1) Mechanisms of neurotransmitter and hormone release. Calcium-triggered fusion of cargo-laden vesicles with the plasma membrane releases neurotransmitters and hormones. The initial ~1-3 nm wide connection between the fusing membranes (the fusion pore), can flicker open-closed before resealing or dilating. Pore flickering impacts releasable cargo size, release kinetics, and vesicle recycling. Components of the release machinery are known, but how calcium entry drives membrane fusion and mechanisms regulating fusion pore dynamics are poorly understood. We are studying mechanisms contributing to fusion pore regulation and release kinetics in conventional synapses, and the unique features of hair cell synaptic transmission which, different from conventional synapses, relies on a large protein called Otoferlin for neurotransmitter release by poorly-understood mechanisms.

2) Dynamics of cell membrane tension and membrane turnover. Exocytosis and endocytosis generate and are regulated by membrane tension gradients. Such gradients drive membrane flows, but how rapidly plasma membrane flow can relax tension gradients is controversial.

We discovered that membrane tension can propagate at vastly different speeds depending on cell type. In bipolar neuronal terminals specialized for rapid vesicle turnover, membrane tension equilibrates within seconds, whereas it does not propagate in neuroendocrine adrenal chromaffin cells. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Thus, membrane flow and tension equilibration may be adapted to distinct membrane recycling requirements.

We are currently trying to elucidate the molecular and biophysical mechanisms regulating cell membrane tension propagation and membrane flows and how such flows affect the spatio-temporal coupling between exocytosis and endocytosis.

3) Novel sub-cellular protein localization and membrane fission mechanisms. How do cellular components localize to specific sites and how does membrane fission occur in bacteria? Only a few mechanisms have been proposed for the former and virtually nothing was known about the latter.

We discovered a new mechanism utilized by a protein called FisB to localize to a highly curved membrane neck in B. subtilis cells during formation of stress-resistant spores, relying solely on its tendency to form trans-complexes bridging membranes, homo-oligomerization, and the cell geometry. After localizing to the membrane neck, FisB promotes membrane fission, but only if the membrane tension is sufficiently high. High membrane tension is provided by energy-intensive DNA packing into the small forespore compartment that inflates it. In future studies, we will explore how lipids and other components are transported across the membrane compartments that form during sporulation.

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