Recovery of ESCRT-III Filaments Subjected to Force: An ‘Invasive Mode’ HS-AFM Study

Abstract

Endosomal sorting complex required for transport III (ESCRT-III) proteins are crucial to membrane sculpting processes, including cytokinesis and biogenesis of multivesicular bodies. How ESCRT-III polymerization generates membrane curvature remains debated. Using High-Speed Atomic Force Microscopy (HS-AFM), a versatile technique with unprecedented spatial and temporal resolution, we acquired insights into how Snf7 assemblies, the major component of the ESCRT-III system, changed architecture in the presence of divalent cations and how they recover after being dissected by applying increased forces to well-defined delimited areas of the sample surface. The dissected assemblies present free ends of broken filaments onto which new monomers from the imaging solution can polymerize. After initial perturbation, the recovering assemblies show a tendency toward maximization of interfilament contacts, manifesting as nascent filaments or elongation of broken filaments along pre-existing filaments that act as scaffolds, as well as reparation of broken filaments. Based on these results, we hypothesize about a novel mechanism by which lateral interactions between ESCRT-III filaments drive constriction of the assemblies in order to induce membrane deformation.