The cell membrane continuously undergoes fusion and fission in order to transport cargoes in and out of the cell. The endosomal sorting complexes required for transport (ESCRT) proteins are an ancient and ubiquitous membrane remodeling machinery present in all three branches of life. ESCRTs drive membrane scission of various biological processes where the limiting membrane invaginates and bud away from the cytosol such as in intraluminal vesicle formation in multivesicular bodies, cytokinesis, HIV-1 viral release. The core ESCRT machinery is comprised of ESCRT-0, -I, -II, -III, ALIX, and the AAA+ ATPase VPS4 whereby the ESCRT-III proteins are most responsible for membrane constriction. ESCRT-III proteins form heteropolymers and act on membranes that have negative curvature, and together with VPS4, catalyze scission possibly by active subunit treadmilling. In Chapter one and two of this dissertation we provide a general introduction to the function and biophysical basis for membrane scission by ESCRT proteins. These chapters discuss the myriad of biological processes that take advantage of the host cell’s ESCRT proteins as well as unresolved questions in the field.

Recent work has shown that a subset of ESCRT-III subunits are capable of binding and constricting positively-curved membrane tubules thereby establishing that ESCRTs can also perform normal-topology scission. The microtubule-severing AAA+ ATPase spastin that has been found to colocalize with ESCRT-III enriched sites in cells could also perhaps play a role in membrane scission. Here we used biochemical and biophysical approaches using giant unilamellar vesicles and optical tweezers to directly probe ESCRT function as it constricts and severs the membrane. Chapter three of this dissertation describes the construction of an optical tweezer and fluorescence microscopy setup to observe ESCRT protein function on single membrane nanotubes in vitro. This instrument is a general setup that can be used to interrogate protein functions on the surface of membranes.

Finally, in Chapter four we focused on the role of ESCRT-III subunits CHMP1B and IST1 as well as the AAA+ ATPases VPS4 and spastin in normal-topology membrane scission using optical tweezers. We show that CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but do not disassemble the CHMP1B-IST1 coat from the membrane while VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.