Loss of CHMP2A implicates an ordered assembly of ESCRT-III proteins during cytokinetic abscission

The ESCRT machinery mediates membrane remodeling in fundamental cellular processes including cytokinesis, endosomal sorting, nuclear envelope reformation, and membrane repair. Membrane constriction and scission is driven by the filament-forming ESCRT-III complex and the AAA-ATPase VPS4. While ESCRT-III-driven membrane scission is generally established, the mechanisms governing the assembly and coordination of its twelve mammalian isoforms in cells remain poorly understood. Here, we examined the spatial organization and interdependence of ESCRT-III subunits during mammalian cytokinetic abscission by depleting CHMP2A, a core ESCRT-III component. Using live cell imaging, structured illumination microscopy (SIM) and correlative light-electron microscopy (CLEM), we show that CHMP2A knockout cells display a significant delay—but not failure—in abscission, accompanied by distinct mislocalization phenotypes across ESCRT-III subunits. While IST1 and CHMP2B were minimally disrupted, CHMP4B, CHMP3, and CHMP1B display progressively severe organization defects at the abscission site. Dual- protein imaging reveals disrupted coordination between ESCRT-III subunits in individual CHMP2A-deficient cells, supporting an ordered assembly of ESCRT-III subunits in cytokinetic abscission. Together, our findings provide the first in vivo evidence for hierarchical assembly of ESCRT-III subunits during ESCRT-mediated membrane remodeling and identify CHMP2A as a key organizer of ESCRT-III architecture essential for timely membrane abscission.