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ERC

MEM_FIZZ Report Summary

Project ID: 261104
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Final Report Summary - MEM_FIZZ (Mechanics of ESCRT-III mediated membrane scission)

Cellular processes such as cytokinesis, the budding of enveloped retrovirus (e.g. HIV-1), and multivesicular biogenesis have direct links to several human diseases including carcinogenesis and neuro-degeration etc. While seemingly unrelated, these processes all involve membrane abscission for generating two newly formed membrane bound structures - a process aided by the cytosolic proteins collectively termed ESCRT-III and VPS4. Understanding these processes for therapeutic intervention has so far focused on identification of the factors involved, their structures, and the interactions between them. Due to fast and highly localised transformations, protein mediated membrane remodelling has proven difficult for quantitative mechanistic scrutiny. Understanding the molecular mechanism of such processes requires bottom-up reconstitution of the process in vitro and the ability to monitor the process. We thus proposed (1) production and labelling of functional human ESCRT-III and VPS4 complexes (2) development of appropriate model membranes, (3) detection systems and methodologies for monitoring protein polymerisation/depolymerisation, membrane protein interaction and membrane remodelling.
We successfully achieved rapid and efficient production of different ESCRT-III proteins in bacterial expression systems and succeeded in specifically and quantitavely labelling them in a cysteine and/or histidine tag specific manner. Using purified and labelled ESCRT-III and VPS4 proteins we addressed the molecular mechanism through which human VPS4 depolymerises ESCRT-III polymers in an ATP dependent manner. We find, VPS4A as well as VPS4B from ATP induced dynamic homo and hetero hexamers which, as opposed to monomers, are the dominant ATP hydrolysing species and that hexamer disassembly is an integral part of the ATPase cycle. The two orthologs have different specificities toward different ESCRT-III polymers, notable CHMP2A-3 copolymer exclusively recruits VPS4B. Furthermore, hexameric VPS4B discriminates between soluble monomeric CHMP2A and CHMP3 from membrane bound polymeric CHMP2A-3 copolymer through multivalent interaction. We further find that while individual sub-unit within the VPS4 hexamer independently and stochastically hydrolyse ATP, ESCRT-III depolymerisation requires coordinated ATP hydrolysis between the sub-units within the hexamer.
In order to address ESCRT-III-membrane interaction we developed a number of novel model membranes such as polymer supported bilayer, surface adsorbed vesicle-clusters and membrane nanotubes for addressing ESCRT-III binding and scission. In order to monitor binding kinetics we successfully replicated RIF-TIRF - a measurement platform for simultaneous label-free and fluorescence detection in real time. We further developed a bespoke microscope for carrying out electro-optical measurement of horizontally-extruded lipid bilayer nanotubes where ESCRT-III polymerisation and membrane scission can be monitored in a time resolved manner with single molecule precision. Using these novel model membranes and detection platforms measurement of ESCRT-III binding kinetics and scission is work in progress.

Reported by

UNIVERSITY OF BRISTOL
United Kingdom
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