Impact de la protéine CHMP2B et de ses variants pathogènes sur le modelage des membranes biologiques

This work focused on CHMP2B, an ESCRT-III complex subunit. This protein complex is involved in several membrane fission processes with an inversed topology (virus budding, multivesicular body genesis, cytokisesis, autophagy). Mutations in the gene coding for CHMP2B have been linked to neurodegenerative diseases such as fronto temporal demencia (FTD) and Spinal Lateral Amyotrophy (SLA). This work consisted in understanding molecular mechanisms of CHMP2B's action in order to better understand it' s links with these pathologies. The project was organized around three research axis: - The potential involvement of CHMP2B in the mitochondrial dynamics. - The role of CHMP2B and the impact of the FTD linked mutations on the morphology and the physiology of dendritic spines. - The study of helical CHMP2B containing complexes that deform the plasma membrane into tubular structures. Our experiments led us to the following observations: Extinction of CHMP2B inhibits mitochondrial fission by an unknown mechanism. The FTD linked CHMP2B mutants disrupt the normal pattern of spine development. This alteration could participate to the progressive appearance of FTD. CHMP2B is thought to be involved in the remodeling of cytoplasmic organelles such as late endosomes, autophagosomes, centrosome... Our work demonstrate that the protein is manly recruited at the plasma membrane where it assembles in rigid tubular structure that can deform the lipid bilayer.Ce travail de thèse s'est concentré sur l'étude de la protéine CHMP2B. Cette protéine fait partie du complexe ESCRT-III, impliqué dans divers processus de fissions membranaires à topologies inversées (genèse ces corps multivésiculés, cytokinèse, bourgeonnement des virus enveloppés, autophagie). Des mutations dans le gène codant pour CHMP2B ont été très fortement corrélées à la survenue de maladies neuro-dégénératives (démences fronto-temporales et amyotrophie latérale spineuse). Ce travail a consisté à préciser les mécanismes moléculaires d'action de cette protéine afin de mieux cerner les causes potentielles des ces dysfonctionnements. Il se divise en trois sous parties: - L'étude du l'import potentiel de CHMP2B dans les mitochondries, et de son rôle dans la dynamique mitochondriale. - Le rôle de CHMP2B et l'impact de mutants pathogènes dans la morphogénèse des épines dendritiques. - L'étude de la formation de d'hyper-complexes tubulaires de CHMP2B déformants la membrane plasmique. Nous avons ainsi pu montrer les propriétés suivantes: L'extinction de CHMP2B semble inhiber la fission des mitochondries par un mécanisme qui reste à préciser. L'expression des mutants de CHMP2 liés aux démences fronto-temporales perturbe la morphogénèse des épines dendritiques. Cette altération pourrait participer à l'émergence progressive des symptômes de la FTD. La littérature suggère que les fonctions que CHMP2B concernent des organelles cytoplasmiques (endosomes tardifs, autophagosomes,centrosome). Nous avons montrés que cette protéine est principalement recrutée à la membrane plasmique, où elle se polymérise en une structure tubulaire rigide capable de déformer cette bicouche lipidique

Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane.: Plasma membrane deformation by CHMP2B

International audienceThe endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation

Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity.

International audienceExosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system