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Endocytotic mechanisms in synapses Nadine Jarousse and Regis B Kelly* Nerve terminals are highly enriched in proteins needed for endocytosis. Although constitutive and ligand-stimulated endocytosis take place in nerve terminals, the primary type is compensatory endocytosis — the process by which a cell retrieves the additional membrane added to cell surface by a regulated secretory event. This process has been extensively characterized using electrophysiological techniques. Except for an unusual form of coupled exo- and endocytosis called kissand-run release, compensatory endocytosis appears to use basically the same clathrin-mediated mechanisms as the constitutive and ligand stimulated type. The remarkable speed and selectivity of compensatory endocytosis may be achieved by concentrating the machinery at specialized sites in the nerve terminal adjacent to exocytosis sites and by the use of neuronal isoforms of the proteins that mediate endocytosis. Addresses Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448, USA; *e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:461–469 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations Ab antibody AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid AP adaptor protein ENTH epsin NH2-terminal homology eps15 epidermal growth factor pathway substrate 15 GLUT4 plasma membrane glucose transporter isoform type 4 Hsc 70 heat shock cognate protein 70 kDa NSF N-ethylmaleimide sensitive factor N-WASP neuronal Wiskott-Aldrich syndrome protein PH pleckstrin homology PI phosphoinositide PI(4,5)P2 phosphatidylinositol 4,5-biphosphate PM plasma membrane PRD proline-rich domain RSOs regulated secretory organelles SH3 src-homology region 3 STG synaptotagmin STG-TM STG transmembrane domain

Introduction Regulated secretory cells, such as neurons, exocrine and endocrine cells, mast cells, neutrophils and many egg cells, have homeostatic mechanisms for maintaining their cell surface area following stimulated exocytosis of their regulated secretory organelles (RSOs). Reinternalization of the membrane of the RSO is a rapid and efficient process, usually called compensatory endocytosis to distinguish it from the constitutive endocytosis of empty receptors for proteins such as transferrin and the ligand-stimulated endocytosis of G-protein-coupled receptors, for example. In general, the molecular machinery required for compensatory endocytosis is the same as for constitutive and ligand-stimulated but

Figure 1 Exocytotic event

∆C

A

B

Fusion pore

Endocytotic event

∆C

A

B

Fission pore Current Opinion in Cell Biology

Capacitance flicker during exocytosis and endocytosis implies pore formation. Rapid fluctuations between two membrane configurations, A and B, cause membrane capacitance flicker (∆C step followed by another of similar amplitude but opposite direction) as indicated in the ∆C traces and capacitative current flow through the transient fusion or fission pore (red). As capacitance flicker has now been seen for endocytotic events (downward step), as well as exocytotic events (upward step), and for constitutive secretory as well as regulated secretory cells [3]. Thus, the formation of a transient fusion/fission pore may be a universal event in membrane traffic.

adapted to allow tighter spatial and temporal regulation. Because compensatory endocytosis can be so readily quantified, however, its study has yielded insights into aspects of endocytosis that were missed by other approaches.

Kiss-and-run exocytosis/endocytosis A possible exception is considered first in which conventional endocytotic machinery may not be used for compensatory endocytosis. This suggests the existence of ‘fission pores’ in endocytosis. After compensatory endocytosis, the cell recovers only the membrane added by the preceding exocytosis event. One way cells can achieve this selective recovery is by aborting exocytosis immediately after the formation of a fusion pore (Figure 1). By reversing fusion pore formation, the secretory vesicle membrane is recovered without mixing its lipid or proteins with the plasma membrane. Thus, conventional endocytotic machinery is not required for membrane recovery after what is frequently called ‘kiss-and-run’ exocytosis. Evidence for kiss-and-run membrane recycling is strongest for endocrine cells. Opening and closing of fusion pores

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Figure 2

Glutamate receptor clusters

Postsynaptic plasma membrane Endocytosis zone

Synaptic vesicles

Active zone

In this schematic of a synaptic varicosity in the neuromuscular junction of a third instar Drosophila larva, active zones of exocytosis are indicated by clusters of synaptic vesicles already docked on the presynaptic plasma membrane. Opposite the active zones of exocytosis on the postsynaptic plasma membrane are arrays of receptors that detect the neurotransmitter, in this case glutamate, released by synaptic vesicle fusion. Surrounding the active zones, in a region often called the ‘periactive zone’ are domains in which the endocytotic machinery (green) is anchored by some unknown mechanism to the presynaptic plasma membrane. (Reprinted with permission from [11]).

Current Opinion in Cell Biology

gives rise to transient increases in cell surface area, which can be detected electrophysiologically as a quantal change in capacitance (Figure 1). Sometimes the fusion pore opens and closes many times in succession giving rise to the phenomenon called ‘capacitance flicker’ (Figure 1). Capacitance flicker implies that formation of the fusion pore is reversible. As small neurotransmitter molecules can exit through the fusion pores of endocrine secretory granules, this is a feasible mechanism for neurotransmitter release at synapses. Although no direct evidence for kiss-and-run recycling has been found in synapses, its existence has been suggested from discrepancies between the amount of dye released from prelabeled nerve terminals and the amount of neurotransmitter released. Lipophilic dyes such as FM1-43 dissociate from membranes with a half time of about 2 s and so will not diffuse through a transient, proteinaceous fusion pore as rapidly as neurotransmitters. In one well known example of such a discrepancy (in frog neuromuscular junctions treated with staurosporine) the data have now been attributed to a failure of synaptic vesicle equilibration rather than to kiss-and-run [1]. In hippocampal neurons, less dye is released than expected from neurotransmitter release, and this result has been used to calculate that about 20% of normal exocytotic events could be of the kiss-and-run type [2•]. Two other types of measurement have suggested that kiss-and-run exocytosis takes place at nerve terminals. Endocytosis has two kinetic time constants, one of about 1 s and another a factor of 10 slower [3]. The faster component, which is inhibited by prolonged synaptic activity, could be kiss-and-run endocytosis, whereas the slower could be conventional clathrin-mediated endocytosis. In addition, a fast type of endocytosis that is dynamin-dependent and clathrin-independent [4] has also been interpreted as kiss-and-run. Since both these measures are again indirect it is probably wise to keep an open mind on kiss-and-run exocytosis in synapses until its existence can be verified directly.

Electrophysiologists now have strong evidence that even the fusion of constitutive secretory vesicles with plasma membranes involves the reversible formation of a pore [5••]. What is even more exciting about this work was the discovery of capacitance-flickering during endocytosis, implying a reversible ‘fission pore’. Cell biologists are comfortable with constricted coated pits as intermediates in endocytosis. If the neck of a constricted pit can open and close again several times before fission, this could explain the endocytotic capacitance flicker (Figure 1). Thus the fusion pore formed during exocytosis could have an analogue in the ‘fission pore’ formed during endocytosis.

Non-mixing of regulated secretory organelles and plasma membranes In some types of exocytosis, the RSO membrane merges transiently with the plasma membrane but the two do not mix. This is seen very dramatically when fertilization induces the fusion of cortical granules with the plasma membranes of sea urchin eggs [6••]. If the plasma membrane proteins are labeled before fusion, the labeled proteins do not mix with the proteins of the cortical granule. These results are consistent with earlier studies on neuroendocrine cells [7]. A non-mixing type of exocytosis at the synapse could help maintain the composition of synaptic vesicles during very rapid recycling.

Speed of endocytosis The rates of internalization of proteins via constitutive or ligand-stimulated endocytosis are usually in the range of 1–10% per min at 37°C. This is comparable with the time taken to recover cortical granule membranes after fertilization. In contrast, internalization of synaptic vesicle membranes can occur in as little as 4 s [8•]. Since the molecular machinery used for synaptic vesicle internalization has been shown by genetics, morphology and in vitro reconstitution to be very similar to the machinery for slow constitutive endocytosis, high speeds can only be achieved by overexpressing the proteins in the brain and concentrating them in synapses close to sites of exocytosis. Recent morphological observations using both

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Table 1 Molecular requirements for endocytosis. Step

Inhibitory reagent (IR)

Potential targets of the IR

Likely affected event

References

PI(4,5)P2-binding peptide AP2, PI-binding defective peptide AP180, PI-binding defective peptide DLL motifs from AP2 or AP180 AP180, auxilin RNAi to auxilin STG-TM Synprint of calcium-channels

PI(4,5)P2

Clathrin Clathrin Auxilin STG, STG-interacting proteins STG

Coat recruitment to the PM AP2 recruitment to the PM AP180 recruitment to the PM Clathrin assembly Association of clathrin to the PM Clathrin distribution and dynamics STG oligomerization STG–AP2 interaction

[16] [14] [17••] [18] [79] [80] [23•] [21••]

Invagination

Anti-endophilin Ab

Endophilin

Lipid or actin-mediated curvature (?)

[9]

Constriction

Amphiphysin SH3 Intersectin SH3A GTPase– dynamin mutants (K44A, T65A) Synaptojanin PRD (PP19)

PRD-containing proteins PRD-containing proteins Endophilin

Recruitment of dynamin Unknown Dynamin concentration to the neck Endophilin–dynamin interaction

[24,34] [37] [34,36] [32•]

Amphiphysin SH3 Endophilin SH3 Syndapin SH3 Intersectin SH3 C, E

PRD-containing proteins PRD-containing proteins PRD-containing proteins PRD-containing proteins

Amphiphysin–dynamin interaction Endophilin-dynamin interaction Unknown Unknown

[37] [32•,37] [37] [37]

Synaptojanin PRD (PP19) Anti synaptojanin Ab Hsc ATPase–

Endophilin Synaptojanin

Synaptojanin recruitment PI-dephosphorylation

[32•] [32•] [44••]

Coat recruitment

Scission

Uncoating

Endocytosis can be broken down into several steps that have different molecular requirements. Most of these requirements have been established using proteins or peptides that inhibit a step, causing an intermediate to accumulate (see Figure 3). Please refer to the numbered references for further details. If a study was done in vivo bold face is used

for the corresponding reference. A recent study argued that the T65A dynamin mutant, previously described in [34], oligomerizes normally around invaginated neck but is defective instead in the scission step [35]. Ab, antibody; PM, plasma membrane; PRD, proline-rich domain; SH3, src-homology region 3; STG, synaptotagmin; TM, transmembrane.

lamprey and snake synapses have clearly shown that endocytosis occurs in a ‘periactive zone’ immediately adjacent to and surrounding the active zone of exocytosis [9,10•]. Immunochemistry on Drosophila nerve terminals has revealed that these periactive zones are packed with the endocytotic machinery already assembled on the plasma membrane (Figure 2; [11]). Thus endocytosis might be accelerated during compensatory exocytosis by having the necessary machinery concentrated exactly where it is needed.

and overexpression studies (Table 1) now require us to subdivide endocytosis into several distinct steps: coat assembly on membranes, invagination, fission and finally uncoating.

A second way to speed up vesicle recycling is to sort the synaptic vesicles directly on the plasma membrane, thus avoiding an endosomal step. The membranes or synaptic vesicles are not diluted by endosomal membrane as they recycle, implying that synaptic vesicles are equivalent to what are usually called primary endosomes [12,13••].

Molecular mechanisms of compensatory endocytosis in nerve terminals Nerve terminals are enriched in some proteins known to be involved in constitutive endocytosis, such as AP2, clathrin, epsin, eps15 (epidermal growth factor pathway substrate 15), amphiphysin and synaptojanin. They also enriched in neuronal-specific forms of other proteins, such as AP180, dynamin 1, syndapin 1 and intersectin. Before considering how such proteins might be adapted for compensatory endocytosis, it is useful to summarize current ideas of how they function in non-neuronal cells. Inhibitor

Assembly of clathrin coats on membranes

After exocytosis, synaptic vesicle proteins are recognized by the AP2 adaptor complex which, with clathrin and the neural specific AP180, forms a clathrin-coated vesicle of uniform diameter. Their endocytosis is coupled (Figure 3; Table 1) to the presence of phosphorylated phosphoinositides (PIs) in the plasma membrane. AP2 (Figure 3), AP180 and epsin share the ability to bind both clathrin and PIs, and so they could link the clathrin coat to a phosphoinositide-enriched region. Mutations that prevent either AP2 or epsin binding to PI affect receptor-mediated endocytosis [14,15•], whereas constitutive endocytosis of transferrin is inhibited by a phosphatidylinositol 4,5-biphosphate (PI[4,5]P2)-binding peptide [16]. AP180 binding to PI(4,5)P2 is essential for recruiting clathrin to liposome membranes [17••]. As AP180 binds clathrin through repeated motifs [18], it could potentially bind several clathrin molecules and may therefore facilitate their transfer to AP2 heterotetramers. AP2, AP180 and clathrin appear to be sufficient by themselves for initiation of coated pit invagination on PI(4,5)P2-containing liposomes, with AP2 providing the curvature [17••]. Membrane proteins are also involved in nucleating coat formation [19]. At the plasma membrane AP2 is recruited to a

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Figure 3

(a) Coat recruitment

Figure 3 legend

PI(4,5)P2 AP2, AP180, clathrin Synaptotagmin

(b) Invagination

(c) Constriction

(d) Scission

Endophilin

Dynamin Amphiphysin Endophilin (?) Intersectin (?) GTP

Dynamin Amphiphysin (?) Endophilin (?) Intersectin (?) Syndapin (?)

Hsc70 ATPase (e) Uncoating

Synaptojanin Auxilin

Current Opinion in Cell Biology

component of the fusion machinery, synaptotagmin, which binds AP2 [20]. This binding is likely to be physiologically relevant because expression of a peptide that prevents the binding of AP2 to synaptotagmin inhibited transferrin uptake [21••]. Synaptotagmin and endocytotic cargo proteins with a tyrosine-based internalization motif bind to different sites on the µ subunit of AP2 [21••], and binding of a peptide containing a tyrosine-based motif enhances association of AP2 with synaptotagmin [22], implying a coordinated interaction between the three elements (Figure 4). Inhibition of transferrin uptake could mean that a synaptotagmin molecule is

Molecular requirements for the different steps of clathrin-mediated endocytosis. (a) Recruitment of coat proteins to form a curved membrane can be reconstituted in vitro with PI(4,5)P2-containing liposomes, AP2, AP180 and clathrin [17••]. The role of the synaptotagmin–AP2 interaction in coat formation was also recently shown in vivo [21••,23•]. (b) Inhibition of endophilin function can block internalization at the shallow invagination level thus identifying a second step in the process [9]. Endophilin appears to function again at a later stage (scission). The next intermediate is deep invagination, before the formation of the characteristic dynamin neck structure (brown rectangles). Deeply invaginated coated pits accumulate in vitro when terminals contain the SH3 domain of amphiphysin, supporting a role for amphiphysin in dynamin recruitment. A possible role for endophilin in constriction was suggested by Ringstadt et al. [9] showing that endophilin is associated with dynamin tubules and stimulates or stabilizes them in a coating assay using purified synaptic membranes [24]. (c) Constriction and closure of the neck require GTP-bound dynamin [35], although it remains controversial whether GTP hydrolysis by dynamin is required for constriction, or fission, or for any of these steps [33–36]. SH3 groups from several proteins, such as amphiphysin and intersectin, block the constriction or fission steps (see Table 1), although these, steps may be mediated by a somewhat non-specific inhibition of a PRD-containing protein (probably dynamin), which is needed for these steps to occur. We should note, however, that a direct role for amphiphysin in constriction and (d) scission is further supported by data showing that amphiphysin can stimulate dynamin-mediated tubulation and fragmentation of liposomes [25]. (e) Synaptojanin may be recruited by endophilin [32•] to participate in the uncoating reaction [39], in cooperation with auxilin and the hsc70 ATPase [44••]. Since steps in the fission reaction appear to be reversible (Figure 1) the intermediate that accumulates may not define the point of action of an inhibitor exactly.

even involved in constitutive endocytosis. Consistent with this idea, transmembrane domains of synaptotagmin can inhibit endocytosis of LDL (low density lipoprotein) receptors by fibroblasts, although the mechanism for this is not yet known [23•]. As synaptotagmin can also bind phosphorylated phosphatidylinositols, coat recruitment by synaptotagmin and the PI lipids may be synergistic. Formation of a constricted pit and fission

Dynamin is the protein most commonly implicated in the fission step. Although we now have clues to how it is recruited to clathrin coated pits, crucial molecular details are still lacking. We know that amphiphysin can interact with AP2, clathrin and the GTPase dynamin, thus providing a potential link between the clathrin coat and dynamin [24–26]. In addition, binding of dynamin to PI through its pleckstrin homology (PH) domain is essential for clathrin-mediated endocytosis [27–29]. As coating proceeds, the complex of amphiphysin and dynamin may be displaced from its binding site on AP2 and concentrated at the neck connecting the clathrin-coated pit to the plasma membrane. GTP binding to dynamin and dynamin’s binding to amphiphysin have been proposed to trigger redistribution of dynamin to the neck and formation of a constricted pit [24,25,30]. Amphiphysin and dynamin can form helical arrays along membrane tubules [25], and endophilin can stimulate or stabilize dynamin-coated tubules in vitro [9], suggesting the

Endocytotic mechanisms in synapses Jarousse and Kelly

existence of rings composed of dynamin, amphiphysin and perhaps endophilin. Elongated necks surrounded by electron-dense rings can be trapped on clathrin-coated pit intermediates in vitro by incubating lysed synaptosomes in the presence of GTPγS [31] or in vivo after microinjection of the SH3 domain of endophilin at the lamprey synapse [32•]. In most models, the ring structure around the neck of a constricted pit generates a membrane fission event, conceivably through dynamin’s GTPase activity, although this is controversial [33–36]. Amphiphysin and endophilin may be cofactors for dynamin-mediated fission [25,32•,37]. This attractive model is not consistent, however, with in vitro evidence that amphiphysin only binds to dissociated dynamin and may inhibit ring formation by dynamin [38]. Although we do not know the details of the steps outlined in Figure 3, it is now clear from inhibitor studies that three independent events take place (Table 1). Antibodies to endophilin trap a curved intermediate that has the form of a shallow pit [9]; amphiphysin SH3 domains [24] or dynamin mutant K44A [36] produce deeply invaginated pits that do not undergo constriction, and SH3 fragments of several proteins inhibit the conversion of constricted coated pits to coated vesicles.

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Figure 4

PI(4,5)P2

Phospholipid AP2 -KKSTG

STG

-YCargo

Current Opinion in Cell Biology

The heterotrimeric AP2 complex uses separate domains to bind to a dilysine motif (-KK-) in synaptotagmin (STG) and simultaneously to the tyrosine-based internalization motif on a cargo protein [22]. Cargo binding enhances synaptotagmin binding, implying a synergistic interaction. Both synaptotagmin and AP2 can bind phosphorylated phosphatidylinositols (PI[4,5]P2), which could help concentrate components of the internalization complex. To internalize cargo, synaptotagmin oligomerizes through the region near its transmembrane domain with another protein, most likely a second synaptotagmin [23•].

Uncoating of the clathrin-coated vesicle

If PIs trigger coat and neck assembly, then their dephosphorylation should reverse the process. Synaptojanin, which has two phosphatase domains, has such a role, demonstrated by its knockout in mice leading to the accumulation of clathrin-coated vesicles [39]. A peptide that disrupts the interaction between endophilin and synaptojanin, and an antibody to synaptojanin, also inhibits uncoating in vivo [32•]. One role for endophilin therefore may be to anchor synaptojanin, although it may also modify lipids [40] or act at the earlier step of invagination [9]. As phosphorylated phosphoinositides regulate a number of cellular events, altering synaptojanin function should have a pleiotropic effect. Indeed the mouse knockout also affects the actin cytoskeleton, and the synaptojanin mutation in C. elegans (unc-26) perturbs many steps in nerve terminal function [41•]. The actual removal of the clathrin coat requires auxilin, a component of the coated vesicle that also binds to the appendage domain of AP2. Auxilin recruits the heat shock protein hsc70 to the clathrin-coated vesicle and stimulates its ATPase activity [42,43]. Overexpression of ATPase-deficient hsc70 mutants inhibits the uncoating of clathrin-coated vesicles in vivo [44••]. Auxilin regulation of uncoating has not yet been linked to dephosphorylation by synaptojanin.

Linking endocytosis to exocytosis in the nerve terminal In compensatory endocytosis, the endocytotic machinery must be regulated so that it immediately recovers only the membrane added by the exocytotic events. When a small region of a cell surface needs to perform a specialized function

it usually involves phosphorylation of lipids and proteins at that site, which then recruit phosphate-binding proteins. Phosphorylation of lipids occurs during compensatory endocytosis, because inhibiting the phosphatase synaptojanin perturbs vesicle recycling [39,41•]. Such regulation is not, however, unique to compensatory endocytosis, as phosphorylated PIs have a role in constitutive endocytosis [14,15••,16,27–29]. It is likely that PI kinases, either on synaptic vesicles [45] or near the active zones, trigger endocytosis at nerve terminals. Endocytotic retrieval of synaptic vesicle membranes would also be facilitated if the membrane proteins of synaptic vesicles, like those of egg cortical granules [6••], do not disperse into the nerve terminal plasma membrane but instead stay clustered. Evidence that synaptic vesicle proteins might be in a specialized ‘raft’ domain was obtained by showing that synaptic-like microvesicle formation in PC12 cells is much more sensitive to cholesterol depletion than transferrin uptake was [46]. In addition, the GPIlinked protein Thy-1 is efficiently targeted to this class of vesicles [47•]. Since regulated exocytosis is triggered by the elevation of cytoplasmic calcium levels, it is not surprising that compensatory endocytosis is stimulated the same way. The cortical granules of sea urchin eggs are enriched in P-type calcium channels, and entry of calcium through these channels is essential for membrane retrieval [6••]. In nerve terminals, calcium influx causes a massive dephosphorylation of dynamin, amphiphysins 1 and 2 and synaptojanin,

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and dephosphorylation is critical for coat assembly in vitro [48–52]. The calcium-activated phosphatase calcineurin, which binds to dynamin, has been identified as the calcium sensor in this case [53,54]. To inactivate the phosphatase and turn off the endocytotic process, cells may use the calcineurin inhibitor, Cain, which binds amphiphysin [55•]. Like regulation via phosphoinositides, regulation of endocytosis via calcineurin does not appear to be restricted to neuronal cells [55•], implying again that a ubiquitous regulatory mechanism is adapted for compensatory endocytosis at the nerve terminals.

Synaptic modulation through endocytosis Cells often have endocytotic organelles containing a reserve supply of surface components. For example, glucose uptake can be stimulated by mobilizing GLUT4 (plasma membrane glucose transporter isoform type 4) transporters from intracellular endosomes. Nerve cells can also modify their surface composition and thus the properties of their synapses. Endocytosis of a class of glutamate receptors, the AMPA (alpha-amino-3-hydroxy-5-methyl-4isoxale-propionic acid) class, generates long term synaptic depression via a mechanism that requires calcium activation of the phosphatase calcineurin, AP2, clathrin, dynamin and the function of the NSF (N-ethylmaleimide sensitive factor) ATPase [56,57,58••,59–65]. AMPA receptors are not the only synaptic surface protein to be modulated. Substance P receptors [66] and proline transporters [67], for example, may have intracellular storage pools affected by synaptic activity.

Linking endocytosis to other synaptic functions Endocytosis in nerve terminals is coupled to exocytosis, as well as to synaptic adhesion, the actin cytoskeleton and to signalling. The exocytosis/endocytosis machinery must be precisely coupled to receptors in the postsynaptic membrane by some adhesion proteins. In larval Drosophila neuromuscular junctions, the fasciclin and integrin adhesion proteins are excluded from active zones, and they cluster in a ‘periactive zone’ distribution [68]. Although the endocytotic machinery and the adhesion mechanisms both have periactive zone locations, they do not codistribute ([68]; J Roos, RB Kelly, unpublished data).

implicated in constitutive endocytosis, the link to the actin cytoskeleton cannot be nerve-terminal-specific [74]. The proteins that couple endocytosis to actin polymerization, dynamin, intersectin and syndapin all have neuronal-specific isoforms. If we think of the cell surface as a combination of plasma membrane and subcortical actin cytoskeleton, then it is not too surprising that the internalization machinery interacts with both membrane and cytoskeletal components. The links between endocytosis and the processes of exocytosis, adhesion and actin polymerization are not, however, the end of the story, because endocytosis is becoming increasingly associated with cell signaling mechanisms [75]. The past year has seen the publication of reports that epsin traffics to and from the nucleus where its epsin amino-terminal domain (ENTH) binds the promyelocytic leukemia zinc finger protein, a transcription factor [76]. Furthermore, the SH3A domain of intersectin binds the ras GTP exchange factor mSOS1, and thus regulates the MAP kinase pathway [77•,78•]. Although we as yet know little about the physiological significance of these findings, they create considerable excitement among neurobiologists seeking to link synaptic activity with gene expression.

Conclusions The function and plasticity of the synapse depend greatly on membrane traffic — endocytosis as well as exocytosis. In recent years, we have come to a much deeper understanding of the molecular mechanisms of endocytosis and, perhaps more importantly, of the large number of reagents that perturb endocytosis at known steps (Table 1). We can look forward to explaining the rapidity of membrane traffic at the synapse, where pulses of exocytosis occur as fast as every 2 ms, and to learning what locates the endocytotic machinery next to exocytotic sites and how it is regulated by exocytotic events. Any molecular model that is generated will have to survive precise scrutiny by the powerful technologies of the electrophysiologists.

Acknowledgements The work of RBK is supported by funds from the National Institutes of Health (NS 09878 and NS 15927), from Johnson & Johnson Focussed Giving Program and by the Albert Bowers Chair. NJ is supported by postdoctoral funds from NARSAD.

References and recommended reading Periactive zones have also been reported to be sites of extensive actin polymerization [69,70•]. Although the actin polymers could be part of the adhesion mechanism, strong evidence now links actin to the endocytosis process. Amphiphysin 1 and dynamin 1 affect actin polymerisation in neuronal growth cones [71]. Dynamin 2 and endophilin are crucial for podosome invagination, suggesting that these two proteins may use the actin cytoskeleton to deform the plasma membrane [72•]. Two other dynamin-associated proteins, syndapin and intersectin, bind the actin regulator N-WASP and link, in some as yet obscure way, the processes of membrane invagination to the actin cytoskeleton [73]. As dynamin, amphiphysin, syndapin and intersectin have all been

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Becherer U, Guatimosim C, Betz WJ: Effects of staurosporine on exocytosis and endocytosis at frog motor nerve terminals. J Neurosci 2001, 21:782-787.

2. Stevens CF, Williams JH: ‘Kiss and run’ exocytosis at hippocampal • synapses. Proc Natl Acad Sci USA 2000, 97:12828-12833. This paper is an example of the way electrophysiologists compare dye and neurotransmitter release to infer secretion through a fusion pore. In this case, to explain the results the authors postulate either a proteinaceous pore (but see [5]) or a pore that remains open less than 6 ms. 3.

Neves G, Lagnado L: The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells. J Physiol 1999, 515:181-202.

Endocytotic mechanisms in synapses Jarousse and Kelly

4.

Palfrey HC, Artalejo CR: Vesicle recycling revisited: rapid endocytosis may be the first step. Neuroscience 1998, 83:969-989.

5. ••

Henkel AW, Meiri H, Horstmann H, Lindau M, Almers W: Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells. EMBO J 2000, 19:84-93. The existence of fusion pores during constitutive exocytosis and of fission pores during endocytosis demonstrates the importance of a narrow-necked intermediate during many if not all budding and fusing events. 6. ••

Smith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS: Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis. J Cell Biol 2000, 148:755-767. After exocytosis, the lipids of the cortical granule membranes and the plasma membrane mix but the proteins do not. Compensatory endocytosis requires the influx of calcium through channels in the cortical granule membrane. 7.

Nordmann JJ, Artault JC: Membrane retrieval following exocytosis in isolated neurosecretory nerve endings. Neuroscience 1992, 49:201-207.

8. •

Sankaranarayanan S, Ryan TA: Real-time measurements of vesicle SNARE recycling in synapses of the central nervous system. Nat Cell Biol 2000, 2:197-204. Instead of using dye to measure synaptic vesicle recycling rates, the authors use a pH-sensitive form of GFP–VAMP. They confirm the kinetics obtained from dye studies and show that endocytosis is saturable, with a maximum rate of one synaptic vesicle per second per active zone. 9.

Ringstadt N, Gad H, Low P, Di Paolo G, Brodin L, Shupliakov O, De Camilli P: Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 1999, 24:143-154.

10. Teng H, Wilkinson RS: Clathrin-mediated endocytosis near active • zones in snake motor boutons. J Neurosci 2000, 20:7986-7993. In agreement with [11], sites of endocytosis are closely associated with active zones of exocytosis. Synaptic vesicle recovery takes place both by plasma membrane vesiculation and by the formation of large deep invaginations. 11. Roos J, Kelly RB: The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr Biol 1999, 9:1411-1414. 12. Murthy VN, Stevens CF: Synaptic vesicles retain their identity through the endocytic cycle. Nature 1998, 392:497-501. 13. Zenisek D, Steyer JA, Almers W: Transport, capture and exocytosis •• of single synaptic vesicles at active zones. Nature 2000, 406:849-854. This beautiful paper primarily records single synaptic vesicle exocytotic events. It is pertinent to endocytosis because it shows that exocytosis uses a fusion pore that must be lipidic, because it allows lipid dyes to diffuse laterally. Secondly from the measurements of dye molecules per vesicle it is very unlikely that synaptic vesicle formation involves an endosomal intermediate, in agreement with [12]. 14. Gaidarov I, Keen JH: Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits. J Cell Biol 1999, 146:755-764. 15. Itoh T, Koshiba S, Kigawa T, Kikuchi A, Yokoyama S, Takenawa T: Role • of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 2001, 291:1047-1051. In agreement with [17••] this paper shows that the ENTH domain, found in epsin, huntingtin interacting protein 1 and AP180, is another PI(4,5)P2-binding site. 16. Jost M, Simpson F, Kavran JM, Lemmon MA, Schmid SL: Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation. Curr Biol 1998, 8:1399-1402. 17. ••

Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A, Hopkins CR, Evans PR, McMahon HT: Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 2001, 291:1051-1055. In addition to establishing the function of ENTH domains (see [15•]), the authors are able to reconstitute coat formation on a lipid surface with AP180 and clathrin. The addition of AP2 allows formation of deeply invaginated, coated pits of about 80 nm diameter. 18. Morgan JR, Prasad K, Hao W, Augustine GJ, Lafer EM: A conserved clathrin assembly motif essential for synaptic vesicle endocytosis. J Neurosci 2000, 20:8667-8676. 19. Salem N, Faundez V, Horng JT, Kelly RB: A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex. Nat Neurosci 1998, 1:551-556.

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