Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis

Abstract

The Endosomal Sorting Complex Required for Transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in presence of Vps4. Continuous ESCRT-III remodeling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…The cytokinetic abscission reaction differs considerably from ILV formation in both scale and timing because the ESCRT machinery accumulates and acts over a much longer time window (~80 min) and the ESCRT-III-lined bud neck within the midbody is nearly 1 μm in length and 1.25 μm in diameter (Elia et al 2011, 2012). Moreover, the ESCRT-III filaments within the midbody appear to be much wider (~17 nm) than those formed during vesiculation reactions (~5 nm) and may therefore comprise bundles of ESCRT-III filaments and potentially other proteins (Guizetti et al 2011, Mierzwa et al 2017). …”

“…ALIX (Bro1) binding may thereby weaken autoinhibitory interactions, consistent with the observation that CHMP4 autoinhibition can be relieved by removing the terminal helix (Lata et al 2008b, 2009; Tang et al 2015, 2016). ALIX can dimerize (Pires et al 2009), and both ESCRT-II and ALIX may, therefore, help activate ESCRT-III subunits to form two filaments or even double-stranded filaments (Henne et al 2012, McCullough et al 2015, Mierzwa et al 2017, Teis et al 2010). This activity is reminiscent of the activities of actin and microtubule nucleators, which bind and orient two actin monomers (Rottner et al 2017) or heterodimeric tubulin subunits (Kollman et al 2011).…”

“…The highly interlocked and domain-swapped nature of the CHMP1B strand of the IST1 NTD /CHMP1B filament—in which each monomer interacts with eight neighbors by virtue of a CHMP1B i to CHMP1B i +4 domain swap—suggests that this strand will be unusually robust to bending (Chiaruttini & Roux 2017, McCullough et al 2015, Mierzwa et al 2017). By contrast, actin also polymerizes through noncovalent interactions, but the F-actin filament contains interactions between globular nearest neighbors only.…”

“…Recent studies have demonstrated that ESCRT-III subunits exchange rapidly at sites of filament action, although the role of Vps4 in mediating this exchange is debated (Adell et al 2017, Mierzwa et al 2017). Vps4 activity is required for virus budding (Baumgartel et al 2011), abscission (Mierzwa et al 2017), and MVB vesicle formation (Adell et al 2017), and the enzyme appears to perform an essential role in promoting the fission reaction (rather than simply recycling ESCRT-III subunits after fission).…”

“…Vps4 activity is required for virus budding (Baumgartel et al 2011), abscission (Mierzwa et al 2017), and MVB vesicle formation (Adell et al 2017), and the enzyme appears to perform an essential role in promoting the fission reaction (rather than simply recycling ESCRT-III subunits after fission). In vitro, Vps4-mediated subunit exchange facilitates subunit exchange into highly dynamic arrays of growing, shrinking, and spiraling ESCRT-III filaments (Mierzwa et al 2017). However, ESCRT-III subunit exchange can continue at sites of yeast MVB formation even in the absence of an active Vps4 enzyme (Adell et al 2017).…”