Abstract
Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca2+-binding C2 domains that tether membranes via interactions with anionic phospholipids. It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them. As quantitative information on membrane gaps is key to understanding general cellular mechanisms, including the role of syt-1, we developed a fluorescence-lifetime based inter-membrane distance ruler using membrane-anchored DNAs of various lengths as calibration standards. Wild-type and mutant data provide evidence that full-length syt-1 indeed regulates membrane gaps: without Ca2+, syt-1 maintains membranes at distances of ~7–8 nm. Activation with 100 μM Ca2+ decreases the distance to ~5 nm by binding the C2 domains to opposing membranes, respectively. These values reveal that activated syt-1 adjusts membrane distances to the level that promotes SNARE complex assembly.
Highlights
Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1, resulting in fusion of synaptic vesicles with the presynaptic membrane
The SNAREs do not assemble before arrival of the Ca2 þ signal, syt-1 may already be in contact with the plasma membrane via the KKKK–PIP2 electrostatic interaction[10]
The calibrated fluorescence-lifetime-based membrane distance ruler with membrane-anchored DNAs of well-defined lengths allowed for direct quantitative determination of membrane gaps controlled by wild-type and mutants of the full-length neurotransmission trigger syt-1
Summary
Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca2 þ -binding C2 domains that tether membranes via interactions with anionic phospholipids It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them. Before the formation of fully zippered SNARE complexes, which exert forces on the membranes and lead to fusion, the synaptic vesicles are first primed to the presynaptic plasma membrane and await the Ca2 þ activation of syt-1. Following Ca2 þ triggering, the SNAREs rapidly progress through zippering and fusion[17] In line with the latter scenario, recently, it has been proposed that syt-1 triggering is based on Ca2 þ -dependent regulation of the gap between the vesicle and presynaptic membranes. Experimental support for this model was still elusive, primarily because of the lack of experimental evidence for distance variations at the appropriate length scale
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