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 viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common 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 microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, 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 developments 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. 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 viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common 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 microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, 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 developments 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. Research into the endosomal sorting complex required for transport (ESCRT) family of proteins is tightly linked to two questions that arose regarding the mechanism by which proteins are sorted in the endosomal and biosynthetic pathways, namely: (i) what is the signal that determines the fate of cargo proteins which enter these pathways and (ii) what constitutes the machinery that mediates this sorting? Initial genetic and biochemical investigations showed that mutations in a set of budding yeast genes led to aberrations in the endosomal protein sorting pathway (1Rothman J.H. Stevens T.H. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway.Cell. 1986; 47: 1041-1051Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 2Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.Mol. 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The identification of ESCRT proteins as an evolutionarily conserved and physiologically important membrane-remodeling machinery gave rise to numerous proposals on the molecular mechanism that governed ESCRT assembly, ESCRT-mediated membrane remodeling, Vps4-mediated ESCRT disassembly, and ESCRT-mediated membrane scission. Initial models suggested that homopolymeric assemblies of ESCRT-III components acted as loaded spiral springs that release accumulated energy in an out-of-plane buckling deformation of the membrane-bound protein assembly (Fig. 1C) (37Lenz M. Crow D.J. Joanny J.F. Membrane buckling induced by curved filaments.Phys. Rev. Lett. 2009; 103038101Crossref Scopus (44) Google Scholar). 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As the bending energy required to shape a hemispherical membrane dome from a flat membrane is on the order of ∼160 kBT, the energy accumulated in the filament would be sufficient to bend the membrane (42Helfrich W. Elastic properties of lipid bilayers: theory and possible experiments.Z. Naturforsch. C. 1973; 28: 693-703Crossref PubMed Scopus (5097) Google Scholar). However, it remained unclear whether the loaded spiral springs model of membrane deformation was applicable in a physiological context and how it could account for the variety of membrane geometries (both in terms of curvature as well as length scales) that ESCRT-III must adapt to. Thus, the limitations of the monocomponent-loaded spiral spring model led to the elaboration of an expanded model, in which a sequential, Vps4-mediated, ATP-fueled polymerization–depolymerization sequence of different ESCRT-III components leads to gradual changes in the composition and mechanical properties of ESCRT-III assemblies, concomitant with gradual changes in the geometry of the underlying membrane (Fig. 1D) (33Mierzwa B.E. Chiaruttini N. Redondo-Morata L. Moser von Filseck J. König J. Larios J. et al.Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis.Nat. Cell Biol. 2017; 19: 787-798Crossref PubMed Scopus (151) Google Scholar, 43Chiaruttini N. Roux A. Dynamic and elastic shape transitions in curved ESCRT-III filaments.Curr. Opin. Cell Biol. 2017; 47: 126-135Crossref PubMed Scopus (34) Google Scholar, 44Pfitzner A.K. Moser von Filseck J. Roux A. 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We extend our discussion regarding the unique possibilities offered by HS-AFM to characterize structure and dynamics of biomolecular assemblies. The biggest hurdle in understanding the molecular mechanism of the ESCRT system is the study of both its structure and its dynamics, parameters that are in this case inextricable from each other, because the ESCRT system has no fixed structure but is in a continuous turnover and rearrangement throughout its functional cycle. The simplest approach, that also provides the most protein-specific results, is the bottom-up reconstitution of the system with a limited number of purified components (46Liu A.P. Fletcher D.A. Biology under construction: in vitro reconstitution of cellular function.Nat. Rev. Mol. Cell Biol. 2009; 10: 644-650Crossref PubMed Scopus (169) Google Scholar, 47Ganzinger K.A. Schwille P. More from less - bottom-up reconstitution of cell biology.J. Cell Sci. 2019; 132jcs227488Crossref PubMed Scopus (40) Google Scholar). Such isolated versions of the system allow to verify whether purified components behave as predicted and how they relate to ESCRT-III structural assemblies in cells. EM provided information about the structure of ESCRT components in isolation or in combination with other components (48Henne William M. Buchkovich Nicholas J. Zhao Y. Emr Scott D. The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices.Cell. 2012; 151: 356-371Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 49McCullough J. Clippinger A.K. Talledge N. Skowyra M.L. Saunders M.G. Naismith T.V. et al.Structure and membrane remodeling activity of ESCRT-III helical polymers.Science. 2015; 350: 1548-1551Crossref PubMed Scopus (172) Google Scholar). This technique identified the formation of various supramolecular structures, planar as well as three-dimensional. Yet, electron micrographs represent snapshots of the system frozen in time and do not provide information about the path the system took to arrive at a given state nor how it will develop from that state. Crucially, questions regarding the transition from a single-component planar configuration to a nonplanar structure by addition of other components remain unanswered—in other words, using EM, the dynamics of protein assemblies can only be inferred. Shortly after the first description of the ESCRT system (5Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting.Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar, 6Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 7Katzmann D.J. Babst M. Emr S.D. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar), the ability of several ESCRT-III components to homopolymerize or heteropolymerize was first assessed by EM, resulting in observations of diverse higher-order structures. Recombinant Saccharomyces cerevisiae Vps24 was found to polymerize into helical structures, while Snf7 formed sheet-, ring-, and string-like structures (27Ghazi-Tabatabai S. Saksena S. Short J.M. Pobbati A.V. Veprintsev D.B. Crowther R.A. et al.Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24.Structure. 2008; 16: 1345-1356Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, similar EM studies resulted in contrasting outcomes, requiring either point mutations or large-scale deletions of the autoinhibitory C-terminal region in ESCRT-III subunits to facilitate polymerization (48Henne William M. Buchkovich Nicholas J. Zhao Y. Emr Scott D. The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices.Cell. 2012; 151: 356-371Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 50Lata S. Roessle M. Solomons J. Jamin M. Gőttlinger H.G. Svergun D.I. et al.Structural basis for auto-inhibition of ESCRT-III CHMP3.J. Mol. Biol. 2008; 378: 818-827Crossref PubMed Scopus (102) Google Scholar). On the other hand, fluorescence microscopy (FM) allows to observe how reconstituted biological systems change over time (28Wollert T. Wunder C. Lippincott-Schwartz J. Hurley J.H. Membrane scission by the ESCRT-III complex.Nature. 2009; 458: 172-177Crossref PubMed Scopus (489) Google Scholar, 33Mierzwa B.E. 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FM takes advantage of fluorescently labeled membrane and protein components to observe the dynamics of different ESCRT proteins polymerizing or copolymerizing on the membrane. In this configuration, the action of the protein is observed using a lipid vesicle or a planar lipid bilayer as substrate on which the ESCRT proteins can assemble and interact. The information derived from this approach depends on the choice of the geometry of the lipid substrate: using intact liposomes in combination with purified ESCRT components, confocal FM provided information on how the protein and membrane colocalize over time and how the shape of the membrane changed with addition of proteins to the system (28Wollert T. Wunder C. Lippincott-Schwartz J. Hurley J.H. Membrane scission by the ESCRT-III complex.Nature. 2009; 458: 172-177Crossref PubMed Scopus (489) Google Scholar, 51Wollert T. Hurley J.H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.Nature. 2010; 464: 864-869Crossref PubMed Scopus (568) Google Scholar). Using planar supported lipid bilayers (SLBs), no information about changes in the membrane geometry could be obtained, but observation of the colocalization of individual ESCRT components could be used to derive information about the interaction of ESCRT proteins (33Mierzwa B.E. Chiaruttini N. Redondo-Morata L. Moser von Filseck J. König J. Larios J. et al.Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis.Nat. Cell Biol. 2017; 19: 787-798Crossref PubMed Scopus (151) Google Scholar, 45Pfitzner A.K. Mercier V. Jiang X. Moser von Filseck J. Baum B. Saric A. et al.An ESCRT-III polymerization sequence drives membrane deformation and fission.Cell. 2020; 182: 1140-1155.e18Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The benefit of obtaining such dynamic information comes at a cost—the loss of information about the structure of ESCRT-III assemblies on lipid bilayers. Rigorously, no statement regarding the structure and assembly of the ESCRT proteins can be made based on the measurement of fluorescence signals emerging from attached fluorescence tags. Also, the typical spatial resolution of these FM experiments is in the hundreds of nanometer range, larger than the size of an individual ESCRT-III spiral and orders of magnitude larger than the thickness of a filament. Thus, neither EM nor FM allows to fully appreciate the ESCRT-III system's interaction between its constituents and with lipid bilayers. Covering this blind spot to understand the structure-dynamics duality of ESCRT assemblies, HS-AFM provides movies with nanometer lateral and subsecond temporal resolution. AFM makes use of a nanometer sharp tip attached to a flexible microcantilever to scan the surface of a sample (Fig. 2A). 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