| Summary: | Retrieval of synaptic vesicles can occur 1–10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone. The subsequent loss of clathrin from these regions reflected endocytosis because the application of a potent clathrin inhibitor Pitstop2 dramatically slowed down this phase by ~3 fold. These results indicate that clathrin-dependent retrieval of synaptic vesicles is unusually fast, most probably because of a “priming” step involving a state of association of clathrin with the docked vesicle and with the endosomes and cisternae surrounding the ribbons. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) showed that the majority of clathrin is moving with the same kinetics as synaptic vesicle proteins. Together, these results indicate that the fast endocytic mechanism operating to retrieve synaptic vesicles differs substantially from the classical mode of CME operating via formation of a coated pit.
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