The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks

Abstract

Endosomal sorting complexes required for transport (ESCRT)-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It has been proposed that stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at the post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of giant unilamellar vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, highlighting a potential important function of CHMP2B in maintaining synaptic spine structures.

The ESCRT-III Isoforms CHMP2A And CHMP2B Display Different Effects On Membranes Upon Polymerization

“…As an example, the CHMP4B isoform is likely present in the ESCRT-III spirals formed at the mid-body during cytokinesis 64,65 whereas CHMP4C is implicated in abscission control 15 . The increased rigidity imposed by the CHMP2B network might be important for dendritic spine maintenance 66 where it might limit protein diffusion, in agreement with experiments showing that CHMP2B forms a diffusion barrier at membrane necks reconstituted in vitro 44 . It might also significantly contribute to the mechanical property of the ESCRT-III spirals at the cytokinetic bridge that become very loose when CHMP2B is depleted 65 .…”

“…Second, we confirm that CHMP2B displays a stronger binding for PI(4,5)P2 containing membranes as compared to other phosphoinositides and DOPS lipids 44 . In contrast CHMP2A and CHMP3 require only negatively charged membranes for binding with no preference for specific lipid head groups.…”

“…A previous in vitro study 44 has shown that the interaction of CHMP2B proteins with membrane is significantly enhanced in the presence of PI(4,5)P2 lipids in comparison with DOPS or PI(3,5)P2-membranes. Thus, we compared the preferential binding of CHMP2A versus CHMP2B on GUVS doped with 10% PI(4,5)P2 using confocal imaging.…”

“…2-A). It has been shown that the displacement or truncation of the C-terminal region of CHMP proteins results in the activation of the protein required for polymerization on membranes 19,21,22,44 . Thereupon, the constitutively active version of CHMP2A and CHMP2B, i.e.…”

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

“…In vivo, CHMP4 or CHMP2B over-expression leads to membrane tube formation with CHMP4 and CHMP2B filaments inside the tube, respectively [27][28][29] . Polymerization is guided by conformational changes that stabilize the filaments via domain exchange, thereby generating basic surfaces for interaction with positively curved 30 or negatively curved membranes 31 carrying a negative net charge 30,32,33 . Although Snf7 (CHMP4) polymerizes on supported lipid bilayers 22 , preformed membrane curvature was suggested to favor Snf7 (CHMP4) membrane interaction 34,35 .…”