Les cellules multiciliées jouent un rôle essentiel dans la propulsion des fluides physiologiques. Leur dysfonctionnement provoque des maladies chroniques. Contrairement à la plupart des cellules de mammifères qui possèdent un centrosome composé de deux centrioles, les cellules multiciliées possèdent une centaine de centrioles qui servent de base à la nucléation des cils motiles. Les mécanismes d'amplification de centrioles ou de régulation du nombre de centrioles dans ce type cellulaire étaient jusque-là inconnus. Les centrioles nouvellement formés étaient considérés comme apparaissant " de novo ". Une approche de vidéomicroscopie et de microscopie de super-résolution corrélative nous a d'abord permis de déterminer que tous les procentrioles sont générés à partir du centrosome préexistant. Nous démontrons que le centriole fils du centrosome est le site principal de nucléation de 95% de centrioles nouvellement formés dans les cellules multiciliées. Ces résultats réfutent par conséquent l'origine " de novo " des centrioles dans ce type cellulaire. Puis, nous montrons que la machinerie mitotique orchestre la progression spatio-temporelle de la dynamique centriolaire dans ces cellules post-mitotiques et en phase terminale de différentiation. L'amortissement de l'activité de Cdk1 empêche la rentrée en mitose tout en permettant la coordination du nombre de centrioles, leur croissance, et leur désengagement par des transitions phasiques nécessaires à la nucléation de cils motiles. Cette thèse aide à mieux comprendre la différentiation des cellules multiciliées, les ciliopathies, ainsi que l'amplification centriolaire pathologique associée avec le cancer et la microcéphalie. / Multiciliated mammalian cells play a crucial role in the propulsion of physiological fluids. Their dysfunction causes severe chronic diseases. In contrast to the strict centriole number control in cycling cells, multiciliated cell differentiation is marked by the production of up to several hundred centrioles, each nucleating a motile cilium. The mechanisms of centriole amplification or centriole number control in these cells were unknown and new centrioles were thought to appear de novo in the cytoplasm. First, videomicroscopy combined with correlative super-resolution and electron microscopy has enabled us to determine that all procentrioles are generated via runs of nucleation from the pre-existing progenitor cell centrosome. We show that the daughter centriole of the centrosome is the primary nucleation site for 95% of the new centrioles in multiciliated cells and thus refute the de novo hypothesis. Then, we provide evidence of an activation of the mitosis regulatory network during the centriole dynamic. With single cell live imaging and pharmacological modulation of mitosis regulators, we show that the mitosis machinery orchestrates the spatiotemporal progression of centriole amplification in terminally differentiating multiciliated cell progenitors. The fine-tuning of Cdk1 activity prevents mitosis while allowing the timely coordination of centriole number, growth, and disengagement through checkpoint-like phase transitions necessary for subsequent functional motile ciliation. This PhD provides a new paradigm for studying multiciliated cell differentiation, cilia-related diseases and pathological centriole amplification associated with cancer and microcephaly.

Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.