AbstractThe 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.