Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Tuesday Speaker Abstracts

MECHANICAL REGULATION OF MEMBRANE FUSION, EXOCYTOSIS AND ENDOCYTOSIS STUDIED BY COARSE-GRAINED SIMULATION AND EXPERIMENTAL DATA ANALYSIS

Ben O'Shaughnessy 1 ; 1 Columbia University, New York, NY, USA 2 Columbia University, chemical engineering, New York, NY, USA

Cells fuse membranes for synaptic transmission and other fundamental processes using a multi molecular machinery that responds to calcium to fuse vesicle and plasma membranes (PMs) for secretion. Key components include the SNARE proteins and the calcium sensor, synaptotagmin. Computationally, since physiological timescales (> milliseconds) are far beyond atomistic approaches, coarse-graining is necessary to uncover collective mechanisms. We use coarse grained computational simulation methods to render the cellular fusion machinery on physiological timescales. We found membrane fusion is driven by entropic forces among the bulky SNARE complexes (SNAREpins) which clear the fusion site, squeeze membranes so the outer leaflets fuse (hemifusion stalk), and expand the stalk into a growing hemifusion diaphragm (HD). Finally, the HD nucleates a hydrophobic simple pore that yields to an unstable growing hydrophilic pore for HD rupture and fusion. Thus, SNARE zippering energy does not drive fusion, but assembles SNAREpin rods with maximal entropic volume. Enveloped viruses such as SARS-CoV-2 appear to use the same entropic mechanism. In simulations, synaptotagmin mediated unclamping and membrane fusion are parallel processes: calcium stimulates progressive SNAREpin unclamping, increasing the fusion rate since more snarepins generate higher entropic forces. Consequently, action potential-evoked release probabilities are higher (lower) with more (fewer) available SNAREs, in agreement with experimental studies. During stimulated exocytosis, such evoked membrane fusion and release events occur repeatedly. How are they spatiotemporally regulated? We analyzed dense core vesicle exocytosis in chromaffin cells from data collected by Ling-Gang Wu (NIH), revealing repeated release hotspots, a reservoir of PM-fused vesicles, endocytic removal of reservoir vesicles and budding of new reservoir vesicles from the PM. With a mathematical model, we conclude repeated exocytosis lowers the PM tension and mechanically stablilizes a reservoir. The reservoir regulates exocytosis by blocking release sites, and provides vesicles for fast endocytic internalization that short-circuits the slow clathrin pathway.

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