Mathematical Modeling Supports the Hypothesis that Synaptotagmin Rings Clamp Fusion

Abstract

Synaptotagmin (Syt) is a component of the cell's machinery that mediates membrane fusion during synaptic release and other fundamental processes. Syt is an integral membrane protein in synaptic vesicles with calcium binding domains C2A and C2B and binds SNARE proteins whose assembly drives fusion. SYT is the calcium sensor during evoked release, but the mechanisms are unclear. Recently, electron microscopy (EM) showed Syt C2AB domains spontaneously forming rings on negatively charged phospholipid monolayers. Ca2+disassembled the rings, suggesting Syt rings could serve as membrane spacers that clamp fusion by preventing SNARE assembly until the Ca2+ pulse, when their disassembly would trigger fusion (Wang et al, 2015).Here we used mathematical modeling to test the hypothesis that SYT rings form on target membranes and clamp fusion. We developed a coarse-grained model that incorporates features from the Syt crystal structure and treats the membranes in a dynamic triangulation scheme.The model showed that Syt monomers self-assemble into rings on monolayers, with poly-lysine SYT patches on the inner edge of the ring attracting and buckling the charged membrane. The model reproduced experimental ring size distributions and structures seen in EM: smaller rings produced dome-shaped membrane deformations, larger rings produced volcanoes, reflecting competition between membrane bending energy and unbinding energy from the substrate. Further, we find that docking of a SYT-containing vesicle will generate SYT rings on the target membrane (trans-binding) due to the charged PS and PIP2 components, consistent with the ring spacer clamping hypothesis. Interestingly, rings buckled the membrane towards the synaptic vesicle, a deformation that might be expected to promote fusion. Overall, we find that charged membranes promote formation of Syt rings that may clamp fusion, but the rings simultaneously deform membranes into potentially fusion-promoting shapes.