期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：46
页码：13051-13056
DOI：10.1073/pnas.1615885113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceFusion of membranes plays an important role in many biological processes such as vesicle trafficking and viral infection. However, without assistance from specialized proteins such as SNAREs the energy barriers associated with merging of two bilayers are too high to achieve fast kinetics as required for the release of neurotransmitters. We devised a method based on colloidal probe microscopy that allows monitoring the distance of two membranes at a defined contact pressure, providing access to the lifetime of intermediate states of fusion. Changing the contact pressure gives access to the energy landscape and kinetic rates of fusion in the presence of neuronal SNARE proteins. This allows one to explore fusion pathways as a function of molecular composition and environmental cues. 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 atomic-force 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.
