Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin‑1 -PI(4,5)P₂ Interaction in Constrained Geometries

A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca²⁺ the binding of syt-1 to membranes depends on the presence and content of PI­(4,5)­P₂. Addition of Ca²⁺ switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein. Fusion of single unilamellar vesicles equipped with syt-1 and synaptobrevin 2 with planar pore-spanning target membranes containing PS and PI­(4,5)­P₂ shows an almost complete suppression of stalled intermediate fusion states and an accelerated fusion kinetics in the presence of Ca²⁺, which is further enhanced upon addition of ATP.