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Table of contents
Chapter 1. Introduction
Chapter 2. Physics of Biomembranes
2.1 Structure of lipids
2.1.1 Phospholipids
2.1.2 Sterols
2.1.3 Phosphoinositides
2.2 Membrane mechanics
2.3 Membrane tension
2.4 Membrane fission
Chapter 3. The ESCRT-dependent membrane remodelling processes
3.1 ESCRT machinery in Saccharomyces Cerevisiae
3.2 ESCRT machinery in Homo sapiens
3.2.2 ESCRT role in HIV-1 budding
3.2.3 ESCRT role in neuronal pruning
3.3 ESCRT-III crystal structure and cycling
3.4 ESCRT-III polymer structures in vivo and in vitro
3.4.1 ESCRT-III polymers form flat spirals
3.4.2 ESCRT-III polymers form helices and tubes
3.5 Theoretical models for membrane scission by the ESCRT-III polymers
3.5.1 Spiral spring (buckling) Model for ESCRT-III mediated membrane scission
3.5.2 Theoretical Dome Model for ESCRT-III mediated membrane scission
3.6 Objective: characterization of CHMP2B and determination of its role within the ESCRT-III machinery
3.6.1 CHMP2B is specific to higher organisms
3.6.2 CHMP2B is implied in the diversification of ESCRT functions
3.6.3 CHMP2B mutation leads to a neurological disorder: Fronto-Temporal Dementia
3.6.4 Thesis objective: study of CHMP2B using model membranes in vitro
Chapter 4. Material and methods
4.1 Protein purification
4.2 Model membrane systems
4.2.1 Reagents
4.2.2 Lipid mixtures
4.2.3 GUVs preparation
4.2.4 Making LUVs and SUVs
4.2.5 Making SLBs
4.3 Fluorescence microscopy
4.3.2 Spinning disk confocal microscopy
4.3.3 Fluorescence recovery after photobleaching assay (FRAP)
4.3.4 Fluorescence-activated cell sorting (FACS)
4.4 Cryo-electron microscopy
4.4.1 Cryo-EM principle
4.4.2 Experimental conditions
4.5 Micropipette aspiration assay
4.5.1 Micropipette aspiration principle
4.5.2 Experimental conditions
4.6 Quartz Crystal Microbalance with Dissipation monitoring
4.6.1 QCM-D principle
4.6.2 Typical experiment
4.7 Atomic Force Microscopy (AFM)
4.7.1 Principle of AFM
4.7.2 Experimental conditions
Chapter 5. Results
5.1 Optimization and characterization of CHMP2B protein interaction with model membranes
5.1.1 Study of CHMP2B protein stability
5.1.2 CHMP2B proteins bind preferentially to PI(4,5)P2-containing membranes
5.1.3 Encapsulation of CHMP2B proteins inside GUVs to mimic ESCRTs inverted topology .
5.1.4 CHMP2B proteins interaction with PI(4,5)P2 lipids is irreversible
5.1.5 CHMP2B proteins form a reticular-like structure on GUVs
5.1.6 CHMP2B assembles into ring-like structures at the nanoscale
5.2 CHMP2B polymers modulate membrane elastic properties
5.2.1 Investigation of CHMP2B mechanical properties by applying osmotic shocks
5.2.2 Study of CHMP2B mechanical properties by micropipette aspiration
5.2.3 Study of CHMP2B mechanical properties by AFM
5.2.4 Mobility of CHMP2B supramolecular assembly on GUVs
5.2.5 Diffusion of membrane-associated protein on GUVs covered by CHMP2B assemblies
5.3 CHMP2A and CHMP2B display opposite properties on model membranes
5.3.1 Study of CHMP2A protein interaction on model membrane
5.3.2 CHMP2A and CHMP2B proteins display opposite mechanical properties on membrane
5.3.3 CHMP2A + CHMP3 supramolecular assembly on membrane is dynamic in contrast with CHMP2B
5.4 CHMP3 perturbs CHMP2B polymerization and assembly on membranes
5.4.1 CHMP3 blocks CHMP2B polymerization on membranes
5.4.2 CHMP2B + CHMP3 supramolecular assembly is not dynamic
5.4.3 CHMP3 modulates the mechanical properties of CHMP2B polymers
5.5 CHMP2A and CHMP2B modulate CHMP4B assembly on membranes
5.5.1 CHMP4B assembly on membranes
5.5.1.1 CHMP4B alone forms spirals on flat membranes
5.5.1.2 Mechanical properties of GUVs coated with CHMP4B
5.5.2 CHMP2B disorganizes CHMP4B spirals on flat surfaces
5.5.3 CHMP2A and CHMP2B induce deformations on CHMP4 assembly on membrane tubes
Chapter 6. Conclusions and perspectives
Bibliography
