Abstract
Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially "tethered" to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the "primed" vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.
Highlights
Neuronal communication is largely achieved by the controlled release of neurotransmitters stored in synaptic vesicles (SVs) into the synaptic junctions [1,2,3,4]
Vesicle fusion is catalyzed by the membrane-bridging complexes formed by the synaptic SNARE proteins, VAMP2 on the SV (v-SNARE) and Syntaxin1/SNAP25 (t-SNAREs) on the plasma membrane (PM) [6,7,8]
We find that there is a symmetric arrangement of exactly six protein densities at the docking interface, suggesting the fusion machinery is well ordered and prearranged for fast and precise release of neurotransmitters
Summary
Neuronal communication is largely achieved by the controlled release of neurotransmitters stored in synaptic vesicles (SVs) into the synaptic junctions [1,2,3,4]. In vitro reconstitution experiments show that the fusion of an isolated SV (or SV mimetics) with planar bilayers by the purified synaptic SNARE proteins takes 20 to 100 ms after the SNAREs engage to form a half-zippered (RRP-like) state [6, 17,18,19]. While this is more than ample to explain membrane fusion in intracellular protein transport and endocytosis [20,21,22], the speed of SNAREs alone is 100 to 1,000 times too slow to explain synchronous neurotransmitter release.
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