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Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins 1 – 3 . The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles 4 , 5 . Here we report that the plant ESCRT component FREE1 6 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization. Plant ESCRT component FREE1 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation.

Key Points Endosomal sorting complex required for transport (ESCRT) proteins carry out scission of membrane necks with a topology (or sidedness) opposite to that of the better-understood process carried out by coat proteins, dynamin and BAR (Bin, amphiphysin and Rvs) domain proteins. ESCRT-mediated reverse-topology membrane scission is initiated by two upstream branches: the first comprising ESCRT-I and ESCRT-II, and the second comprising ALIX. The ESCRT-III protein family has 12 different subunits in humans. ESCRT-III monomers are about 200 amino acids in length and have open and closed conformations. ESCRT-III proteins can assemble into flat spirals, helical tubes or conical funnels. ESCRT-III assemblies are taken apart by the AAA+ ATPase vacuolar protein sorting-associated 4 (VPS4), which unfolds ESCRT-III monomers and threads them through a central pore of the VPS4 hexamer. It is currently unresolved whether scission is mediated by the drawing-together of membrane necks by a tapered dome, buckling by the mechanical spring-like action of curved ESCRT filaments or some other means. New observations of ESCRT-mediated reverse-topology membrane scission are building towards a structural and biophysical explanation of the mechanism involved. The narrow membrane necks formed during viral, exosomal and intra-endosomal budding from membranes, as well as during cytokinesis and related processes, have interiors that are contiguous with the cytosol. Severing these necks involves action from the opposite face of the membrane as occurs during the well-characterized formation of coated vesicles. This 'reverse' (or 'inverse')-topology membrane scission is carried out by the endosomal sorting complex required for transport (ESCRT) proteins, which form filaments, flat spirals, tubes and conical funnels that are thought to direct membrane remodelling and scission. Their assembly, and their disassembly by the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively studied, but the mechanism of scission has been elusive. New insights from cryo-electron microscopy and various types of spectroscopy may finally be close to rectifying this situation.

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.Cryo-EM structures of human ESCRT-III proteins forming membrane-bound and membrane-free filaments show how CHMP1B and IST1 polymerize sequentially, driving membrane tubulation, constriction and bilayer thinning, leading to membrane fission.

The ESCRT protein complexes are essential for cell division, the release of HIV from infected cells via budding, and other cell processes involving the scission of narrow membrane necks from their inner surface. The unusual inside-directed membrane cutting has made it hard to recapitulate this reaction and understand its mechanism. Schöneberg et al. encapsulated ESCRTs inside lipid vesicles and used optical tweezers to pull out membrane nanotubes. In the presence of adenosine triphosphate, clusters of ESCRTs generated force and constricted the nanotube, eventually severing it. This approach provides a window into the molecular mechanisms involved in the activities of ESCRTs. Science , this issue p. 1423 Reconstituted ESCRT-III and Vps4 can harness ATP-dependent force production for membrane scission. The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 and Vps4 puncta within nanotubes whose presence followed ATP release, correlated with force generation and nanotube constriction, and preceded scission. These observations directly verify long-standing predictions that ATP-hydrolyzing assemblies of ESCRT-III and Vps4 sever membranes.

The biogenesis and maintenance of thylakoid membranes require vesicle-inducing protein in plastids 1 (VIPP1). VIPP1 is a member of the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, whose members form diverse filament-based supramolecular structures that facilitate membrane deformation and fission. VIPP1 cryo-electron microscopy (EM) structures in solution revealed helical rods and baskets of stacked rings, with amphipathic membrane-binding domains in the lumen. However, how VIPP1 interacts with membranes remains largely unknown. Here, using high-speed atomic force microscopy (HS-AFM), we show that VIPP1 assembles into right-handed chiral spirals and regular polygons on supported lipid bilayers via ESCRT-III-like filament assembly and dynamics. VIPP1 filaments grow clockwise into spirals through polymerization at a ring-shaped central polymerization hub, and into polygons through clockwise polymerization at the sector peripheries. Interestingly, VIPP1 initially forms Archimedean spirals, which upon maturation transform into logarithmic spirals through lateral annealing of strands to the outermore low-curvature spiral turns. Using high-speed atomic force microscopy, the authors reveal the polymerization mechanism of VIPP1 into ESCRT-III-like filaments that adopt spiral and polygonal supramolecular structures.

The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems. Using AFM and cryo-EM, the authors investigate how ESCRT-III-like protein Vipp1 transitions between polymorphic forms of planar sheets and helical filaments, providing insights into membrane-repair processes.

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the endosomal sorting complex required for transport (ESCRT) complexes 0, I, II and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inwards by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, -I, -II and -III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. Here we show that ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and ESCRT-II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and ESCRT-II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction. The path to degradation Internalized proteins destined for degradation are delivered to lysosomes via multivesicular bodies (MVBs). In this pathway, cargo proteins are ubiquitinated and sorted by the ESCRT (endosomal sorting complex required for transport) complexes 0, I, II, and III. Thomas Wollert and James Hurley have reconstituted MVB biogenesis using giant unilamellar vesicles and all the ESCRT complexes. They find that ESCRT-0 is required for clustering of cargo proteins, while ESCRT-I and II in combination deform the membrane into buds, in which cargo is confined. ESCRT-III subunits localize to the bud neck and are required for scission of the membrane to form intralumenal vesicles. These results explain how ESCRT complexes sequester cargo proteins into MVBs. Here, multivesicular body (MVB) biogenesis is reconstituted using giant unilamellar vesicles and all of the ESCRT complexes. ESCRT-0 is required for clustering of cargo proteins, whereas ESCRT-I and -II in combination deform the membrane into buds, in which cargo is confined. ESCRT-III subunits localize to the bud neck and are required for scission of the membrane to form intraluminal vesicles. These results explain how ESCRT complexes sequester cargo proteins into MVBs.

Key Points Endosomal sorting complexes required for transport (ESCRTs) are required for the lysosomal degradation of plasma membrane proteins, budding of most enveloped viruses, cytokinesis and autophagy. ESCRT-I and ESCRT-II work together to bud membranes away from the cytosol by stabilizing the neck of the bud. ESCRT-III forms helical assemblies that sever membrane necks from within. The AAA+ ATPase vacuolar protein sorting 4 (Vps4) forms a dodecameric assembly together with Vta1 that solubilizes and recycles membrane-bound ESCRT-III following membrane scission. The endosomal sorting complex required for transport (ESCRT) machinery catalyses membrane budding in the endolysosomal pathway, which differs from other budding events in that it is directed away from the cytosol. Recent studies have elucidated a mechanism whereby ESCRT-I and ESCRT-II stabilize the bud neck and ESCRT-III mediates neck cleavage. The endosomal sorting complexes required for transport (ESCRTs) catalyse one of the most unusual membrane remodelling events in cell biology. ESCRT-I and ESCRT-II direct membrane budding away from the cytosol by stabilizing bud necks without coating the buds and without being consumed in the buds. ESCRT-III cleaves the bud necks from their cytosolic faces. ESCRT-III-mediated membrane neck cleavage is crucial for many processes, including the biogenesis of multivesicular bodies, viral budding, cytokinesis and, probably, autophagy. Recent studies of ultrastructures induced by ESCRT-III overexpression in cells and the in vitro reconstitution of the budding and scission reactions have led to breakthroughs in understanding these remarkable membrane reactions.

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises \"open\" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of \"closed\" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

The endosomal sorting complex required for transport (ESCRT) is a multicomplex machinery comprising proteins that are conserved from bacteria to humans and has diverse roles in regulating the dynamics of cellular membranes. ESCRT functions have far-reaching consequences for cell biological processes such as intracellular traffic, membrane repair, cell signalling, metabolic regulation, cell division and genome maintenance. Here we review recent insights that emphasize the pathophysiological consequences of ESCRT dysfunctions, including infections, immune disorders, cancers and neurological diseases. We highlight the possibilities of using our knowledge about ESCRT structures and functions for drug discovery. This Review examines recently gained insights into the roles of ESCRT complexes in viral infection, immunity, cancer and neurological disease.