Solution Single Vesicle Fusion Assay Reveals PIP2 Mediated Sequential Actions of Synaptotagmin-1 onto SNAREs

Abstract

Synaptotagmin-1 (Syt1) is a major Ca2+ sensor for fast synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs. Syt1 is known to interact with target membrane (t-) SNARE, ternary SNARE complex, and anionic phospholipids. However, how Syt1 utilizes its diverse interactions to regulate vesicle fusion remains illusive. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out all subpopulations of fusion intermediates in bulk solution, particularly the docking stage before lipid mixing. The results show that membrane-anchored Syt1 undergoes at least three distinct steps prior to lipid mixing. First, in the absence of Ca2+, Syt1 mediates vesicle tethering by directly binding to t-SNARE, which requires PIP2. Second, synaptobrevin-2 binding to t-SNARE to form the ternary complex displaces Syt1 from the SNARE complex. Third, in the presence of Ca2+, Syt1 rebinds to the SNARE complex, which again requires PIP2. Thus in the absence of Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP2 to help ternary SNARE complex formation and then, upon Ca2+-influx, it may rebind to the ternary complex with the aid of PIP2, which may trigger fast synchronous fusion.