Elucidate the role of VPS4 isoforms in cytokinetic abscission

The AAA-ATPase, VPS4, is part of the ESCRT machinery that drives membrane constriction and fission in numerous processes in cells. The ESCRT complex is comprised of over 20 proteins in mammalian cells that are divided into 5 sub-families, ESCRT 0-III and VPS4. VPS4 is the most evolutionary conserved and is indispensable for the fission reaction. Notably, mammalian cells encode for two VPS4 isoforms – VPS4A and VPS4B, but the role of each isoform is unknown. Here, we set to characterize the role of VPS4 isoforms in ESCRT mediated membrane fission, employing the well documented ESCRT mediated cytokinetic abscission as a model.

To this end, we generated CRISPR/cas9 knock out cell lines of VPS4A, VPS4B and VTA1, a cofactor that stabilizes the hexameric, active form of VPS4. Our results show that depletion of VPS4A causes a considerably more severe delay in abscission compared to VPS4B depletion. STORM imaging revealed that lack of VPS4A leads to over-accumulation of the ESCRT-III protein IST1, at early abscission stages, suggesting a role for VPS4A in early abscission stages that precedes the fission reaction itself. Unexpectedly, a VPS4A mutant locked in the monomeric, inactive, state was able to partially rescue the abscission delay in VPS4A KO cells, and interacted with known abscission checkpoint proteins. Moreover, depletion of VTA1, shifted endogenous VPS4A proteins to their monomeric resulting in accelerated abscission. Collectively, our data highlight a role for monomeric VPS4A in regulating the abscission checkpoint, which is independent from its ATP hydrolysis activity.