The Fusion Machine · The nanomechanical mechanism of exocytotic fusion pore formation

Cells release neurotransmitters, hormones and other compounds stored in secretory vesicles by a process called exocytosis. In this process, the molecules are released upon stimulation by a nanomachine forming a fusion pore that connects the vesicular lumen to the extracellular space. Similar fusion events are also essential for intracellular transport mechanisms and virus-induced fusion.Here I propose a multidisciplinary approach using highly innovative techniques to determine the nanomechanical mechanism of fusion pore formation. The proposal is based on the hypothesis that the vesicle fusion nanomachine is formed by the mechanical interactions of the SNARE proteins synaptobrevin, syntaxin, and SNAP-25 and that the fusion pore is opened by intra-membrane movement of the transmembrane domains. I will combine fluorescence resonance energy transfer microscopy with detection of individual fusion events using microfabricated electrochemical detector arrays to demonstrate that fusion pore formation is produced directly by a conformational change in the SNARE complex. I will estimate the energies that are needed to pull the synaptobrevin C terminus into the hydrophobic membrane core and the forces that are generated by the SNARE complex for wild type and a set of specific mutations using molecular dynamics simulations. I will determine how these energies and forces relate to inhibition and facilitation of experimentally observed fusion, performing patch clamp capacitance measurements of vesicle fusion in chromaffin cells expressing wild type and mutated SNARE proteins. Based on these results I will develop a detailed picture of the molecular steps, the energies, and the forces exerted by the molecular nanomachine of fusion pore formation and will ultimately generate a molecular movie of this fundamental biological process. Understanding cellular and viral fusion events will likely lead to novel treatments from spasms and neurodegeneration to cancer and infectious disease