Periodic Reporting for period 1 - ESCRT model (A biophysical model for ESCRT-III mediated membrane scission) Periodo di rendicontazione: 2017-04-01 al 2019-03-31 Sintesi del contesto e degli obiettivi generali del progetto The ESCRT-III complex is an evolutionary conserved protein machinery that mediates membrane remodeling and scission in many important physiological and pathological cellular contexts, such as cell division, HIV virus release, multivesicular bodies formation, nuclear membrane repair, dendritic spines regulation, and others. A subset of ESCRT-III proteins, namely CHMP4B, CHMP3 and CHMP2A/B, appear to be strictly required in all these processes, indicating that they might constitute the minimal scission machinery. ESCRT mis-function has been linked with the pathogenesis of several diseases, including the neurodegenerative disease Fronto-Temporal Dementia (FTD). All these cellular processes involve membrane remodeling/scission activities that are topologically equivalent, and are characterized by the so-called “inverse topology”, in which the ESCRT-III complex assemble and function inside a membrane tube or neck. Thus, the complex is expected to display a membrane curvature preference, in particular it has been proposed to assemble preferentially on negatively curved membranes. This, however, is not supported by convincing experimental evidence, and despite several model being proposed over the past decade, the molecular mechanism of ESCRT-III function is still obscure. This lack of experimental data is mainly due to the technical challenge of reconstituting the ESCRT-III complex on a negatively curved membrane. This question is very relevant, since ESCRT-III might represent potential therapeutic targets for treatment of a number of diseases. The original aim of this proposal was to develop a novel in vitro approach to reconstitute and characterize the assembly and mechanism of function of the ESCRT-III complex on a negatively curved membrane, thus reproducing the correct membrane topology present in vivo. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti • I identified the endogenous signaling lipid PI(4,5)P2 as a specific membrane component able to recruit CHMP2B to the membrane and nucleate its polymerization. Moreover, I showed that PI(4,5)P2 is able to recruit also the other components of the ESCRT-III complex. • I performed micropipette aspiration experiments on different ESCRT-III subunits, with a particular focus on CHMP2B, in order to investigate their effect on the mechanical properties of the membrane.• I set up a novel experimental system in order to reconstitute the ESCRT-III complex on a relevant membrane topology (i.e. inside a membrane nanotube and membrane neck). This technique allow to study dynamic recruitment and curvature sorting of each ESCRT-III components, separately or in combination, on complex membrane geometries which were previously unaccessible.• I reconstituted CHMPB, CHMP2A, CHMP2B and CHMP3 inside GUVs by this fusion technique, alone and in combination, and carried out a thorough investigation of their membrane curvature preference by comparing their sorting between flat, negatively curved and catenoid-like shaped membrane geometries.• I also reconstituted all these ESCRT-III protein outside GUVs, in a well-established tube pulling assay, and carried out a thorough investigation of their membrane curvature preference by comparing their sorting between flat and positively curved membrane geometries.• I performed FRAP experiments on CHMP2B reconstituted at membrane neck, showing that it can act as a diffusion barrier for membrane-associated components.• With our collaborators, we performed Cryo-EM experiments revealing the molecular arrangement of the ESCRT-III complex on both flat and positive membrane geometries.• During my secondment, I performed membrane reconstitution experiments with the aim of reconstructing the assembly of the ESCRT-III complex on the correct membrane geometry (inverted topology) and image it at high resolution by negative staining and Cryo-EM. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) Thanks to the novel methodology that I set up, I obtained very surprising and provocative results, which collectively lead us to question the current accepted model of ESCRT-III function. In particular, and contrary to the expectations, none of the ESCRT-III subunits have a preference for negative curvature; some preferentially assemble on a flat membrane geometry, while others even prefer a positive membrane curvature. This led us to hypothesize that the ESCRT-III complex initially assembles in a metastable, high-energy state, which could be release by the action of Vps4, an ATPase that work in concert with ESCT-III. Moreover, I opened a new line of investigation on the role of ESCRT proteins in modulation of mechanical properties of the membrane. I also unveiled a novel putative function for CHMP2B as diffusion barrier on membrane necks. While this finding requires further investigation, it might contribute to explain the molecular basis for the neurodegenerative disease Fronto-Temporal Dementia.During my secondment, I managed to reconstitute an ESCRT-III-membrane complex in the correct “inverted” topology and image it at high resolution using negative staining EM and Cryo-EM. This represent a long-sought achievement in the ESCRT field. Moreover, I unveiled a complex interplay of positive and negative feedback loops between CHMP2A, CHMP3 and lipids, which regulate ESCRT-III assembly.Collectively, my results shed a new light on the mechanism of function of ESCRT-III, challenging current models from a fresh prospective. In addition, the novel technique that I developed could find application in other fields to study interaction with membrane of complex geometries for proteins other than ESCRTs. Reconstitution of CHMP2B by fusion and its function as diffusion barrier
