Nano-mechanics and functional consequences of synaptotagmin·membrane interactions

Rapid communication between neurons primarily relies on release of neurotransmitters from the presynaptic neuron to the synaptic cleft, where the transmitters bind and activate specific receptors on the postsynaptic neuron. Neurotransmitter release is mediated by Ca2+-triggered synaptic vesicle exocytosis, which occurs on sub-millisecond time scales and is strongly Ca2+ dependent. During exocytosis, fusion of synaptic vesicles with the presynaptic membrane is catalyzed by SNAREs and a handful of regulatory proteins. The Ca2+ sensitivity and extraordinary speed of this process is at least in part conferred by the vesicle protein synaptotagmin-I (syt-I). Syt-I regulate membrane fusion through engaging target membranes and SNAREs. The main goal of thesis is to understand the molecular mechanism of syt-I synaptic vesicle exocytosis, with a primary focus on the nano-mechanics and functional consequences of syt-membrane interactions.;The cytoplasmic portion of syt-I is comprised of two tandem C2 domains that are able to bind Ca2+, the target membrane, and the core fusion apparatus, SNAREs. In this work, we show that the two C2 domain of syt-I simultaneously penetrate into one monolayer of the target membrane with diffusion-limited kinetics. Removal of Ca2+ results in rapid dissociation of syt-I from the membrane. Some isoforms of syts exhibit much slower membrane unbinding kinetics than syt-I, suggesting that these syt isoforms might function during delayed, sustained neurotransmitter release after the collapse of Ca2+ microdomains.;Partial membrane insertion of the C2 domains drives local deformation of the target membrane. Using SNARE-bearing liposomes with different membrane curvatures, we directly demonstrate that local bending of the plasma membrane is essential for syt-I to regulate membrane fusion. Membrane bending deficient syt-I mutant fails to stimulate fusion between relatively flat membranes, while the wild-type promotes such fusion efficiently in response to Ca 2+. Furthermore, addition of a membrane bending module involved in the endocytic pathway efficiently rescues the functional defect of the membrane bending deficient syt-I mutant. These findings support a model in which syt-I drives localized invagination of the plasma membrane towards individual synaptic vesicles in response to Ca2+, providing a focal point for membrane fusion that significantly reduce the energy cost of this process.