Endosomal sorting complexes required for transport (ESCRT) are involved in many cellular membrane remodelling processes including membrane deformation and fission. Examples of such processes are the biogenesis of endosomal vesicles, virus budding, cytokinesis and nuclear envelope closure. These machineries are also vulnerable to cancerous growth and neurodegenerative diseases. In membrane fission, first a budded neck is formed assisted by ESCRT III filaments that stabilize the highly curved membrane neck. In the final stage, these filaments accumulate to set the stage for membrane fission, in cooperation with vacuolar protein sorting-associated protein 4 (VPS4). The small size and the dynamic nature of these machineries present challenges for scientists to gain a comprehensive understanding of these processes. Therefor, we sought to understand the role of such ESCRT III machineries at the single-molecule level. Specifically, we scrutinize the first and the last steps of membrane budding and fission using High Speed -Atomic Force Microscope (HS-AFM). The unique ability of HS-AFM to study bio-molecules in near-to physiological conditions and with high spatio-temporal resolution makes it a successful tool to study the dynamics of single molecules at millisecond resolution. Our results reveal, for the first time at nm resolution, the dynamics of CHMP2A/CHMP3 constriction by Vps4, as a stepping stone to membrane scission. Furthermore, we scrutinize the role of CHMP2B and CHMP4B in membrane remodelling and discuss possible pathways for the initiation of vesicle budding.
2559-PosDriving Forces Stabilizing Cellular Prion Protein (Prp C ) Monomers and Dimers on the Cell Surface The conversion of the prion protein PrP C to its infectious form PrP Sc via autocatalytic misfolding is critical to the development of a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). To gain insight on the factors that influence the PrP C to PrP Sc conversion, it is pertinent to identify how PrP C interacts with its environment. PrP C is attached to the extracellular side of the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and can be found in monomer or homodimer form. To investigate the driving forces that stabilize PrP C monomers and dimers on the cell membrane surface, we used molecular modeling techniques. From our analysis, we identified stable dimer conformations and characterized the dimer interface using residue interaction network analysis and residue contact maps. We then implemented a series of molecular dynamics simulations to mimic the effects of changing membrane lipid composition on the protein-lipid interface. Our results indicate that PrP C dimers are stabilized by hydrophobic interactions along the b-sheets and that dimer stability is affected by the orientation of PrP C a-helices on the cell membrane surface. We will propose a mechanism by which PrP C dimers with hydrophobic interfaces inhibit PrP Sc propagation due to the mobility constraints that GPI anchors plac...
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