Cleaving membrane necks by ESCRT-III – Neck4Fission

The "endosomal sorting complex required for transport" (ESCRT) catalyzes a wide range of physiological and pathological membrane remodeling processes including budding of vesicles and enveloped viruses, cell division and many others involving topologically similar inside-out budding processes. The eukaryotic ESCRT machinery is composed of 5 complexes, ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III and the ATPase VPS4. ESCRT-III and VPS4 constitute the machinery that executes membrane fission and both are recruited to all ESCRT-catalyzed remodeling processes. Evidence suggests that ESCRT-III members CHMP4, CHMP3 and CHMP2 play an essential role in membrane constriction and fission and could represent with VPS4 a minimal fission machinery. Because ESCRTs operate inside membrane necks, the question how ESCRT filaments organize to achieve membrane constriction and cleavage in conjunction with VPS4 remains a major challenge. The objective of our proposal is to understand the structural basis and the mechanisms of ESCRT-III and VPS4-catalyzed membrane remodeling processes leading to membrane fission.
ESCRTs operate in systems such as the cytokinetic midbody that requires constriction from a large diameter (µm) down to complete constriction and membrane fission while vesicle or enveloped virus budding requires membrane neck constriction starting from smaller diameters of about 50 nm. Here we will focus on the reconstitution of constriction and membrane fission from smaller diameters resembling those of vesicles or enveloped viruses. We hypothesize that functional ESCRT-III recruitment/polymerization on membranes requires optimal membrane geometries such as a full catenoid (dumb-bell) or a truncated one, like in a budding tube.
The aim of NECK4FISSION is to set up model membranes shapes that recapitulate native membrane neck structures to allow reconstitution of membrane fission with a minimal set of ESCRTs and VPS4. We will develop four different in vitro systems to reconstitute ESCRT-III/VPS4 function. Two are based on membrane tubes preformed around ESCRT-III polymers or pulled from GUVs and two systems are based on the reconstitution of artificial virus-like budding systems employing GUVs and LUVs. We will employ high-resolution imaging techniques and micromanipulation to establish conditions for fission and we will devise state of the art cryo-EM imaging to visualize the effect of ESCRT-IIIs and VPS4 on membrane tubes and on the neck of membrane buds wrapped around virus-like particles inside LUVs and GUVs. These in vitro assays will be complemented by Cryo-TM imaging of HIV-1 budding site employing FIB-SEM and cryo-ET. Finally, all data will be considered to develop a novel membrane fission physical model based on elastic coupling of the ESCRT machinery to the local membrane shape and on membrane viscous stresses created by ATP-dependent remodeling of ESCRT-III, which has not been considered yet. Thus, we will unveil how ESCRT-III and VPS4 contribute to membrane deformation and mechanical stress under different geometrical constraints.
Together our data will provide novel important insight into the function, structure and dynamics of ESCRT-III/VPS4-catalyzed membrane fission and on the underlying mechanisms.