Final Report Summary - CYTOCUT (Cytokinesis’ final cut: mechanics of abscission and ESCRT-III mediated membrane fission.) ESCRT-III is a protein complex recently discovered, processing an unusual but essential membrane remodeling event: membrane fission from the inside of a membrane neck. This complex is involved in several fission mechanisms (abscission, HIV budding, endocytic membrane trafficking), and is highly conserved among organisms.Despite a lot of indirect insights, no membrane fission event has been observed live, and the proposed mechanism for ESCRT-III remains elusive. This project aimed at studying ESCRT-III fission events with live imaging and quantitative methods.Using a combination of fluorescence microscopy, electronic microscopy (EM) and high speed atomic force microscopy (HS-AFM) (collaboration S. Scheuring lab, U1006 Inserm, Université de la Méditerranée), this project has led to very original and compelling results that shed light into the mechanism of ESCRT-III.1 - The full sequence of ESCRT-III nucleation – polymerization – depolymerization was reconstituted on supported membranes, showing the functionality and sequential actions of 8 proteins (Escrt-II: Vps25, Vps22, Vps36; Escrt-III: Vps20, Snf7, Vps2, Vps24; Vps4 + ATP), in a fully controlled in vitro assay. 2 – We focused on Snf7, the major component of this complex. We showed the formation of micron sized spirals on supported membranes that were studied in details by fluorescence, EM and HS-AFM. The precise dynamics of Snf7 spiral formation was studied and a theoretical model (collaboration M. Lenz, Soft biophysics group, Université Paris-Sud) was built that fully agreed to the observed structure and dynamics, and membrane deformation. A publication is in preparation regarding Snf7 mechanics.3 – A clear comprehension of Snf7 has emerged, which is not sufficient to understand ESCRT-III fission mechanism. Qualitative data have been acquired on downstream (Vps24, Vps2, Vps4) and upstream (ESCRT-II) proteins. A second publication is also in preparation regarding all ESCRT-III proteins co-assembly.Understanding the mechanism of proteins transiently acting on membranes is challenging. We manage to build membrane assays that are simple and which provide useful quantitative data. Thus this work is fruitful and straightforward prolongations are under process: 1 - acquiring better structural data with cryo-EM 2 – repeating the quantitative analysis done with Snf7 with other proteins 3 – continuing the induced membrane deformation study.+ ATTACH FIGURES (pdf in attachment) Related documents final1-14-08-20-figures-report.pdf
