PROJECT SUMMARY Many basic processes rely on secretion of bioactive molecules by exocytosis, when membrane-enclosed vesicles containing neurotransmitters (NTs), hormones or other molecules fuse with the plasma membrane (PM) and release their contents through fusion pores. Neurotransmission relies on NT release at neuronal synapses, when a multicomponent machinery senses Ca!"influx triggered by an action potential and fuses small synaptic vesicles with the plasma membrane on sub-millisecond timescales, releasing NTs into the synaptic cleft to elicit a post-synaptic response. Other regulated exocytosis is slower, such as hormone release from neuroendocrine cells when a stimulus provokes large dense core vesicles to release contents after seconds or longer. In all cases, the membrane fusion step is accomplished by the SNARE proteins, when vesicle-associated VAMP (the v-SNARE) and two PM-associated t-SNAREs syntaxin and SNAP 25 zipper into a ternary complex, pulling the membranes together and fusing them. However, the mechanism of membrane fusion remains unclear. Other components in the machinery block (“clamp”) SNARE-mediated fusion, until the Ca!"signal releases the clamp. Synaptotagmin (Syt) is the Ca!"sensor for synchronous release, but the molecular identity of the clamp and the Ca!"-triggered unclamping mechanism are not established. Mutations in SNARE proteins and other NT release machinery components are associated with neurodevelopmental and neurodegenerative disorders, and impaired fusion pore dilation is associated with reduced insulin secretion by β-cells of type-2 diabetics. The proposed research aims to use mathematical modeling to establish the mechanisms of regulated membrane fusion and the mechanisms that regulate the vesicle and its pore for controlled contents release following fusion. From the previous funding period, we have working molecular dynamics (MD) simulations of SNARE-mediated membrane fusion and of the NT release machinery incorporating the core SNARE and Syt components. The simulations used sufficiently coarse-grained (CG) representations to achieve the computationally demanding millisecond physiological timescales of fusion and release. Aim 1 is to advance the SNARE-mediated fusion simulations with more realistic SNAREs, and to use machine learning to catalogue the pathways to membrane fusion as a function of the size of the fusing vesicles and other key variables. Mutated SNAREs will be simulated and compared to experiments by collaborators. Aim 2 is to use continuum mathematical modeling to establish the structure, energetics and evolution of the fused vesicle-PM complex and its fusion pore, and the mechanisms of SNARE- and Syt-mediated pore dilation. Aim 3 is to advance the MD simulations of the NT release machinery by introducing molecularly explicit representation of the membranes, and by incorporating additional components as their interactions become experimentally characterized, toward a long-term goal of “rec...