Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster. S ynaptic communication depends on local recycling of synaptic vesicles in nerve terminals. A major recycling pathway involves retrieval by means of clathrin-coated pits (1-3). The basic features of this process are shared with that of clathrin-dependent endocytosis in nonneuronal cells (4). It involves recruitment of clathrin by adaptors to the membrane followed by polymerization of the clathrin coat. A clathrin-coated pit grows progressively along with an invagination of the coated membrane. After a deeply invaginated coated pit has formed, the base of the pit is cut off resulting in formation of a free coated vesicle. A range of accessory proteins have been identified, which are thought to regulate different aspects of the endocytic process (2). Many of these proteins interact with components of the actin-regulating machinery, which has pointed to a coupling between endocytosis and the actin cytoskeleton (2, 5, 6). Such a coupling is also supported by the observation of actin tails at moving endocytic vesicles (7-10). Disruption of actin function has been shown to inhibit endocytosis in some but not all cell types (11)(12)(13)(14).In nerve terminals clathrin-mediated budding occurs in an endocytic zone of the plasma membrane, which surrounds the release site (1, 3). Several observations indicate that actin filaments are present in this synaptic compartment. Studies of the denervated frog neuromuscular junction have shown that phalloidin labeling accumulates around rather than within release sites (15). In cell-free synaptosome preparations actin was identified by immunogold labeling in regions containing endocytic intermediates a...

The giant reticulospinal synapse in lamprey provides a unique model to study synaptic vesicle traffic. The axon permits microinjections, and the active zones are often separated from each other, which makes it possible to track vesicle cycling at individual release sites. However, the proportion of reticulospinal synapses with individual active zones ("simple synapses") is unknown and a quantitative description of their organization is lacking. Here, we report such data obtained by serial section analysis, intermediate-voltage electron microscopy, and electron tomography. The simple synapse was the most common type (78%). It consisted of one active zone contacting one dendritic process. The remaining synapses were "complex," mostly containing one vesicle cluster and two to three active zones synapsing with distinct dendritic shafts. Occasional axosomatic synapses with multiple active zones forming synapses with the same cell were also observed. The vast majority of active zones in all synapse types contained both chemical and electrotonic synaptic specializations. Quantitative analysis of simple synapses showed that the majority had active zones with a diameter of 0.8-1.8 microm. The number of synaptic vesicles and the height of the vesicle cluster in middle sections of serially cut synapses correlated with the active zone length within, but not above, this size range. Electron tomography of simple synapses revealed small filaments between the clustered synaptic vesicles. A single vesicle could be in contact with up to 12 filaments. Another type of filament, also associated with synaptic vesicles, emerged from dense projections. Up to six filaments could be traced from one dense projection.

Chemical synapses convey impulses at high frequency by exocytosis of synaptic vesicles. To avoid failure of synaptic transmission, rapid replenishment of synaptic vesicles must occur. Recent molecular perturbation studies have confirmed that the recycling of synaptic vesicles involves clathrin-mediated endocytosis. The rate of exocytosis would thus be limited by the capacity of the synaptic clathrin machinery unless vesicles could be drawn from existing pools. The mobilization of vesicles from the pool clustered at the release sites appears to provide a mechanism by which the rate of exocytosis can intermittently exceed the rate of recycling. Perturbation of synapsins causes disruption of vesicle clusters and impairment of synaptic transmission at high but not at low frequencies. Both clathrin-mediated recycling and mobilization of vesicles from the reserve pool are thus important in the replenishment of synaptic vesicles. The efficacy of each mechanism appears to differ between synapses which operate with different patterns of activity.