Molecular Determinants of Synaptic Vesicle Exocytosis and Endocytosis Coupling

Synaptic vesicle recycling is essential for maintaining normal synaptic function. The reuse of vesicles maintains the size of the presynaptic terminal and ensures the availability of synaptic vesicles for subsequent exocytosis. The coupling of exocytosis and endocytosis allows for continued rapid synaptic transmission; however, the molecular mechanisms of this process are not well understood. This coupling is assumed to be Ca2+-dependent but the exact role of Ca2+ and its key effectors in the regulation of endocytosis are not clear. Using a genetically encoded pH-sensitive GFP tag expressed in cultured hippocampal neurons, I analyzed synaptic vesicle trafficking in high resolution optical experiments. By manipulating the expression of various effectors of vesicle fusion I was able to dissect out the relationship between exocytic pathway and subsequent endocytic kinetics. My results showed that the slowed endocytosis phenotype previously reported after synaptotagmin 1 loss-of-function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss-of-function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles towards slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin7. This divergence in Ca2+ sensor function supports findings that VAMP4 selectively drive asynchronous release through a population of vesicles that do not interact with synaptotagmin 1 or complexins. At the single synaptic vesicle level, synaptotagmin 1 acted as an essential determinant of synaptic vesicle endocytosis time course by increasing the kinetics of vesicle retrieval in response to increasing Ca2+ levels. In contrast, synaptotagmin 1 did not affect the rapid retrieval of spontaneously fused vesicles. Taken together, these results suggest that exocytic pathways dictate endocytic kinetics as asynchronously fused vesicles are retrieved slowly while spontaneously fused vesicles are rapidly retrieved. These mechanisms may diversify the molecular compositions of synaptic vesicles regenerated after fusion to provide presynaptic terminals with a wide range of synaptic vesicle populations with distinct biogenesis properties and exo-endocytosis kinetics.