Abstract
ESCRT proteins catalyze the function of membrane budding and scission from the inside of the cytosol to the outside. This process is fundamental in cell biology, comprising multivesicular endosome biogenesis, cytokinesis, viral budding (e.g. HIV, Ebola, Dengue) and other pathways. The mechanism underlying ESCRT-III-mediated membrane budding and scission remains elusive. We have encapsulated within giant unilamellar vesicles (GUVs) a minimal ESCRT module consisting of ESCRT-III subunits and the AAA+ ATPase Vps4. Using optical tweezers, membrane nanotubes reflecting the correct topology of scission can be pulled from these GUVs. Upon photo-uncaging of ATP, surprisingly large forces in the tens of piconewtons were recorded and tube scission could be observed. ESCRT subunit composition and concentration alter force generation and scission behavior. In combining confocal fluorescence microscopy and optical tweezers, the scission events can be observed in both force and fluorescence and studied in detail. For the first time, the biophysics of ESCRT membrane budding and scission are revealed.
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