Human ESCRT-III Polymers Assemble on Positively Curved Membranes and Induce Helical Membrane Tube Formation

Abstract

Endosomal sorting complexes required for transport-III (ESCRT-III) are thought to assemble in vivo inside membrane structures with a negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and conversely how ESCRT-III polymers shape membranes is still unclear. Here, we used human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron microscopy, cryo-electron tomography and highspeed AFM. We show that CHMP4B filaments bind preferentially to flat membranes or to membrane tubes with a positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes, the latter winding around the tubes. Although combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes, they also reshape large unilamellar vesicles into helical membrane tubes with a pipe surface shape. Sub-tomogram averaging reveals that the filaments assemble parallel to the tube axis with some local perpendicular connections, highlighting the particular mechanical stresses imposed by ESCRT-III to stabilize the corkscrew-like membrane architecture. Our results thus underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature of ESCRT-III required for all or selected cellular membrane remodeling processes.

Membrane Constriction and Thinning by Sequential ESCRT-III Polymerization

“…Recent in vitro studies of the yeast ESCRT-III proteins Vps2-Vps24-Snf7 50 and their human homologs 58 have also demonstrated positive curvature membrane remodeling activity -the same membrane orientation seen in our structures -despite their well-characterized negative membrane curvature deformation activity in vivo. While the significance of those in vitro observations remains to be fully elucidated, it appears that ESCRT-III assemblies are more versatile than previously realized.…”

Principles of membrane remodeling by dynamic ESCRT-III polymers

“…They can take a variety of shapes on membranes in vitro and in vivo, including rings, flat spirals, open and closed helices, as well as cones. Most intriguingly, different ESCRT-III subunits can copolymerize, and the shapes of single-subunit polymers can vary significantly from those of multisubunit polymers, even if they contain the same subunits [9,10,15,[20][21][22][23][24][25] (Figure 1C). Of note, the ESCRT-III cones are often observed in vivo in various functions [26][27][28], while in vitro, helices, rings, and spirals are more common.…”

“…Several recent studies reported the formation of double-stranded and three-stranded ESCRT-III filaments [9,10,16,20,24,31,34]. The most intuitive consequence following the transition from a single-stranded to a multistranded filament is an increase of the overall polymer rigidity.…”

Rules for the self-assembly of ESCRT-III on endosomes

“…6a, c): (1) The growing Snf7 protofilament allows the lateral co-assembly of a Vps24 - Vps2 heterofilament. The two filaments presumably correspond to parallel filamentous structures seen in vitro (Mierzwa et al, 2017, Pfitzner et al, 2020, Moser von Filseck et al, 2020, Bertin et al, 2020, Banjade et al, 2019). (2) Vps24 and Vps2 both use positively charged amino acid residues in their helices α1 to interact with negatively charged amino acids in helix α4 of Snf7 protomers in the protofilament.…”

“…In vitro Snf7, Vps24, Vps2 and other ESCRT-III proteins, when combined with each other, or on their own, assemble into polymers with a range of different shapes: short filaments, rings, long spirals, helical filaments, sheets, tubular and dome like structures. Some of these polymers consist of parallel ESCRT-III filaments with the capacity to deform membranes (Henne et al, 2012, Pfitzner et al, 2020, Moser von Filseck et al, 2020, Bertin et al, 2020, Maity et al, 2019, Schoneberg et al, 2018, Goliand et al, 2018, McCullough et al, 2015, Shen et al, 2014, Cashikar et al, 2014, Guizetti et al, 2011, Bajorek et al, 2009, Ghazi-Tabatabai et al, 2008, Lata et al, 2008b, Hanson et al, 2008, Banjade et al, 2019). These experiments unambiguously demonstrate that ESCRT-III subunits self-assemble into heteropolymers with different shapes and distinct properties that can remodel membranes.…”