Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs

摘要

Synaptotagmin-1 (Syt1) is a major Ca²⁺ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca²⁺, Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP2) complex and increases the docking rate by 10³ times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca²⁺, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca²⁺, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP2 to help SNAREpin formation and then, upon Ca²⁺ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.