Visualization of SNARE-Mediated Organelle Membrane Hemifusion by Electron Microscopy
SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have revealed many of the fundamental mechanisms that drive the membrane fusion process, as well as features unique to organelle fusion. However, until recently, it has not been amenable to electron microscopy methods that have been invaluable for studying hemifusion in other model systems. Herein, we describe a method to visualize hemifusion intermediates during homotypic vacuole membrane fusion in vitro by transmission electron microscopy (TEM), electron tomography, and cryogenic electron microscopy (cryoEM). This method facilitates acquisition of invaluable ultrastructural data needed to comprehensively understand how fusogenic lipids and proteins contribute to SNARE-mediated membrane fusion-by-hemifusion and the unique features of organelle versus small-vesicle fusion.
Key wordsMembrane fusion Hemifusion SNARE Lysosome Vacuole Transmission electron microscopy (TEM) Cryogenic electron microscopy (cryoEM) Tomography Lipid bilayer merger
We thank K. Basu and staff members at the Facility for Electron Microscopy Research at McGill University (Montreal, Canada) for technical assistance. S.M. was supported by a Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Award and a Fonds de Recherche du Québec Summer Research Scholarship. This work was supported by Natural Sciences and Engineering Research Council of Canada grants RGPIN/403537-2011 and RGPIN/2017-06652 to C.L.B.