Real-Time Visualization of Dynamin-Catalyzed Membrane Fission and Vesicle Release

Abstract

The GTPase dynamin assembles at the necks of budded vesicles in vivo and functions in membrane fission. We have developed fluid supported bilayers with excess membrane reservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Consistent with previous studies, in the absence of GTP, dynamin assembles in spirals, forming long membrane tubules. GTP addition triggers disassembly, but not membrane fission, arguing against models in which fission is mediated by concerted and global GTP-driven conformational changes. In contrast, under physiological conditions in the constant presence of GTP, dynamin mediates membrane fission. Under these conditions, fluorescently labeled dynamin cooperatively organizes into self-limited assemblies that continuously cycle at the membrane and drive vesicle release. When visualized at the necks of emergent vesicles, self-limited dynamin assemblies display intensity fluctuations and persist for variable time periods before fission. Thus, self-limited assemblies of dynamin generated in the constant presence of GTP catalyze membrane fission.