Determining the composition of the ESCRT-III filament in cytokinesis abscission of mammalian cells

The endosomal sorting complex required for transport (ESCRT) participates in membrane constriction and fission in a variety of processes in cells including abscission of the intercellular membrane bridge connecting two daughter cells at the end of cytokinesis. The ESCRT machinery is composed of five different subfamilies: ESCRT 0, I, II, III and the AAA ATPase VPS4. Early ESCRT components (ESCRT 0, I, and II) are essential for recruiting cytosolic ESCRT-III monomers to the membrane. Membrane-bound ESCRT-III and VPS4 are thought to induce membrane constriction and fission through polymerization and remodeling of ESCRT-III helical filaments. Recently, we resolved the organization of the ESCRT-III component, IST1 at different stages of abscission using 3D STORM. However, there are over 11 ESCRT-III subunits in the human genome (named CHMPs) and therefore it is unclear whether the localization pattern observed for IST1 reflects the overall organization of the ESCRT-III complex. To decipher the organization of all ESCRT-III components at the intercellular bridge, we are generating a series of knock-in cell lines that carry an HA epitope tagged versions of different ESCRT-III components using the CRISPR/Cas9 technology. Using endogenous expression levels will avoid artifacts associated with over expression of specfic ESCRT-III components. By performing a series of two-color 3D STORM datasets, one using IST1 antibodies and one using antibodies against the epitope tag in each knock-in cell line, in cells undergoing abscission we plan to generate a map of the organization of ESCRT-III components at the intercellular bridge.