov and Keen, 1999; Haucke and De Camilli, 1999; Kirchhausen, 1999; Haucke et al., 2000; Ford et al., 2001). In nerve terminals, the clathrin APs associated with coated vesicles are the synapse-specific AP180 and ubiquitously-expressed AP-2 (Maycox et al., 1992; Zhou et al., 1992; Zhou et al., 1993; Takei et al., 1996; McMahon, 1999). Both AP180 and AP-2 bind to membrane phospholipids

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Fig. 2. Molecular mechanisms of clathrin-mediated synaptic-vesicle recycling in the nerve terminal. CCV formation in the nerve terminal can be divided into several morphologically and functionally distinct steps—recruitment, clathrin assembly, fission, and uncoating—each of which requires a subset of clathrin-associated proteins. The initiation of CCV formation begins with the recruitment of the clathrin assembly proteins, AP180 and AP-2, to synaptic-vesicle proteins and phospholipids in the plasma membrane. Next, clathrin assembly occurs when AP180 and AP-2 bring together clathrin molecules into a polygonal cage in a process that induces inward invagination of the plasma membrane and that requires endophilin. Following coated bud formation, dynamin and amphiphysin are recruited to form a ring around the neck of the bud. The actions of dynamin, amphiphysin, and endophilin somehow lead to the separation, or fission, of the coated vesicle from the plasma membrane. Finally, CCV uncoating occurs when Hsc70 is recruited to the coat. Uncoating also requires auxilin and synaptojanin, although the precise timing of their recruitment to the CCV is still unclear. Following uncoating, the synaptic vesicle is then refilled with neurotransmitter and reused during a subsequent bout of exocytosis.

in an interaction that is important for targeting the APs to the plasma membrane and marking the site for coated vesicle formation (Hao et al., 1997; Gaidarov and Keen, 1999; Cremona and De Camilli, 2001; Ford et al., 2001). Once at the membrane, APs also interact with membrane proteins and target them for internalization. In synapses, the interaction of AP-2 with synaptotagmin and SV2 is likely to play a role in recycling these synaptic vesicle proteins (Haucke and De Camilli, 1999; Haucke et al., 2000). Similarly, AP180 may target synaptobrevin for recycling because this synaptic vesicle protein is selectively mislocalized in C. elegans AP180 mutants (Nonet et al., 1999). Thus, it is likely that AP180 and AP-2 each target a subset of synaptic vesicle proteins for recycling, substantiating the idea that synaptic vesicles are rapidly recycled directly from the plasma membrane rather than being sorted through an endosomal compartment. Following AP recruitment to the plasma membrane, AP180 and


AP-2 assemble individual clathrin molecules into a regular polygonal cage in a process that induces inward invagination of the budding coated pit (Fig. 2) (Ye and Lafer, 1995; Kirchhausen, 1999; Brodin et al., 2000; Pearse et al., 2000; Slepnev and De Camilli, 2000). This process of clathrin assembly may also require the regulatory actions of other proteins, such as Eps15, epsin, and endophilin (Tebar et al., 1996; Chen et al., 1998; Benmerah et al., 1999; Ringstad et al., 1999; Scheele et al., 2001). Genetic and acute perturbations of AP180 and AP-2 indicate that clathrin assembly represents a crucial step in recycling synaptic vesicles (Gonzalez-Gaitan and Jackle, 1997; Zhang et al., 1998; McMahon, 1999; Morgan et al., 1999; Nonet et al., 1999; Morgan et al., 2000). For example, synapses in Drosophila AP180 and AP-2 mutants and C. elegansAP180 mutants all have fewer synaptic vesicles than controls, a phenotype that is consistent with blocking synaptic vesicle recycling (Gonzalez-Gaitan and Jackle, 1997; Zhang et al.,

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Synaptic Vesicle Endocytosis 1998; Nonet et al., 1999). It is possible that these defects are caused by blocking some other AP function, such as membrane recruitment, rather than from a specific defect in the clathrin-assembly activity of these proteins. However, specific peptide inhibitors of AP180 and AP2-mediated clathrin assembly in vitro also inhibit neurotransmitter release, dramatically reduce the numbers of synaptic and coated vesicles, and cause corresponding expansions of the plasma membrane in squid giant synapses, indicating that clathrin assembly is essential for recycling synaptic vesicles in vivo (Morgan et al., 1999; Morgan et al., 2000). In addition to AP180 and AP-2, formation of the coated vesicle may also require the lipid-modifying activity of the clathrin accessory protein endophilin to induce inward curvature of the plasma membrane (Schmidt et al., 1999). Consistent with this notion, acute and genetic disruptions of endophilin result in a buildup of clathrin-coated structures at various stages of inward curvature and block synaptic vesicle recycling (Ringstad et al., 1999; Guichet et al., 2002; Verstreken et al., 2002). Together, these results indicate that the formation of CCVs is important for recycling synaptic vesicles. Following clathrin assembly, the functions of other CCV accessory proteins, including dynamin and its binding partners amphiphysin and endophilin, are necessary for processes that constrict the neck of the coated bud and lead to fission, thus creating a free CCV (Fig. 2). Dynamin, amphiphysin, and endophilin all cause the deformation of round liposomes into tubules in vitro by oligomerizing into regularly spaced rings around membranes, indicating one possible mechanism for how the neck of the coated bud becomes constricted (Takei et al., 1995; Warnock et al., 1996; Takei et al., 1999; Farsad et al., 2001). Dynamin is critical for CCV fission because perturbation of its GTPase activity induces deeply invaginated membrane tubules and prevents the separation of coated buds from the plasma membrane both in vitro and in vivo (Damke et al., 1994; De Camilli et al., 1995; Takei et al., 1995; Takei et al., 1996; McNiven, 1998; Stowell et al., 1999; Sever et al., 2000; Marks et al., 2001). The precise mechanism of dynamin GTPase activity—whether through a pinching or springing action—is still unclear. However, a temperaturesensitive mutation in Drosophila dynamin, known as shibire, results in a complete loss of synaptic vesicles from the nerve terminal, inhibits synaptic transmission, and causes reversible paralysis in the flies, indi-


107 cating the importance of this CCV-associated protein in synaptic vesicle recycling (Poodry and Edgar, 1979; Koenig and Ikeda, 1989; De Camilli et al., 1995; Koenig and Ikeda, 1996; Kuromi and Kidokoro, 1998). The actions of amphiphysin, endophilin, and synaptojanin are also crucial for the fission process because acute disruptions in their recruitment to the neck of the coated bud or genetic disruptions of these proteins arrest CCV formation at a late stage and block synaptic vesicle recycling (Shupliakov et al., 1997; Gad et al., 2000; Di Paolo et al., 2002). Together, these results indicate that fission of the CCV is an important step in recycling synaptic vesicles, providing further evidence in support of the coated vesicle pathway as a predominant mechanism. Following their formation, CCVs must be uncoated before the vesicle is refilled with neurotransmitter for another round of fusion (Fig. 2). Uncoating of CCVs in vitro requires the ATPase Hsc70 and the clathrin-associated protein auxilin (Schlossman et al., 1984; Chappell et al., 1986; Ahle and Ungewickell, 1990; Ungewickell et al., 1995). Perturbation of Hsc70 or auxilin in vivo causes defects in clathrinmediated endocytosis in yeast, C. elegans, and mammalian cells (Greener et al., 2000; Pishvaee et al., 2000; Cremona, 2001; Newmyer and Schmid, 2001). In neurons, acute disruption of the interaction between Hsc70 and auxilin in squid giant synapses inhibits synaptic transmission, decreases synaptic vesicles, and increases the number of CCVs, a phenotype consistent with blocking synaptic vesicle recycling by perturbing CCV uncoating (Morgan et al., 2001). CCV uncoating is also blocked by disruptions of synaptojanin, a synaptic inositol phosphatase, suggesting that phospholipid metabolism is important for synaptic vesicle recycling (Cremona et al., 1999; Gad et al., 2000; Harris et al., 2000; Cremona and De Camilli, 2001). Further research is likely to focus on the precise sequence of reactions that lead to CCV formation, and how these reactions are spatiotemporally coordinated to be vectorial within the nerve terminal. Similarly, studies of phosphoinositide metabolism and protein phosphorylation will further elucidate the mechanisms and regulation of CCV-mediated endocytosis (Cousin and Robinson, 2001; Cremona and De Camilli, 2001). However, based on a wealth of data it is already clear that the coated vesicle pathway represents a predominant mechanism for recycling synaptic vesicles.

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108 In contrast to the tremendous progress that has been made in elucidating the molecular mechanism underlying the coated vesicle pathway for synaptic vesicle recycling, the potential mechanisms underlying kiss-and-run are poorly understood. Kiss-andrun in neurosecretory cells is regulated by intracellular calcium, PKC, and the PKC-substrate Munc-18 (Ales et al., 1999; Graham et al., 2000; Burgoyne et al., 2001; Fisher et al., 2001). In general, it is assumed that fast endocytosis and kiss-and-run are clathrin-independent. In neuro-secretory cells, this appears to the be the case because fast endocytosis is unaffected by clathrin antibodies (Artalejo et al., 1995). However, rapid endocytosis is dynamindependent. Similarly, a morphologically identified rapid pathway for recycling synaptic vesicles has been identified in Drosophila dynamin mutants that is mediated by the formation of uncoated structures, suggesting that it is clathrin-independent (Koenig and Ikeda, 1996). However, it can be argued that these structures somehow became uncoated prior to separation from the plasma membrane. In squid nerve terminals, a form of synaptic vesicle recycling has been reported to be clathrin-independent, which requires an interaction between dynamin and synaptophysin (Daly et al., 2000; Daly and Ziff, 2002). However, in these experiments there was an increase in CCVs, suggesting a block in the clathrin pathway. Although the molecular mechanisms underlying fast endocytosis in nerve terminals are still unclear, recent use of green fluorescent protein (GFP)-clathrin to observe coated pit formation in fibroblasts suggests the possibility that fast endocytosis may be clathrin-mediated. In these experiments, a few clathrin-coated pits appeared within 1–2 s in regions of the plasma membrane where they did not exist previously (Gaidarov et al., 1999). This sudden appearance is unlikely to be a result of the movement of a previously formed coated pit from a distant location into the field of view, because coated pits move very little once they are formed (Gaidarov et al., 1999). Future experiments using GFP-clathrin are needed in order to unequivocally establish whether CCVs form in synapses at rates consistent with the kinetic measurements of fast endocytosis.

The Location of Synaptic Vesicle Endocytosis The classical studies of synaptic vesicle recycling suggest that kiss-and-run endocytosis may occur at


Morgan et al. the active zone, and clathrin-mediated endocytosis may occur beyond active zones (Fig. 1) (Ceccarelli et al., 1972; Ceccarelli et al., 1973; Heuser and Reese, 1973; Ceccarelli et al., 1979; Heuser, 1989). Recent suggestions are consistent with recycling at both locations. For example, the plasma-membrane invaginations that mediate rapid endocytosis in Drosophila dynamin mutants emanate directly from the active zone, whereas similar structures mediate slower endocytosis that arises at regions away from the active zone (Koenig and Ikeda, 1996). In these mutants, the active-zone endocytotic structures are uncoated, as expected from the kiss-and-run model. However, in contrast to the kiss-and-run model, these structures were much larger than single synaptic vesicles. Other experiments place clathrinmediated endocytosis in regions of the synapse much closer to the active zone than originally imagined from the original Heuser and Reese experiments (Fig. 3). At the snake NMJ, CCVs are found within hot spots of synaptic vesicle endocytosis, and these hot spots have a distribution similar to active zones (Teng et al., 1999; Teng and Wilkinson, 2000). Thus, synaptic vesicle recycling occurs both at and away from active zones, and the coated vesicle pathway may be able to mediate recycling at both locations.

Does the Coated Vesicle Pathway Predominate Under Physiological Levels of Activity? The classical experiments demonstrating a role for coated vesicles in synaptic vesicle recycling were performed under conditions of high levels of nerve activity (10 Hz stimulus frequency, 15 min duration) (Heuser and Reese, 1973). In contrast, coated vesicles were reported to be rare under lower levels of stimulation (Ceccarelli et al., 1973). No correlation was detected between coated vesicles and transmitter release or estimated synaptic vesicle turnover during more intense stimulation (Ceccarelli et al., 1973; Torri-Tarelli et al., 1987). As a result, there has been doubt about whether the clathrin pathway is utilized under conditions of physiological synaptic activity. More recent studies in the squid giant presynaptic nerve terminal indicate that the clathrin pathway predominates when the synapse is undergoing physiological levels of activity (1 stimulus every

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Fig. 3. The coated vesicle pathway is the predominant mechanism for synaptic vesicle recycling. Recent modifications of the original models have led to the suggestion that the coated vesicle pathway is the primary means for recycling synaptic vesicles based on demonstrations of its speed, molecular mechanisms, and location. First, demonstrations that many apparent endosomes are long extensions of the plasma membrane offers the possibility of quickly recycling synaptic vesicles through the coated vesicle pathway without going through a slower, endosomal compartment. Second, perturbing clathrin and many of its associated coat proteins blocks synaptic vesicle recycling and completely inhibits neurotransmitter release, indicating a critical functional role for the coated vesicle pathway. Finally, coated vesicles have now been observed both at and away from AZs. In contrast, the molecular mechanisms underlying kiss-and-run are poorly understood, making it much more difficult to establish whether this pathway plays any physiological role in synaptic vesicle recycling.

30 s) (Morgan et al., 1999; Morgan et al., 2000; Morgan et al., 2001). In these studies, reagents that perturbed either the interactions between clathrin and the APs or the recruitment of clathrin-uncoating proteins were injected into nerve terminals. Even at a low frequency of synaptic activity, neurotransmitter release is completely blocked by such reagents, suggesting that the clathrin pathway can account for all synaptic vesicle trafficking. In addition, the size of the synaptic vesicle pool is dramatically reduced. However, in these experiments and others in which coated vesicle pathway is acutely blocked, there are always at least a few synaptic vesicles remaining in the nerve terminal (Shupliakov et al., 1997; Morgan et al., 1999; Ringstad et al., 1999; Gad et al., 2000; Morgan et al., 2000; Morgan et al., 2001). Using this approach, it is impossible to determine whether the remaining synaptic vesicles originated from clathrin-independent endocytosis or whether their presence is somehow caused by a secondary block in exocytosis. Asecondary block in exocytosis could


result from the buildup of synaptic vesicle proteins in the plasma membrane, causing an improper formation of the SNARE protein complexes necessary for synaptic vesicle fusion (Augustine et al., 1999; Lin and Scheller, 2000). Nonetheless, these experiments show that the coated vesicle pathway is not simply a compensatory mechanism used under intense synaptic stimulation. Rather, these experiments indicate that the coated vesicle pathway is the predominant mode of recycling synaptic vesicles under physiological levels of synaptic activity and that no other clathrin-independent mechanisms compensate when the clathrin pathway is blocked.

Conclusion Since the original demonstrations of synaptic vesicle recycling by Heuser, Reese, Ceccarelli, and colleagues, it has been established that synaptic vesicle recycling can be both fast and slow, depending

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110 on the amount of membrane added during a period of exocytosis. Observations of coated-pit formation in non-neuronal cells suggest that this process may be capable of keeping up with the rapid speeds necessary for fast endocytosis. This is made possible by the fact that coated vesicles do not need to proceed through an endosomal step in order to recycle synaptic vesicles (Fig. 3). Further, it has been established that the coated vesicle pathway is mediated by clathrin and its many accessory proteins, the functions of which are crucial for synaptic vesicle recycling, even when the synapse is undergoing modest, physiological levels of activity. Finally, it is now clear that synaptic vesicle recycling occurs both near and far from active zones, and that the coated vesicles may account for the recycling in both locations. In contrast to the evidence supporting the coated vesicle pathway, it is still unclear whether true kiss-and-run exists at the synapse (Fig. 3). Moreover, despite suggestions of clathrin-independent synaptic vesicle recycling, it is clear that the clathrindependent pathway is prevalent under physiological conditions because perturbations of the clathrin pathway can completely block neurotransmitter release. Whether fast endocytosis employs an entirely separate molecular pathway from the coated vesicle pathway has not yet been determined. These and questions of the spatiotemporal regulation of membrane trafficking are likely to be central issues during future explorations of synaptic vesicle recycling.

Acknowledgments Preparation of this review was supported by NIH grants NS-29051 and NS-21624, the Muscular Dystrophy Association and a Yale University Alexander Brown-Coxe Fellowship.

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