SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

Abstract

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.embrane fusion plays a pivotal role in many fundamental biological processes, comprising viral infection, cell-cell fusion during fertilization, tissue formation, and intracellular transport during exo-and endocytosis (1). Among the best-studied biological examples is the Ca 2+ -regulated fusion of synaptic vesicles with the presynaptic plasma membrane in neurons and chromaffin cells (2, 3). Fusion in these cells is catalyzed by concerted action of SNARE proteins through formation of a tetrameric coiled-coil complex that releases a sufficient amount of free energy to lower the barriers for membrane merging. One major energy barrier is associated with the strong repulsive hydration forces when two smooth bilayers approach each other. Other energy-costly contributions originate from lipid splaying as the initiation of stalk formation, expansion of the stalk structure driven by the strong curvature associated with membrane destabilization, hemifusion diaphragm expansion, and, finally, pore formation (4-8). The free energy associated with complex formation and folding of SNAREs was reported to be ∼35 k B T, close to the energy required to create the highly curved transition structures (40-50 k B T) (9, 10). Albeit a part of this free energy might be dissipated as heat, this coincidence of matching energy is also supported by experimental studies showing that only very few SNARE complexes are sufficient to induce fusion in artificial systems (5,6,(11)(12)(13). Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) m...

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

“…We used a minimal SNARE machinery with Syb reconstituted in the LUVs and the ΔN-complex displayed by the SLBs or GUVs 31 , 32 . In previous studies we and others could show that this lipid-protein composition is sufficient to accomplish merging of membranes within seconds 33 , 34 .…”

“…A minimal fusion machinery consisting of syntaxin, SNAP 25 and synaptobrevin as described previously was employed to obtain a reasonable fusion efficiency to begin with 33 , 34 . We also used lipids with the headgroup phosphatidylethanolamine that are known to result in negative spontaneous curvature in membranes and thus promote fusion processes.…”

Myomerger promotes fusion pore by elastic coupling between proximal membrane leaflets and hemifusion diaphragm

“…Indeed, many functionally important regions of the proteins that mediate fusion in viral infections and in developmental processes are located in their ectodomains and interact with proximal monolayers. Because of the location of the fusion proteins, most of the suggested mechanisms of protein-mediated fusion have explained only how these proteins drive merger of proximal monolayers resulting in hemifusion by bringing proximal monolayers into very tight contact and bending them 2 , 14 , 15 . Considering that Myomerger is not involved in hemifusion but drives the transition from hemifusion to fusion, this protein presents an important model for analysis of the physical mechanisms underlying an, apparently, indirect effect of Myomerger and other proximal leaflet-associated factors on the fusion pore formation.…”

Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension

“…Furthermore, how mechanical tension in the neuronal SNARE complexes affects the reported effects of Cpx has not been explored so far. It has been presumed that the tension rapidly builds up when a synaptic vesicle approaches a presynaptic membrane because of the electrostatic repulsion, hydration barrier, and steric hindrance between the two fusing membranes 31 , 32 . This tension in a SNARE complex was shown to significantly tilt the energy landscape that governs SNARE zippering processes 33 , 34 .…”