Abstract
Membrane fusion is central to fundamental cellular processes such as exocytosis, when an intracellular machinery fuses membrane-enclosed vesicles to the plasma membrane for content release. The core machinery components are the SNARE proteins. SNARE complexation pulls the membranes together, but the fusion mechanism remains unclear. A common view is that the complexation energy drives fusion, but how this energy is harvested for fusion is unexplained. Moreover, SNAREs likely fully assemble before fusion. Computer simulation is challenging, as even fast neurotransmitter release at neuronal synapses involves fusion on ms timescales, beyond the scope of atomistic or mildly coarse-grained approaches. Here, we used highly coarse-grained representations, allowing simulation of the ms timescales of physiological SNARE-driven fusion under physiological conditions. Due to constant collisions, the rod-like SNARE complexes spontaneously generated entropic forces ∼8 pN per SNARE that cleared the fusion site and squeezed the membranes with forces ∼19 pN per SNARE, catalyzing a hemifused stalk connection. Regrouping, five or more SNARE complexes exerted entropic tensions 2.5 pN/nm or greater, expanding the stalk into a hemifusion diaphragm (HD), followed by HD rupture and fusion. The entropic forces generated tensions ∼17–21 pN in the SNARE linker domains (LDs). Previous optical tweezer measurements suggest that, on the ms timescales of fusion, these LD tensions are sufficient to unzipper the LDs while leaving the C-terminal domain (CTD) marginally intact, which are both required for fusion. Consistent with a recent magnetic tweezers study, we propose that the CTD may be further stabilized by complexin for robust fusion. Our results explain how SNARE-generated forces fuse membranes and predict that more SNARE complexes exert higher net force so that fusion is faster, consistent with experimental electrophysiological studies at neuronal synapses. Thus, entropic forces evolve SNARE complexes into a fusogenic, partially unzippered state, squeeze membranes for hemifusion, and expand hemifusion connections for fusion.