Periodic Reporting for period 1 - ESCRT model (A biophysical model for ESCRT-III mediated membrane scission)
Período documentado: 2017-04-01 hasta 2019-03-31
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.
• 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.
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.