Entropic Forces, Zippering Pressure and Membrane Thinning Drive Snare-Mediated Membrane Fusion

Abstract

We studied SNARE-mediated membrane fusion using ultra coarse-grained (UCG) simulations that achieved fusion of realistically sized vesicles with realistic membrane tensions. Previous simulation approaches primarily used unphysical membrane conditions to force fusion on observable timescales. Our results elucidate fundamental features of the pathway for the first time. Membrane fusion is an essential step during neurotransmitter or hormone release and trafficking. Cells use a multi-component fusion machinery whose core consists of the SNARE proteins. The mechanism of SNARE-mediated membrane fusion is not established, and the intermediate states along the fusion pathway remain controversial. Computer simulations can help uncover mechanisms, but a major obstacle is that physiological and in vitro membrane fusion timescales range from msec to sec, far beyond the range of all-atom or mildly coarse-grained (CG) approaches. Here we used UCG simulations to reveal the pathways to vesicle-vesicle and vesicle-planar membrane fusion on msec timescales, using the Cooke-Deserno force field phospholipid representation (Cooke et al., 2005). SNAREs zippered up between the two vesicles, blocking fusion, but entropic forces among the SNARE complexes and membranes then pushed the SNAREs to the periphery of the membrane-membrane contact zone where zippering forces compressed the membranes and triggered fusion along the pathway: hemifusion to hemifusion diaphragm (HD) expansion to nanopore formation in the HD. Fusion was faster with more SNAREs present, since entropic forces were greater (Mostafavi et al., 2017; McDargh et al., 2018). Transmembrane domains (TMDs) played a surprisingly vital role. With TMDs modeled on wild type SNAREs, fast fusion occurred when the TMDs were pulled deep into the membrane, causing local bilayer thinning and destabilization that triggered the fusion pathway. Modified TMDs simulating mutations failed to sufficiently penetrate the membrane and fusion was not triggered, in agreement with experiment.