The ESCRTs catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated a minimal ESCRT module consisting of ESCRT-III subunits Snf7, Vps24, and Vps2, and the AAA+ ATPase Vps4 such that membrane nanotubes reflecting the correct topology of scission could be pulled from giant vesicles. Upon ATP release by photo-uncaging, this system was capable of generating forces within the nanotubes in a manner dependent upon Vps4 catalytic activity, Vps4 coupling to the ESCRT-III proteins, and membrane insertion by Snf7. At physiological concentrations, single scission events were observed that correlated with forces of ~6 pN, verifying predictions that ESCRTs are capable of exerting forces on membranes. Imaging of scission with subsecond resolution revealed Snf7 puncta at the sites of membrane cutting, directly verifying longstanding predictions for the ESCRT scission mechanism.One Sentence SummaryESCRT-III and Vps4 were reconstituted from within the interior of nanotubes pulled from giant vesicles, revealing that this machinery couples ATP-dependent force production for membrane scission.
Actin filament assembly provides force during clathrin-mediated endocytosis. Here, cryo-electron tomography analysis of actin filament number, organization, and orientation during clathrin-mediated endocytosis in intact human cells revealed that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle of 68 +/- 9° indicate that sufficient force to drive endocytosis can be generated through polymerization, and that assembly is triggered from 4 +/- 2 founding "mother" filaments, consistent with the tomography data. The actin-binding protein Hip1R decorates the entire endocytic invagination, including the neck region. Simulations showed that the unexpected Hip1R neck localization targets filament growth to this region, improving internalization efficiency and robustness. Actin cytoskeleton organization described here allowed direct translation of structural information to mechanism.
provides strong evidence for the plasma membrane being poised to respond to an external stimulus, such that signaling components exploit subtle modulations of membrane organization to facilitate the protein-protein interactions that carry the stimulus across the membrane.
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