ED Sciences Chimiques
Development of Atomic Force Microscopy-related modes for the study of plasma membrane repair
by Carine ASSAF (Institut de Chimie & de Biologie des Membranes & des Nano-objets)
The defense will take place at 9h00 - Amphithéâtre IECB Batiment B13 2 Rue Robert Escarpit 33600 Pessac
in front of the jury composed of
- Michaël MOLINARI - Professeur des universités - Université de Bordeaux - Directeur de these
- Etienne DAGUE - Directeur de recherche - CNRS - Université de Toulouse - Rapporteur
- Andra-Cristina DUMITRU - Professeure - FNRS - Université Louvain-La-Neuve - Rapporteur
- Pierre-Emmanuel MILHIET - Directeur de recherche - CNRS - Université de Montpellier - Examinateur
- Clotilde BILLOTTET - Professeure des universités - Université de Bordeaux - Examinateur
- Anthony BOUTER - Professeur des universités - Université de Bordeaux - CoDirecteur de these
The plasma membrane acts as a crucial barrier for cells, protecting them from various biological, chemical and mechanical stresses. These stresses induce tears in the membrane that are resealed by triggering rapid repair mechanisms. However, understanding the repair process at the nanoscale induced under physiological conditions is challenging. This thesis aims to investigate the mechanisms of plasma membrane repair following a localized mechanical injury under controlled conditions by combining approaches from cellular mechanics and real-time imaging. The main objective of this PhD work is to develop a correlative experimental approach coupling atomic force microscopy (AFM) to confocal microscopy to study the repair mechanism in cancer cells. The first step was to develop an original protocol using the AFM tip to induce, in a controlled and reproducible manner, localized membrane damage while simultaneously following the cell repair. To this end, confocal imaging was performed to monitor in real-time cellular response at the damage site providing information on the recruitment of the molecular markers involved in the repair machinery while AFM was simultaneously used to measure the mechanical properties of the membrane at the damage site. The protocol was successfully set-up using MDA-MB-231 breast cancer cell line, confirming the local restoration of the membrane integrity after damages depending on the physiological conditions. In a second study, the same protocol was applied to another breast cancer cell line, MCF-7, a less invasive phenotype, to compare the repair mechanism. Overall, our work provides a quantitative and dynamic characterization of mechanically induced membrane repair and highlight the potential of AFM and this correlative approach as a powerful tool for investigating membrane repair dynamics. A better understanding of the membrane repair mechanism could allow to identify therapeutic targets in diseases associated with membrane dysfunction, such as cancer.