Soutenance de thèse de Cécile JEBANE

La lamine A/C est un filament intermédiaire et un constituant de la lamina, le réseau de filaments qui sous-tend la membrane nucléaire interne. La lamina est responsable de la forme et de la rigidité du noyau, et joue un rôle important dans la mécanotransduction. Des mutations dans le gène codant pour la lamine A/C (LMNA) ont été liées à une famille de pathologies humaines, collectivement dénommées laminopathies, qui présentent différents degrés de sévérité. Les laminopathies ont plusieurs symptômes communs tels qu’une prédisposition aux maladies cardiovasculaires, des problèmes de répartition des graisses et le vieillissement prématuré. Les laminopathies peuvent être multi-systémique, comme la Progeria qui provoque le vieillissement prématuré de l’organisme entier, ou tissu-spécifiques comme les lipodystrophies (mauvaise répartition des tissus adipeux), ou, à divers degrés, entraîner des problèmes de santé symptomatiques de la vieillesse (problèmes cardiovasculaires, athérosclérose). Si on connaît les conséquences de ces maladies, on ignore encore la relation entre les mutations de LMNA, les altérations mécaniques du noyau et le degré de sévérité. Mon projet porte sur l’étude des mécanismes physiques de vieillissement prématuré lié aux anomalies de la lamina. Pour cela j’utilise une approche microfluidique pour quantifier l’altération des propriétés mécaniques de la cellule et du noyau. Cette étude permettra d’établir la relation entre fragilité du noyau et sévérité de la maladie. Nous espérons qu’à long terme notre système microfluidique pourra être adapté pour élaborer une nouvelle méthode d’exploration des propriétés mécaniques du noyau, simple, rapide et peu coûteuse. Elle permettrait de diagnostiquer des conditions prédisposant au vieillissement prématuré, et constituerait un outil indéniable pour développer des traitements anti-vieillissement.

Lamin A/C is an intermediate filament and a constituent of the nuclear envelope. Lamin A/C contributes to the mechanical properties of the nucleus such as stability, shape, and rigidity. Mutations in the lamin A/C gene LMNA induce pathologies called laminopathies. Laminopathies share common phenotypes such as abnormal shape of the nucleus or premature senescence of the cell (i.e. premature aging). These pathologies display varying severity and can be tissue-specific, like Type 2 Familial Partial Lipodystrophy (FPLD2) where only adipose tissues are affected, or multi-systemic, like Hutchinson-Gilford Progeria Syndrome (HGPS) where the entire body ages prematurely. So far, most studies focused on HGPS, showing an increased rigidity of the cell nucleus. Whether the observed alterations are HGPS-specific or common to all laminopathies is not answered yet. Therefore, the relationship between LMNA mutations, mechanical alterations in cells and laminopathy severity remains unknown, resulting in a lack of diagnosis and treatment. My PhD project aims at investigating such relationship at the cell level, in the second timescale, with strong cellular and nuclear deformations which have been poorly investigated until now. To do so, I developed a two-height microfluidic chip to quantify the deformation dynamics of cells passing through specifically-designed constrictions. We combined high-throughput microfluidic measurements with semi-automated image analysis and a rheological model to extract mechanical properties of human fibroblasts. We studied prematurely senescent fibroblasts issued from two FPLD2 patients carrying the lamin A/C R482W mutation. Additionally, we used a model cell line of premature senescence: fibroblasts from a healthy individual, treated with Atazanavir (AZN), a protease inhibitor used for HIV therapy and known to alter lamin A/C production. We showed that prematurely senescent fibroblasts mainly exhibit a more viscous behavior compared to non-senescent ones. In our set-up, viscosity –but not rigidity– dominates the entry time of the cells in the constrictions. The viscous response of the cell, that we initially thought originating mostly from the nucleus, is also dependent on other components of the cell such as the actin network or the links between the cytoskeleton and the nucleus. In the senescence model, we observed that AZN treatment induces milder phenotypes: from a biological point of view, AZN-treated cells display intermediate signs of senescence, between FPLD2 and HGPS; from a physical point of view, the rheological parameters were less affected than in FPLD2 patient cells. Thus, the cell line under AZN treatment, though it may be considered as a senescence model, does not alter lamin A/C production as much as the R482W mutation. We noticed that cells from the FPLD2 patients, though carrying the same mutation do not behave exactly the same: cells from one patient were much more viscous, and his/her clinical state was incidentally worse. Our experiment may be sensitive to the gravity of the patient state. We believe that our microfluidic device opens the way to progress towards a simple, fast, and inexpensive mechanics-based test to identify predisposition to premature aging or assess the effects of anti-aging drugs.