However, fusion speed in these assays is orders of magnitude slower than at synapses, and spatial precision of exocytosis with its targeting towards postsynaptic receptor domains on target cells cannot be studied in these in vitro systems. Reconstitution assays have revealed that the minimal machinery for Ca 2+-triggered exocytosis consists of SNARE proteins, Munc18, Munc13 and synaptotagmin ( Hu et al., 2003 Ma et al., 2013 Tucker et al., 2004). Our work identifies an 80-kDa β4-Zn protein that bypasses the need for megadalton-sized secretory machines, establishes that fusion competence and docking are mechanistically separable, and defines RIM zinc finger-Munc13 complexes as hubs for active zone function.Įssential insight into the functioning of synaptic exocytotic machinery has come from rebuilding the fusion process in vitro. Action potential-triggering of release was reconstituted by docking these primed vesicles to Ca 2+ channels through attaching RIM zinc fingers to Ca Vβ4-subunits. Re-expression of RIM zinc fingers recruited Munc13 to undocked vesicles and rendered the vesicles release-competent. We here rebuild docking, priming and Ca 2+-secretion coupling in these mutants without reinstating active zone networks. Simultaneous knockout of the active zone proteins RIM and ELKS disrupts active zone assembly, abolishes vesicle docking, and impairs release. The complexity of active zone machinery has made it challenging to determine its mechanisms in release. ![]() They form sites for vesicle docking and priming, and couple vesicles to Ca 2+ entry for release-triggering. Presynaptic active zones are molecular machines that control neurotransmitter secretion.
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