Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

While the role of Synaptotagmin-1 in living cells has been described in detail, it remains a challenge to dissect the contribution of membrane remodelling by its two cytoplasmic C2 domains (C 2 AB) to the Ca 2+ -secretion coupling mechanism. Here, we study membrane remodeling using pairs of optically-trapped beads coated with SNARE-free synthetic membranes. We find that the soluble C 2 AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca 2+ - and protein-dependent manner. A lipid mixing assay with confocal imaging reveals that at low Syt1 concentrations, no hemifusion is observed. Notably, for similar low concentrations of Doc2b hemifusion does occur. Consistently, both C 2 AB fragments cause a reduction in the membrane bending modulus, as measured by an AFM-based method. This lowering of the energy required for membrane deformation likely contributes to the overall Ca 2+ -secretion triggering mechanism by calcium sensor proteins. 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, while hinting to possible preference for protein-protein (and not protein-membrane) interactions for Doc2b. Overall, our study sheds new light on the mechanism of Ca 2+ induced fusion triggering, which is essential for fundamental understanding of secretion of neurotransmitters and endocrine substances.