A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth

Abstract

Neuronal signaling occurs through both action potential-triggered synaptic vesicle fusion and spontaneous release, although the fusion clamp machinery that prevents premature exocytosis of synaptic vesicles in the absence of calcium is unknown. Here we demonstrate that spontaneous release at synapses is regulated by complexin, a SNARE complex-binding protein. Analysis of Drosophila melanogaster complexin null mutants showed a marked increase in spontaneous fusion and a profound overgrowth of synapses, suggesting that complexin functions as the fusion clamp in vivo and may modulate structural remodeling of neuronal connections by controlling the rate of spontaneous release.

Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

“…S4). Thus, the complexin knock-down greatly impaired fast synchronous but not asynchronous synaptic vesicle fusion, and increased spontaneous fusion, thereby reconciling the divergent phenotypes observed in vertebrate autapses, Drosophila neuromuscular junctions, and in vitro fusion assays [7,[10][11][12][13][14][15].We next examined which complexin sequences mediate spontaneous and evoked fusion (Fig. 1C).…”

“…Later responses during the train's asynchronous phase were normal, and delayed release was enhanced (Figs. 3H-3J and S13), thus rendering the WA-mutation a weaker phenocopy of the synaptotagmin-1 KO, the complexin KO, the complexin knockdown, and the synaptobrevin 3A-mutation phenotype (7,15,21;. In contrast to the WA-mutation, the 85-and 86-mutations did not impair rescue of synaptic transmission by synaptobrevin in synaptobrevin-deficient neurons (Fig.…”

“…Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10].…”

“…In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15]. Thus, the role of complexin in fusion is unclear.…”

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo

“…Initial in vitro studies using cell-cell fusion or lipid-mixing assays indicated that Cpx prevents in vitro membrane fusion events, suggesting Cpx may act as a fusion clamp to prevent premature exocytosis in the absence of Ca 2+ (11,14,15). Consistent with the role of Cpx in clamping synaptic fusion, in vivo experiments in Drosophila revealed a dramatic increase in spontaneous synaptic vesicle fusion events (minis) in cpx null mutants (16). In addition to enhanced minis, cpx mutants have reduced evoked release.…”