During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.
Neurotransmitter release is mediated by Ca2+ dependent exocytosis of synaptic vesicles. Neither the amount of transmitter released from individual synaptic vesicles nor the kinetics of this process have yet been directly determined. Using carbon fibres as electrochemical detectors, we have measured release of the neurotransmitter serotonin from cultured neurons of the leech. This technique allowed us to monitor transmitter discharge from single synaptic vesicles as spike-like oxidation currents at high time resolution, providing new insight into the mechanism of neuronal exocytosis. Two types of signals were characterized, corresponding to exocytosis of small clear and large dense core vesicles present in these cells. A small vesicle discharges about 4,700 transmitter molecules with a time constant in the region of 260 microseconds, whereas large vesicles release their content of approximately 80,000 molecules with a time constant of about 1.3 ms. Release from both vesicle types is initiated rapidly, with a rise time of less than 60 microseconds, suggesting an abrupt opening of a preassembled fusion pore.
SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca 2 þ -dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.
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