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SUMMARY:Thesis defense - Maureen Cercy
DESCRIPTION:Venue: CGFB \nDefense in french \n\nMaureen Cercy\nGiannone’s team (IINS) \nThesis supervisor: Grégory Giannone \nTitle\nNanoscale organization of the WAVE regulatory complex in the lamellipodium of\nmigrating cell. \nAbstract\nCell motility is involved in critical biological functions\, and dysregulation of adhesion\, migration and of the actin cytoskeletal can lead to severe disease like cancer. Therefore\, it is essential to study the molecular mechanism driving the formation of sub-cellular structures involved in cell motility. The first step in mesenchymal cell migration is the forward protrusion of the lamellipodium which is a thin sheet of membrane-enclosed actin filaments (F-Actin) networks propelled by actin polymerization.\nThe spatiotemporal coordination of F-actin regulators in the lamellipodium determines the polarity\, architecture and movements of branched F-actin networks. This includes two interacting nanomachines\, the WAVE regulatory complex (WRC) and the Arp2/3 complex. WRC activation is the central molecular event triggering Arp2/3 complex activation and thus\, the initiation and formation of a branched F-actin network in the lamellipodium. WRC activation relies on the exposure of the cryptic Arp2/3-activating WCA domain located at the C-terminal extremity of the WAVE subunit tail. In vitro studies showed that two WCA domains are needed to efficiently activate the Arp2/3 complex.\nBut how the local spatial organization of WRC at the molecular level translate into activation of the Arp2/3 complex triggering the morphogenesis of the lamellipodium is unknown. In other words\, the stoichiometry and the spatial distribution required to translate WRC conformational activation to an efficient activation of Arp2/3 complex are essential missing information.\nThe recent application of super-resolution microscopy (SRM) and single particle tracking (SRM) lead to a drastic rethinking of macromolecular assemblies\, in particular structures involved in cell migration\, including the lamellipodium. By tracking individual proteins and delivering images with spatial resolutions below the diffraction limit of light\, these techniques give access to the nanoscale organization and dynamics of protein complexes in live cells.\nTo reveal the molecular organization of WRC\, we use DNA-PAINT\, a SRM technique which allows spatial resolution below 10 nm. DNA-PAINT is based on hybridization of complementary DNA strands\, one located on the target protein (docking strand) and the other on the dye (imager strand). DNA-PAINT enable absolute molecular counting in protein complexes (Quantitative-PAINT) and multi-color super-resolution imaging (Exchange-PAINT). This allowed us to assess stoichiometries\, colocalizations and composition of nanomachines in the lamellipodium.\nUsing Quantitative-PAINT\, we showed that the stoichiometry of WRC at the lamellipodium tip of migrating mouse melanoma cell (B16) is one; while live super-resolution imaging based on RESOLFT nanoscopy revealed that these single WRC form discrete foci at the lamellipodium tip. Multicolor Exchange-PAINT super-resolution microscopy of the WRC core and its WCA domain\, showed that its conformational activation induces the release of the WCA domain in a radius of 40 nm away from its core. Using stereotyped waveform protrusions\, we correlated WRC molecular organization with the rate of membrane protrusions. We showed that the spatial distribution of individual WRC is below the radius of its conformational unfolding in regions of faster lamellipodial protrusion\, increasing the possibility of WCA domain dimerization and thus activation of the Arp2/3 complex. This way\, the WRC\, functioning as an isolated complex\, must be spaced at a distance less than its conformational unfolding to activate efficiently the Arp2/3 complex in the lamellipodium. Altogether\, our results show that besides biochemical activation of signaling circuitry\, the spatial organization of proteins is crucial for controlling their function in cells. \nKeywords: Actin Regulators\, Lamellipodium\, Cell Migration\, Super-Resolved Microscopy \nPublication\nMehidi A\, Kage F\, Karatas Z\, Cercy M\, Schaks M\, Polesskaya A\, Sainlos M\, Gautreau AM\, Rossier O\, Rottner K\, Giannone G. Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration. Nat Cell Biol. 2021 Nov;23(11):1148-1162. doi: 10.1038/s41556-021-00786-8. Epub 2021 Nov 4. PMID: 34737443. \nJury\nViolaine Moreau (Directrice de Recherche INSERM) – Présidente/Rapportrice\nCécile Leduc (Directrice de Recherche CNRS) – Rapportrice\nChristophe Le Clainche (Directeur de Recherche CNRS) – Rapporteur\nLaetitia Kurzawa (Chargée de Recherche CEA) – Examinatrice\nAlexis Gautreau (Directeur de Recherche CNRS) – Membre Invité\nGrégory Giannone (Directeur de Recherche CNRS) – Directeur de Thèse \n
URL:https://www.bordeaux-neurocampus.fr/en/event/maureen-cercy/
CATEGORIES:Thesis
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