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(264), re19. [DOI: 10.1126/stke.2642004re19]2004Sci. STKEThierry Galli and Volker Haucke (21 December 2004)Cycling of Synaptic Vesicles: How Far? How Fast!

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11. L. M. Traub, Sorting it out:AP-2 and alternate clathrin adaptors in endocyticcargo selection. J. Cell Biol. 163, 203208 (2003).

12. M. G. Ford, I. G. Mills, B. J. Peter, Y. Vallis, G. J. Praefcke, P. R. Evans, H. T.McMahon, Curvature of clathrin-coated pits driven by epsin.Nature419,361366(2002).

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14. C.T.Wang, J. C. Lu, J. Bai, P. Y. Chang, T. F. Martin, E. R. Chapman, M. B.Jackson, Different domains of synaptotagmin control the choice betweenkiss-and-run and full fusion. Nature424, 943947 (2003).

15. T. Virmani, W. Han, X. Liu, T. C. Sudhof, E. T. Kavalali, Synaptotagmin 7splice variants differentially regulate synaptic vesicle recycling. EMBO J.22, 53475357 (2003).

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Citation: T. Galli, V. Haucke, Review update: Cycling of synaptic vesicles.Sci. STKE2004, re19/DC1 (2004).

Citation for the related Review: T. Galli, V. Haucke, Cycling of synapticvesicles: How far? How fast! Sci. STKE2004, re19 (2004).

Citation for the previous version of this Review: T. Galli, V. Haucke, Cyclingof synaptic vesicles: How far? How fast! Sci.STKE2001 (88), re1 (2001).

REV I EW UPDATE


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Synaptic transmission is based on the regulatedexocytotic fusion of synaptic vesicles filled withneurotransmitter. In order to sustain neurotransmit-ter release, these vesicles need to be recycled local-ly. Recent data suggest that two tracks for thecycling of synaptic vesicles coexist: A slow track inwhich vesicles fuse completely with the presynapticplasma membrane, followed by clathrin-mediatedrecycling of the vesicular components, and a fasttrack that may correspond to the transient openingand closing of a fusion pore. In this review, we at-tempt to provide an overview of the components in-volved in both tracks of vesicle cycling, as well as toidentify possible mechanistic links between these

two pathways.

Pathways for the Cycling of Synaptic Vesicles: Fastand Slow Tracks

Neuronal signaling involves the release of neurotransmitter atthe synapse, a specialized site of interneuronal contact. Non-

peptide neurotransmitters are stored in synaptic vesicles, spe-cialized secretory organelles of presynaptic nerve terminals.Synaptic vesicles undergo calcium-regulated exocytosis upondepolarization of the nerve terminal and subsequent entry ofcalcium. The vesicles are formed and recycled locally withinthe presynaptic compartment.

The classical view of synaptic vesicle cycling is a route withseveral stop-and-go steps at which decisions are made to pro-ceed or to wait (1). This track for the cycling of synaptic vesi-

cles takes about 40 to 60 s; thus, it will be referred to as theslow track. In this process, synaptic vesicles move toward the

plasma membrane, are primed, and then docked to the plasmamembrane. Upon stimulation, they fuse with the plasma mem-

brane and release neurotransmitter into the synaptic cleft. Thecomponents of synaptic vesicles that become inserted into the

plasma membrane are subsequently internalized by clathrin-coated vesicles. Finally, these endocytic vesicles shed theircoats and are refilled with neurotransmitter before reenteringthe synaptic vesicle pool [(Fig. 1); Animation 1 (http://stke.sci-encemag.org/cgi/content/full/sigtrans;2004/264/re19/DC2)].The priming of synaptic vesicles may correspond to the ATP(ATP)-dependent reaction preceding the ATP-independent calci-um-mediated triggering step of secretion seen in the case of

granule exocytosis in neuroendocrine cells (2). Priming ofsynaptic vesicles involves proteins and phosphoinositides.Docked synaptic vesicles can be seen by EM in close appositionto the presynaptic plasma membrane of nerve terminals (3).

Surprisingly, these apparently do not correlate with the physio-logically defined readily releasable pool (RRP), which ap-

pears to be dispersed almost randomly throughout the vesiclecluster (4). Terminal fusion of the synaptic vesicle with the

plasma membrane involves the formation of a putative fusionpore and complete bilayer mixing, followed by the endocytoticreinternalization of the vesicular membrane proteins and lipids.Both morphological (57) and genetic (811) experiments haveshown that recycling of synaptic vesicle components occurs byclathrin-mediated endocytosis [reviewed in (12)]. Clathrin-coated pits are frequently observed on the outer margin of theactive zone of a synapse, in particular after stimulation (7).Reinternalization of synaptic vesicles on the slow track occurseither from the plasma membrane itself or from cisternae remi-

niscent of endosomes that emanate from the plasma membrane(5, 13) [(Fig. 1); Animation 1 (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/264/re19/DC2)]. These cisternae,however, do not correspond to classical recycling endosomesthat pass through sorting stations downstream of the plasmamembrane. Indeed, true endosomes are almost completely ab-sent in the synaptic regions of mature differentiated neuronswhere synaptic vesicles constitute the vast majority of membra-nous organelles (2). By contrast, synaptic vesicle proteins, suchas synaptophysin, SV2, and synaptobrevin, are found in recy-cling endosomes in the soma of immature neurons and neuroen-docrine cells (14, 15) or when expressed in nonneuronal cells(1618). Furthermore, a significant pool of synaptic vesicle

proteins is found at the plasma membrane (19), where it couldconstitute a stranded reservoir that may be mobilized by en-

docytosis (20). This slowest version of the slow track is reminis-cent of that followed by the newly synthesized synaptic vesicle

protein synaptophysin, which is found first at the plasma mem-brane after constitutive exocytosis, before entering the synapticvesicle pool (21).

Although this slow-track model conforms with the proper-ties of cycling of synaptic vesicles during intense stimulation ofa nerve, recent advances using membrane-fluorescent styryldyes to measure the kinetics of synaptic vesicle exo- and endo-cytosis (2224 ) suggest that another, fasterless than asecondpathway for cycling may coexist at the synapse. In thisalternative process, a subpopulation of synaptic vesiclesthatcould correspond to the RRP (25)fuses transiently with the

plasma membrane under milder stimulations (less than 30 Hz

stimuli, (26) [(Fig. 2); Animation 2 (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/264/re19/DC2)]. The RRP can beestimated to contain 6 to 10 synaptic vesicles per active zone (4,25, 27) and could preferentially follow the fast track. The fasttrack may use similar mechanisms of exo- and endocytosis asthe slow track but function at a higher rate. Alternatively, thefast track could be limited to only two hypothetical steps: theopening and the closing of an as-yet-undefined proteinaceousor lipid-made fusion pore. This kiss-and-run mechanism ofsynaptic vesicle cycling was first proposed on the basis of an

Cycling of Synaptic Vesicles: How Far? How Fast!

Thierry Galli1* and Volker Haucke2*

(Published 21 December 2004)

(Revised 11 January 2005)

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1Membrane Traffic and Neuronal Plasticity Group, INSERM U536, In-stitut du Fer--moulin, 75005 Paris, France. 2Institut fr Chemie-Bio-chemie, Freie Universitt Berlin, D-14195 Berlin, Germany.

*Corresponding authors. E-mail, [email protected] (T.G.) [email protected] (V.H.)


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incomplete correlation between the number of morphologicallydistinct endocytotic intermediates and the synaptic endocytoticactivity. Mechanistically, the transient opening of a fusion porewould correspond to repetitive swift kisses rather than kiss-and-run, because the synaptic vesicle would stay tightly attached to the

plasma membrane. Successive rounds of transient vesicle fusionwithout complete collapse of vesicle membranes have been observed

by single vesicle tracking in living hippocampal neurons (26). This

fast track would be expected to be so fast that a single vesicle couldrelease and refill in a matter of seconds (24, 28). Such a rapid cy-cling of synaptic vesicles has been observed at synapses of hip-

pocampal neurons in culture (20, 2224, 26, 29) and in neuroen-docrine cells (28). At present, it is, however, unclear whether fast cy-cling involves components of the clathrin-mediated recycling path-way or whether it represents a mechanism similar to kiss-and-run.Cycling by kiss-and-run on the fast track would avoid the delay andthe need for cargo protein sorting that characterizes the slow track,yet retains the identity of the fused vesicles during activity (30). Thisidea, however, is at odds with a recent report showing that the slowtrack is fast enough to compensate for exocytosis during continuousaction potential firing at 10 Hz (31). The time constant of endocyto-sis increases with the strength of the stimulation, so that an increase

in the rate of exocytosis induces a decrease in the rate of endocytosisthat may lead to short-term synaptic depression (32). Therefore, thefast track might allow neurons to react rapidly to mild stimulations,whereas the slow track becomes especially important when neuronsreact to strong repetitive stimuli with a depression if the stimuli lastfor too long. The physiological balance between these two routes re-mains largely unknown, but is likely to differ between differentsynapses and may play a role in presynaptic plasticity.

In this review, we describe the mechanistic details of theslow track and propose a speculative model for the fast track ofsynaptic vesicle cycling. We also postulate hypothetical com-mon mechanisms for the fusion and fission of membranes in-volved in both tracks.

Common Features: Membrane Fusion and Fission

Fusion of membranes. Our understanding of the molecularmechanism of membrane fusion has been greatly advanced inthe last decade through the combination of in vitro assays(3335), yeast genetics (36), and the elucidation of the mecha-nism by which clostridial neurotoxins (that is, botulinum neuro-toxins and tetanus neurotoxin) block exocytosis in neurons(3740). These reports converged to support the SNARE hy-

pothesis of membrane fusion (34, 35) which has guided mostrecent studies (4146). According to this model, synaptobrevin(the v-SNARE on synaptic vesicles), syntaxin, and SNAP-25(the corresponding t-SNAREs on the plasma membrane) form a

parallel four-helix coiled-coil (43) that can zipper from their Ntermini to their membrane-anchored C termini (46). This as-sembled ternary complex is extremely thermostable (47), and

its formation might furnish the energy that is required to effectthe fusion of the two lipid bilayers.Consistent with this view, it has been demonstrated that the

specific assembly of correctly paired SNARE complexes is suf-ficient for the fusion of liposomal membranes (48, 49). This re-constituted fusion system, however, fails to reveal a requirementfor Ca2+ ions, a hallmark of neurosecretion in vivo when fusionwas measured globally among a population of fusing liposomes(48). In contrast, the fusion of single v-SNAREcontaining li-

posomes with t-SNAREcontain ing deposi ted bilayers was

found to be stimulated by calcium and magnesium (50), butthese data are contradicted by another report (51). The rate of

proteoliposome fusion is controlled by the intrinsic propertiesof SNARE molecules (52). The transmembrane domain of syn-taxin has been proposed to line the fusion pore, based on thefinding that mutations of residues within the transmembranesegment of syntaxin affect pore conductance (53). Topologicallyinverted SNARE proteins have also been shown to promote cell

fusion (54).Observations obtained in chromaffin cells treated with bo-

tulinum neurotoxin A (which inhibits exocytosis by proteolyti-cally inactivating SNAP-25) or antibodies directed againstSNAP-25 have led to the idea that the ternary complex can existin two different conformations, a loose and a tight state (55,56). The tight state may correlate with the fast kinetic compo-nent of exocytosis promoted by calcium entry (56). Therefore,fast vesicle fusion could be triggered by the conversion of com-

plexes from the loose to the tight state. The loose state couldcorrespond to a half-zippered or an incomplete SNAREcomplex. In support of the second hypothesis, a partial and re-versible association of the SNARE complex consisting ofsynaptobrevin bound to SNAP-25 and partial association of

syntaxin is seen during priming of secretory granules (57). Thetight state could correspond to a fully zippered and completeSNARE complex (Fig. 1A and Fig. 2) or to higher-orderSNARE oligomers (58). In summary, it is now evident thatSNARE complex formation is crucial for synaptic vesicle fu-sion, but it is not yet clear whether or not additional compo-nents are required to confer Ca2+-dependence and controlspecificity.

Calcium triggering. High intracellular concentrations ofCa2+propel rapid neurotransmitter release and thus are expect-ed to act on late steps of synaptic vesicle fusion, but may alsoact to prepare fusion. Syntaxin 1 and SNAP-25 reversibly pre-assemble after calcium entry (59) and this assembly is de-creased by botulinum neurotoxin E, which cleaves SNAP-25and causes severe blockade of exocytosis (60). Synaptotagmin,

a major calcium-binding protein of synaptic vesicles, is re-quired for fast, synchronous release of neurotransmitter (61).This activity may involve binding of synaptotagmin to phospho-lipids, to t-SNAREs, or most likely to both. Association ofsynaptotagmin with the ternary complex protects the core com-

plex from premature disassembly (34). Calcium enhances theinteraction between synaptotagmin and the ternary SNAREcomplex (62), and the cytosolic domain of synaptotagmin pro-motes calcium-dependent fusion of reconstituted proteolipo-somes in vitro (6365). One might therefore speculate thatsynaptotagmin-mediated conversion of loose into tight SNAREcomplexes may facilitate membrane fusion, perhaps throughsynaptotagmins action on SNAP-25 (64), the SNAP-25/syntaxin1complex (66), or both.

Another set of experiments suggests that SNAP-25 is itselflinked to the control of calcium homeostasis. Relative to gluta-matergic neurons, which express SNAP-25, GABAergic cells,which instead express SNAP-23, show a greater increase in in-tracellular calcium in response to depolarization (67). Further-more, a mutation that interferes with calcium triggering of exo-cytosis has been identified in SNAP-25 (68), which may belinked to its putative role in stabilizing vesicles in the primedstate (69). Thus, SNAP-25 could be involved in the regulationof intracellular calcium levels through yet-unknown partners

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that may include calcium-dependent channels, pumps, or otherligands. These observations are particularly intriguing, given theimportance of the SNAP-25/synaptotagmin interaction and the roleof the latter in the triggering of release. SNARE regulation of calci-um levels has also been described for vacuole fusion in yeast (70).

Just after fusion. Fusion of synaptic vesicles is followed bySNARE complex disassem-

bly. The hexameric AAA-

type ATPase N-ethyl-maleimide sensitive factor(NSF) with the aid of solu-

ble NSF at tachment pro-teins (SNAPs, which are to

be distinguished from the t-SNAREs SNAP-25 andSNAP-23) is able to disso-ciate ternary complexes(44), which allows synapticvesicles to cycle throughfurther rounds of exocytosis(45). Fly mutants with re-duced SNAP activity show

both defects in neurotrans-mitter release and accumu-lation of the synapticSNARE complex (71),which suggests thatSNARE complex disassem-

bly is important for normalsynaptic activity. The ioniclayer of the SNARE com-

plex, corresponding to thecentral arginine (R) andglutamine (Q) residues ofthe v- and t-SNARE motifs,respectively, is a preferen-tial site of action for NSF-

driven disassembly (72 ).Furthermore, mutation of synaptobrevins central arginine into a

proline inhibits disassembly, which leads to mislocalization ofsynaptobrevin to the plasma membrane (73). These data indi-cate that an ionic interaction is essential for SNARE function.

A function of SNAREs before fusion? Although most dataobtained in neuronal model systems are compatible with a ma-

jor role for SNAREs in triggering membrane fusion, studies ofthe slow SNARE-dependent homotypic fusion of yeast vacuoleshave suggested that SNAREs may act before membrane fusionand that the fusion pore could be a proteinaceous channel.Formation of trans-SNARE complexes (the ternary complex) isa transient step that precedes vacuolar membrane fusion in thisassay (74). Biochemical analysis of downstream effectors of

vacuolar SNARE complexes has revealed a requirement for cal-cium and calmodulin (75). The immediate target of calmodulinis the V0 domain of the vacuolar ATPase (V-ATPase), a proteinfound in the membranes of all post-Golgi acidic compartments,including synaptic vesicles, where it associates with synapto-

brevin and synaptophysin (76). The V0 sector is mainly com-posed of the c subunit of the V-ATPase, which is a proteolipidalso known as ductin or mediatophore. V0 sectors from oppos-ing membranes are capable of forming aqueous proteolipidchannels built from trans complexes. These sealed channels be-

tween apposed vacuoles have been proposed to constitute a porethat could radially expand and that eventually could result in thecomplete mixing of the lipid bilayers (77). At present, it is un-known whether V0-like trans complexes constitute the fusion

pore through which neurotransmitter is released or whether thisoccurs solely through lipid bilayer mixing (52).

Additional regulations. Munc18 participates in the cycling ofsynaptic vesicles, because deletion of Munc18 completely blocksrelease of neurotransmitter (78). The biochemical activity ofMunc18 appears to be dependent on its binding to the N-terminalextension of syntaxin, which, like the N-terminal extension of oth-er SNAREs, has been proposed to regulate vesicle docking (79),SNARE assembly (8082), fusion pore dynamics (83), and thetargeting of syntaxin 1 (8486). Other regulators include complex-ins, proteins that bind to SNARE complexes and play a role in thesensitivity of neurotransmitter release to calcium (87, 88).Complexins might promote oligomerization of SNARE complex-es to higher-order macromolecular structures by stabilizing fullyassembled SNARE complexes (89, 90) or by promoting interac-

tion between syntaxin and synaptobrevin transmembrane regions(91). These oligomerized SNARE complexes could form a ringaround the fusion pore (58).

From fusion to fission. Immediately after fusion, the mem-brane of the synaptic vesicle constitutes a microdomain of theplasma membrane that needs to be recycled into a new synapticvesicle to keep the nerve terminals homeostasis. In the case ofcomplete fusion in the slow track, it is not known whether thecomponents of the synaptic vesicle then freely diffuse withinthe plasma membrane. Cholesterol, a lipid that is highly en-

RE V I EW

Synaptotagmin

Dynamin PIP

PIP2

Ca2+ Loose SNAREcomplex

Tight SNAREcomplex

Calcium channel

Start

End

Synaptobrevin

SyntaxinSNAP-25

NSF

Fig. 2. On the fast track, the synaptic vesicles stay in close proximity to the plasma membrane. Thisstate may correspond to vesicle attachment, in part through loose SNARE complexes between synap-tobrevin, syntaxin1, and SNAP-25 or a yet unknown docking system. Entry of calcium could trigger aconformational change in synaptotagmin 1 that would allow further zippering of the SNARE complexinto a tight state, which would lead to the hemifusion stage of lipid bilayer fusion followed by the open-ing of a lipid-made fusion pore. An unknown mechanism would prevent the SNARE complex from pro-ceeding further, thus preventing the membrane anchors of synaptobrevin and syntaxin from dissociat-ing. SNAPs and NSF might catalyze the reversion of tight complexes into incompletely disassembledSNARE complexes, and conformational changes in dynamin could close the lipid-made fusion pore.See Animation 2 (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/264/re19/DC2), showing thesynaptic release through the formation loose and tightly zippered SNARE complexes.


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riched in synaptic vesicles (45), interacts with the synaptic vesi-cle proteins synaptophysin and synaptotagmin (92), and thus,

proteins from newly fused synaptic vesicles could stay concen-trated in cholesterol-rich microdomains at the plasma mem-

brane until they are retrieved by endocytosis in the slow track.Furthermore, syntaxin and SNAP-25 are present in smallcholesterol-dependent clusters at the plasma membrane in liv-ing cells (93, 94), but this targeting is likely not to be intrinsic

(95). These clusters may organize exocytosis, but also may fa-vor the endocytotic retrieval of fused vesicles by restricting the

plasma membrane area where fusion occurs.In addition to cholesterol, phophoinositides may serve as a

spatial landmark for membrane fusion and fission, because theyare required in both exo- and endocytotic reactions (96100).Their cyclic phosphorylation and dephosphorylation could thusconfer directionality to the vesicle cycle (98). Putative targetsfor phosphoinositides in membrane fusion and fission includesynaptotagmin (101, 102), calcium-dependent activator proteinfor secretion (CAPS) (103), dynamin (104), and the inositol-

phosphatase synaptojanin (105).Fission of membranes. Fission of membranes allows the

newly reformed synaptic vesicle to be physically separated from

the plasma membrane. This mechanism is common to the slowand the fast track of vesicle cycling (Figs. 1 and 2). Indeed, theclosing of the fusion pore on the fast track is formally equiva-lent to the fission of the two membranes during the last step inthe budding of coated vesicles on the slow track. The innerleaflet of the narrow neck between the vesicular bud and thedonor membrane becomes constricted such that complete fis-sion of the budding vesicle is triggered (104, 106, 107).

A key participant in the endocytotic fission of membranes isthe large guanosine triphosphatase (GTPase) dynamin, a proteinthat has the capacity to self-assemble into oligomeric rings atthe neck of invaginating coated pits (106, 107). Mutationalanalysis of dynamin in vitro (108) and of the temperature-sensitive mutant shibire from Drosophila melanogasterin vivo(10) revealed that dynamin is essential for a late step in the for-

mation of clathrin-coated vesicles. Dynamin can bind to lipidsthrough its pleckstrin homology domain and, when assembled,has the ability to cause liposomes to form tubules and to frag-ment (109, 110). Therefore, dynamin has been proposed eitherto strangle the neck of an endocytotic intermediate physicallyuntil the neck collapses, forcing membrane fission (pinchase)(109) or to undergo a helical expansion upon GTP hydrolysis,resulting in scission by a spring-like mechanism (poppase)(111). The pinchase model of dynamin action has gained recentsupport from electron microscopic analysis of dynamin rings inthe constricted, as well as the unconstricted, state (112). In ei-ther of these reactions, assembly of dynamin complexes withamphiphysin may assist in the constriction or helical expansionfunctions. Amphiphysin is a cytosolic protein that binds

clathrin, adaptor protein 2 (AP-2), and dynamin (12). In vitro,amphiphysin facilitates dynamin-mediated liposome fragmenta-tion (113). Disruption of the interaction between amphiphysinand dynamin in vivo results in loss of dynamin collars and sub-sequent impairment of clathrin-mediated recycling of synapticvesicles at the stage of invaginated-coated pits (5).

Analysis of dynamin mutants defective in either GTP bind-ing or hydrolysis has stimulated intense debate over whether dy-namin could alternatively function as a molecular switch ratherthan as a mechanochemical enzyme (114, 115). The switch

model postulates that activated GTP-bound dynamin might sig-nal to downstream effectors. One such possible partner for dy-namin is the lysophosphatidic acid acyl transferase (LPAAT)endophilin (116, 117). Endophilin colocalizes with dynamin insitu in the brain (117). It is essential for the formation of synap-tic-like microvesicles in neuroendocrine cells (116) and synap-tic vesicle endocytosis in vivo (118121). Although endophilinhas been implicated in both early (118) and late stages of vesi-

cle budding (116, 122), strong genetic and morphological evi-dence suggests that its main function is in the recruitment andstabilization of the inositol phosphatase synaptojanin (121 ,123), a critical enzyme of the slow track of vesicle cycling (see

below). Another partner of dynamin-GTP is the DnaJ-like pro-tein auxilin (124), a cofactor in the ATP-dependent stripping ofclathrin from coated vesicles (see below).

Clearly, results both in vivo and in vitro have indicated thatdynamin and its partners contribute to the fission of mem-

branes. Accordingly, isoforms of dynamin participate in otherfission processes, such as the fission of mitochondrial tubules(125, 126), vesicle budding from the trans-Golgi network (127),and the formation of caveolae (128). Whether dynamin also

participates in the closing of the putative fusion pore during

fast-track cycling remains to be determined (see below).

Clathrin-Mediated Endocytosis and the Slow TrackEndocytosis of synaptic vesicles mediated by clathrin. In orderto sustain neurotransmitter release under conditions of stimula-tion at high frequency and the corresponding massive insertionof vesicular proteins and lipids into the plasma membrane,synaptic vesicles are recycled by clathrin-mediated endocytosisat the outer margin of the active zone. A strict coupling betweenthe exo- and endocytotic steps is essential for proper signalingat the synapse, because imbalances between the two can lead toexpansion or shrinkage of the plasma membrane and can alterthe size of the pool of synaptic vesicles [reviewed in (12)].Early electron microscopic and physiological studies at the neu-romuscular junction showed that the membranes of synaptic

vesicles can be temporarily fused with the presynaptic plasmamembrane after high-frequency stimulation and subsequentlycan be retrieved by clathrin-coated pits and vesicles (7). Stronggenetic (812, 120, 121, 129, 130), biochemical (116, 118, 131,132), and morphological (57, 10, 123) evidence has corrobo-rated these initial findings and has provided a picture of the en-docytotic machinery in the nerve terminal (12, 133, 134). Al-though the precise requirements for clathrin-based synapticvesicle endocytosis have not been worked out for all types ofsynapses, interfering with this pathway in the giant axon of thesquid results in severe recycling defects even under low, physio-logical rates of stimulation (135)

Clathrin-mediated endocytosis of synaptic vesicles at nerveterminals represents a specialized form of endocytosis. Endocyto-

sis occurs in all higher eukaryotic cells and is involved in a num-ber of cellular functions, including the uptake of nutrients (forexample, by low-density lipoprotein and transferrin) and the inter-nalization of signaling molecules [for example, growth factor re-ceptors (136)]. The reuptake of the constituents of synaptic vesi-cles by endocytosis is essential under conditions of massive stimu-lation and the correspondingly high rates of transmitter release(12). It is, therefore, not surprising that proteins constitutingsynaptic vesicles represent the main cargo of clathrin-coated vesi-cles isolated from rat brain nerve terminals (137, 138). An accu-

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mulating body of evidence suggests that the majority of synapticvesicles found at the synapse were formed by uncoating of suchclathrin-coated vesicles. It is likely that, over the long run, everysynaptic vesicle is eventually emptied by fusion with the plasmamembrane and must be replaced by another that has been re-formed by endocytosis. Elegant work by Teng et al. (139) usingquantitative optical imaging and EM shows that clathrin-mediatedendocytosis appears to be quantitatively the most important mech-

anism for synaptic vesicle recycling.Formation of coated buds. Clathrin-coated vesicles at the

nerve terminal consist of the heavy and light chains of clathrin,the clathrin-associated protein AP180, and the AP-2 adaptorcomplex, which serves as a link for clathrin at the membrane.The AP-2 adaptor complex is a brick-like heterotetramer of !(100 to 110 kD), "2 (110 kD), 2 (50 kD), and #2 subunits (17kD) (134, 140142). Coated pit assembly may be initiated bythe recruitment of AP-2 adaptor complexes along with severalmonomeric adaptors, including AP180, epsin, and HIP1, fol-lowed by the adsorption of clathrin triskelia onto these adaptorsat the active zone (140, 141, 14 3). Lipids, in particular

phoshoinositides (100, 143147), and cargo proteins (12, 132)play an impor tant role in def ining the spat ial and temporal

specificity of coated pit assembly at synapses. Interactions ofAP-2 with phosphatidylinositol 4,5-bisphosphate (PIP2)(141,145, 147), as well as with the C2B domain of the multi-meric synaptic vesicle and plasma membrane protein synapto-tagmin, participate in the AP-2 and clathrin recruitment step(145, 147151). Moreover, it appears that the selection of addi-tional cargo proteins bearing endocytotic motifs is linked to theassembly of coated pits by positively cooperative interactions

between such motifs, the AP-2 adaptor complex, and synapto-tagmin (132, 134). Internalization of synaptotagmin appears torequire additional endocytotic adaptor molecules, such as stonin2, which can bind to AP-2 through unconventional WXXFmotifs (152154) and may be recruited to membranes by its in-teraction with synaptotagmin (155, 156). In agreement with thisscenario, acute inactivation of synaptotagmin I by fluorophore-

assisted light inactivation (FALI) results in severely impairedsynaptic vesicle recycling (157).

In addition to AP-2, monomeric adaptors, such as epsin[which is able to partition into and bend membranes (144)],AP180, and HIP1, could also play an important role in the ini-tial recruitment of clathrin to PIP2-containing sites of endocyto-sis (158 ). AP-2 depletion from cells by RNA interference(RNAi) leads to reduced numbers of clathrin-coated pits, butneither abolishes clathrin recruitment to membranes nor com-

pletely inhibits endocytosis of growth factor receptors, whichsuggests that adaptors other than AP-2, such as AP-180 orepsin, can recruit clathrin and cargo to endocytotic sites(159161).

Formation of endocytotic vesicles may be nested into cycles

of phosphoinositide phosphorylation and hydrolysis (98).Consistent with this proposal, it has been observed that the ac-tivity of PIPKI$, the major PIP2-synthesizing enzyme at thesynapse (162), is regulated by the small GTPase Arf6 (146,163) and by talin (164). Impaired PIP2 synthesis at synapses ofPIPKI$ knockout mice leads to synaptic depression due to im-

paired synaptic vesicle cycling (100). Recruitment of endocyticadaptors to PIP2-enriched membranes is then followed by theassembly of clathrin triskelia into polyhedral cages (142, 165,166). The size of these cages appears to be determined at least

in part by the clathrin-assembly protein AP180 (12), AP-2, andperhaps additional adaptors (167) to polymerize clathrin (168).Genetic disruption of the AP180 gene in D. melanogaster re-sults in death of the animal at a late stage of development, andthe few surviving adults display severe defects in neurotrans-mission, because the synaptic vesicles at their synapses are bothreduced in number and heterogeneous in size (11). Similar datahave been reported for mutants of AP180 in Caenorhabditis el-

egans (169). The effect of the AP180 protein on the size ofsynaptic vesicles strongly suggests that all of the synaptic vesi-cles were at one time formed by direct uncoating of clathrin-coated vesicles formed at the plasma membrane.

During initial stages of coated pit formation, clathrin, theAP-2 adaptor complex, epsin, and AP180 interact with variousadditional factors that themselves are not a part of the final coat[reviewed in (12, 134, 142, 158, 170)]. Elegant structural stud-ies have shown that amphiphysin, endophilin, and several less-well-characterized proteins including arfaptin and sorting nex-ins contain BAR (BIN/amphiphysin/Rvs-homology) domains,which may act as sensors of membrane curvature and therebymonitor invagination or fission reactions (171). Other accessory

proteins, such as syndapin (also termed pacsin) or intersectin

(172, 173) may link nascent coated vesicles to the actin cy-toskeleton (174, 175). The specific role of actin in endocytosisis unclear and both restrictive and propulsive functions of actinhave been proposed [reviewed in (175)].

Uncoating and translocation of the vesicles. As soon as vesi-cles coated with clathrin leave the site of endocytosis, they shedtheir coats in a reaction that is dependent on Mg2+-ATP, thechaperone Hsc70 (176), the DnaJ-like protein auxilin (177), andan as-yet-unidentified protein that catalyzes the release of adap-tors (178). The importance of Hsc70 and auxilin for uncoating(179) and synaptic vesicle cycling has been demonstrated bothin vitro and in vivo (180182). Because free vesicles coatedwith clathrin are rarely observed in electron micrographs, itseems likely that uncoating is coincidental or immediately sub-sequent to the fission of membranes. In support of this, auxilin

and Hsc70 have been shown to bind to dynamin-GTP (124);this complex thus serves as a potential molecular link betweenfission and stripping of the coat. Actin filaments may be in-volved in the translocation of endocytotic vesicles (174), whichhave shed their coat, from their site of endocytosis to the poolof synaptic vesicles (183), and this activity likely is dependenton the function of the peripheral synaptic vesicle proteinsynapsin (184).

Budding from endosomes mediated by the AP-3 coatingcomplex as an alternative to clathrin. Neuroendocrine cells, inaddition to clathrin-mediated endocytosis (116, 133), display asecond pathway for generating the neuroendocrine counterpartof synaptic vesicles that is not mediated by clathrin and that

proceeds by direct budding from endosomes. Unlike the endo-

cytotic recycling pathway in neuronal tissue that relies onclathrin and dynamin, the budding of vesicles from endosomesrequires the AP-3 adaptor complex and a small Ras-likeGTPase of the ARF subfamily (185, 186). Although AP-3 adap-tors are present in a large number of synapses (187), mochamice lacking both AP-3 adaptor A and B complexes are epilep-tic but viable and do not have general defects in synaptic trans-mission or cycling of synaptic vesicles (188). Mice specificallylacking AP-3B show a defect in the formation of inhibitorysynaptic vesicles (189). It remains to be tested to what extent

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AP-3dependent inhibitory synaptic vesicles follow a recyclingpathway that is similar to that of other synaptic vesicles. It is in-teresting that AP-3 interacts with and targets TI-VAMP (190), av-SNARE involved in neuronal differentiation (82, 191, 192)and found in a subset of nerve terminals in the adult brain,where it could mediate specific secretory processes (193).Therefore, AP-3 might have specific roles in synaptic vesiclerecycling in subsets of synapses.

A Speculative Model for the Putative Fast Track ofVesicle CyclingOscillatory fusion and fission. The fast track of synaptic vesiclecycling is proposed to involve oscillatory fusions of a pool ofreadily releasable vesicles docked at or near the plasma mem-

brane (20, 22, 24, 26, 29). This model is closely related to theflickering pores observed in secretory granule exocytosis(194196). In neuroendocrine cells, flickering of a transient

pore precedes complete degranulation and, hence, represents anintermediate step in exocytosis of secretory granules (194). Inthe case of the fast track of synaptic vesicle cycling, all crucialsteps must occur in proximity to the plasma membrane and at arate fast enough to allow for the rapid conversion of an exocy-

tosed synaptic vesicle into a readily releasable one. These stepsinclude closing of the fusion pore, lumenal acidification, andrefilling with neurotransmitter (Fig. 2). The presumed fusion

pore might be formed by phospholipids from the two bilayers,and its opening and closing could be controlled by SNAREs,synaptotagmin, and their partners. Indeed, synaptotagmin af-fects fusion poreopening kinetics (197), and isoforms of this

protein have been implicated in regulating the choice betweentransient incomplete and full fusion (198, 199).

Closing of a lipid-made fusion pore could be mediated bymembrane fission, a process that depends mainly on dynamin,as discussed above. In a hypothetical model [(Fig. 2); Anima-tion 2 (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/264/re19/DC2)], dynamin would mediate the rapid clos-ing of the transient fusion pore, possibly in concert with SNAPs

and NSF. In support of this, dynamin has been implicated in re-capturing secretory granules after exocytosis by a mechanismsimilar to kiss-and-run in broken neuroendocrine cells (200).Furthermore, loss of dynamin in shibire mutants ofDrosophilaleads to a complete depletion of synaptic vesicles in the presy-naptic terminal and a loss of evoked and spontaneous transmis-sion (201, 202). Remaining activity at endophilin A mutant ter-minals may underline the fact that endophilin does not appearto be required for the fast track (120). In order to accomplishthe rapid oscillations, dynamin-mediated fission would need to

be coordinated with SNARE-mediated fusion. The impairmentof fast endocytosis in synaptobrevin 2 knockout mice suggeststhat such coordination may occur and could depend on synapto-

brevin in the fast track (203). Experimental links between dy-

namin and the machinery mediating the fusion of membranesare still scarce and mostly indirect (204, 205). Coordination be-tween fusion and fission could also be achieved by phosphory-lation and dephosphorylation cycles, because dynamin,SNAREs, synaptotagmin, and NSF are the substrates of proteinkinases, such as cyclin-dependent kinase 5 (CDK5), calcium-calmodulin kinase II, protein kinase A, and protein kinase C(PKC), which all play important functions in secretion and vesi-cle cycling (206). In agreement with this model, phosphoryla-tion of NSF by PKC inhibits its binding to the SNAP-SNARE

complex (205), whereas PKC-mediated phosphorylation ofSNAP-25 potentiates vesicle recruitment (207).

Maintenance of a docked pool of synaptic vesicles. If thepool of docked synaptic vesicles corresponds to oscillatorysynaptic vesicles, the members of this pool would have to re-main docked to the plasma membrane or to be rapidly recap-tured after fission has occurred. This could be achieved partial-ly by confinement of t-SNARE proteins to microdomains of the

plasma membrane that may define the docking and fusion sitesfor synaptic vesicles (93, 94), even though their distribution isnot restricted to nerve terminals (208). Maintenance of adocked pool of synaptic vesicles could also involve incompletedisassembly of SNARE complexes, leading to a synapto-

brevinSNAP-25 complex loosely associated with syntaxin, asproposed in the case of the priming step (57). Incomplete dis-assembly of SNARE complexes by NSF could involve regulato-ry mechanisms, such as phosphorylation by PKC (205). Theslow track was found to be PKC-independent, whereas the fasttrack was PKC-dependent (209). This suggests that phosphory-lation and dephosphorylation control the partitioning betweenthe slow and fast tracks of cycling. Another important factor incontrolling the RRP of synaptic vesicles could be Munc13-1, a

plasma membrane phorbol esterbinding protein that interactswith syntaxin and calmodulin (210). Munc13-1 has been pro-posed to prime synaptic vesicles for exocytosis, thereby facili-tating their entry into the RRP (211). In this way, Munc-13could control entry and maintenance of synaptic vesicles intothe fast track.

The SNAREs are unlikely to be the only docking device, be-cause the number of docked synaptic vesicles is not decreasedin nerve terminals treated with botulinum neurotoxin A ortetanus neurotoxin, which cleaves the SNARE proteins SNAP-25 and synaptobrevin, respectively (212, 213). Other factors al-so might participate to ensure that synaptic vesicles recentlyclosed by fission would not be released into the cytoplasm. Arole for synapsin, a peripheral protein of synaptic vesicles capa-

ble of binding actin microfilaments, in exocytosis has been pro-

posed (214). Neutralization of synapsin by antibody injectioninto neurons ofAplysia californica slows the release kinetics incholinergic synapses, suggesting that synapsins may regulatethe competence of docked synaptic vesicles to fuse with the

plasma membrane (215). However, no such effects have beenobserved in synapsin I knockout mice (216). Maintenance ofRRP may also depend on factors involved in membrane fission.Reinvestigation of the onset of theD. melanogaster shibirephe-notype produced by temperature-sensitive mutants of dynaminshowed surprisingly fast synaptic fatigue, even though thesynapse held a full complement of synaptic vesicles (217). Oneinterpretation of this finding is that dynamin, in addition to itsestablished role in endocytotic membrane fission, is also impor-tant for the short-term maintenance of the RRP of synaptic vesi-

cles and thus might play a role in the fast track for cycling ofsynaptic vesicles.

PerspectiveMuch of what we know about cycling of synaptic vesiclescomes from physiological measurements in living cells, ultra-structural studies, and in vitro analysis of synaptic proteins andlipids. Although we hope to know most of the major players inthe slow track for the cycling of synaptic vesicles and have rela-tively detailed knowledge about their functional and structural

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properties, our understanding of the putative fast track lags farbehind. This is mainly because we have not been able to track downmolecular components specifically involved in its regulation. Fur-thermore, increasing the spatial and temporal resolution of our ana-lytical methods should provide important insights into the molecularcomponents involved in the fast track. Most important, we shall beable to find out what determines the decision of a vesicle to followeither pathway. Different types of synapses might utilize preferential-

ly the fast or slow tracks, depending on their mode of release of neu-rotransmitter, the size of the pool of synaptic vesicles, the number ofdocked vesicles, and the efficiency of recycling. It is also conceiv-able that the energetic status of the nerve terminal will determine the

preferred track for the cycling of synaptic vesicles. Further techno-logical advances in imaging will, it is hoped, pave the way for a de-tailed molecular understanding of the life cycle of a synaptic vesicle.

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