Membrane binding, bending, and remodeling by calcium sensor proteins

Synaptotagmin I (Syt1) is a vital protein for neural activity. Despite extensive research, the exact role of Syt1 in membrane fusion remain uncertain. Here we take a novel approach to deciphering the activity of Syt1 and Doc2b (that regulates spontaneous neurotransmitter release), by using complementary single-molecule techniques: Optical tweezers combined with fluorescent confocal fluorescence, and atomic force microscopy (AFM) nanoindentation. Using pairs of optically-trapped beads coated with SNARE-free synthetic membranes, we find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca²⁺ and protein-dependent manner. When comparing symmetrical (both sides) and asymmetrical (one side) presence of protein on the membranes, Syt1 favors an asymmetrical but Doc2b a symmetrical configuration, as inferred from higher tether probabilities and break forces. This provides support for the direct bridging hypothesis for Syt-1. By content mixing and lipid mixing assays, we further reveal that Doc2b readily facilitates hemifusion, while Syt-1 at similar concentrations does not. Using AFM, we find a decrease in membrane bending moduli following protein binding, suggesting these proteins have a role in reducing the energy barrier for fusion. Our approach can be readily extended to explore other membrane-remodelling and membrane-fusion events.