Dynamics, Pathways, and Regulation of Exocytotic Release

Exocytosis involves fusion between a membrane-bound vesicle containing signaling moleculesand the plasma membrane of the cell. Cells use exocytosis to release membrane-impermeant bioactive molecules to the extracellular space, and to deliver lipids and proteins to the plasma membrane. Exocytosis is essential to many fundamental processes including neurotransmission and hormone secretion. Exocytosis is triggered and regulated by a range of cellular components including calcium, SNARE proteins, and the actin cortex. Despite of the accumulative discovery of molecules involved in exocytosis, a unified physical landscape that these molecules act on is missing. The biophysical forces driving exocytosis haven’t been identified, and the mechanisms by which these biophysical forces regulate exocytosis are not established. To address these unsolved questions, we built mathematical model to study exocytosis on the single-vesicle level and the cell level. In the first chapter of the thesis we modeled the shape evolution of dense-core vesicles in chromaffin cells during exocytosis. Emerged from the model, we discovered a novel mechanism that drives vesicles to merge into the plasma membrane. Following fusion, the osmotic pressure of the cell squeezes the vesicle and abolishes the vesicle membrane tension, and the high plasma membrane tension reels the vesicle onto the adjacent cytoskeleton. With no fitting parameters, the model predicted remarkable vesicle shapes consistent with real-time visualizations by super-resolution microscopy from the Wu lab. Interestingly, we predicted vesicles to adopt elongated tubular shapes under mildly high osmotic pressure, which was confirmed by visualizations from the Wu lab, providing a vivid illustration of osmotic squeezing. In the second chapter of the thesis we investigated fusion pores, the membrane connection between a fused vesicle and the plasma membrane. As commonly observed in amperometric traces, the initially small pore may subsequently dilate for full contents release. Here using formalisms of differential geometry, we obtained exact solutions for fusion pores between two membranes. We found three families: a narrow pore, a wide pore and an intermediate tether-like pore. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we showed fusion pores are locked into the narrow pore family in nanodisc-based experiments, powerful systems for the study of individual pores. In the third chapter of the thesis we investigated the mechanism of spatiotemporal regulation of exocytosis on the cell level. By analyzing the spatiotemporal profile of exocytosis events in chromaffin cells observed by confocal microcopy from the Wu lab, we discovered a novel mechanism of exocytosis regulation via release site availability. We found vesicle fusion can happen repeatedly at hotspots, which generated a membrane reservoir consisting of unmerged and slowly merged vesicles that are spatially close to hotspots. In turn, unmerged vesicles occupy release sites and locally suppress exocytosis frequency. We developed a mathematical model to demonstrate that such membrane reservoir requires sufficiently low local membrane tension that abolishes the driving force of vesicle merger. Finally, in the fourth chapter of the thesis we studied virus entry, a process similar to exocytosis but involves membrane fusion between the virus and the host cell. SARS-CoV-2 entry in to host cells is accomplished by the S2 subunit of the spike S protein by capture of the host cell membrane and fusion with the viral envelope. Membrane capture requires the native S2 to transit to its potent, fusogenic form, the fusion intermediate, whose structure is unknown. Here, we computationally constructed a full-length model of the CoV-2 fusion intermediate by extrapolating from known CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the fusion intermediate was remarkably flexible and executed large bending and extensional fluctuations due to three hinges in the C-terminal base. The large configurational fluctuations of the fusion intermediate generated a substantial exploration volume that aided capture of the target membrane. Simulations suggested a host cell membrane capture time of ~ 2 ms. Our simulated structures of the fusion intermediate showed good agreement with cryo-electron tomography data from the Moscona’s lab