Structure and dynamics of ESCRT-III membrane remodeling proteins by high-speed atomic force microscopy

Endosomal sorting complex required for transport (ESCRT) proteins assemble on the cytoplasmic leaflet of membranes and remodel them. ESCRT is involved in biological processes where membranes are bent away from the cytosol, constricted, and finally severed, such as in multivesicular body formation (in the endosomal pathway for protein sorting) or abscission during cell division. The ESCRT system is hijacked by enveloped vi-ruses to allow buds of nascent virions to be constricted, sev-ered, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cyto-solic in their autoinhibited conformation. They share a com-mon architecture, a four-helix bundle with a fifth helix that interacts with this bundle to prevent polymerizing. Upon binding to negatively charged membranes, the ESCRT-III components adopt an activated state that allows them to polymerize into filaments and spirals and to interact with the AAA-ATPase Vps4 for polymer remodeling. ESCRT-III has been studied with electron microscopy and fluorescence mi-croscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respec-tively, but neither approach provides detailed insights into both aspects simultaneously. High-speed atomic force microscopy (HS-AFM) has overcome this shortcoming, providing movies at high spatiotemporal resolution of biomolecular processes, significantly increasing our understanding of ESCRT-III structure and dynamics. Here, we review the contributions of HS-AFM in the analysis of ESCRT-III, focusing on recent de-velopments of nonplanar and deformable HS-AFM supports. We divide the HS-AFM observations into four sequential steps in the ESCRT-III lifecycle: (1) polymerization, (2) morphology, (3) dynamics, and (4) depolymerization.

Automated summary

Endosomal sorting complex required for transport (ESCRT) proteins assemble on membranes and remodel them. ESCRT-III proteins, the downstream components, can polymerize into filaments and spirals upon binding to negatively charged membranes. High-speed atomic force microscopy (HS-AFM) has provided detailed insights into the structure and dynamics of ESCRT-III. It has been used to study the four sequential steps in the ESCRT-III lifecycle: polymerization, morphology, dynamics, and depolymerization.