| Description |
The mammalian Endosomal Sorting Complex Required for Transport (ESCRT) pathway mediates the final membrane fission step of cytokinesis, known as abscission. Prior to abscission, the ESCRT machinery is recruited to the intercellular bridge that connects nascent daughter cells. Once there, subunits of the ESCRT-III complex bind the plasma membrane and oligomerize into membrane-bound filaments. These filaments constrict the membrane, resulting in membrane fission and release of two independent daughter cells. Here, I describe the creation of siRNA/shRNA depletion and rescue assays for testing abscission functions of the ESCRT-III proteins IST1 and CHMP2A. siRNA or shRNA treatments were used to deplete endogenous IST1 or CHMP2A protein levels, and exogenous wild-type or mutant constructs were tested for their ability to "rescue" the resulting abscission defects, as assayed by flow cytometry or morphological assays. These assays confirmed that both IST1 and CHMP2A are required for abscission, and that exogenous wild-type IST1 and CHMP2A can rescue the defects induced by depletion of the cognate proteins. The IST1 and CHMP2A depletion/rescue assays provided a means for testing whether different IST1 and CHMP2A mutants can function in abscission. As an initial proof of principle, I tested whether mutations in conserved basic patches in the N-terminal alpha helix inhibit the ability of IST1 and CHMP2A to function in abscission. Tests were performed because previous studies had implicated these basic patches in ESCRT-III membrane binding. Consistent with this hypothesis, mutations in the basic residues of IST1 abolished abscission functions; however, the CHMP2A data were ambiguous. Thus, my work has confirmed that the ESCRT-III proteins IST1 and CHMP2A are both required for the abscission step of cytokinesis, and have provided an assay system that can be used to test the functionality of mutant proteins. |
