Abstract
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.
Highlights
The classical mode of clathrin-mediated endocytosis (CME) is the major pathway by which cells internalize components of the surface membrane (Conner and Schmid, 2003; Jung and Haucke, 2007; Heerssen et al, 2008)
CLATHRIN IS COMPARTIMENTALIZED WITH SYNAPTIC VESICLES IN A RIBBON SYNAPSE To investigate the role of clathrin in the retrieval of synaptic vesicles we generated transgenic zebrafish expressing the neuronal form of Clathrin Light Chain “a” (LCa) tagged with EGFP, under control of the Ribeye a promoter (Odermatt et al, 2012)
It has been generally assumed that the clathrin-dependent retrieval of synaptic vesicles occurs through mechanisms broadly similar to classical CME of surface receptors, and that recruitment of clathrin occurs after fusion has occurred (Granseth et al, 2007; Mcmahon and Boucrot, 2011; Saheki and De Camilli, 2012)
Summary
The classical mode of clathrin-mediated endocytosis (CME) is the major pathway by which cells internalize components of the surface membrane (Conner and Schmid, 2003; Jung and Haucke, 2007; Heerssen et al, 2008). Imaging methods have allowed CME to be investigated in real-time, in the context of receptor/agonist uptake from the cell surface, which occurs on the time-scale of ∼20–40 s (Loerke et al, 2009). These relatively slow kinetics have caused some to question whether CME is an important mechanism of synaptic vesicle retrieval (Pyle et al, 2000; Harata et al, 2001; Sara et al, 2002). It was shown that clathrin is involved in regenerating synaptic vesicles directly from endosomes (Watanabe et al, 2014) These observations support the idea that a “souped-up” mode of CME acts at the synaptic terminal (Conner and Schmid, 2003).
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