Etude de la fonction du gène rpgrip1l dans les processus de différenciation et de polarité cellulaire chez le poisson-zèbre

Le cil primaire, présent à la surface de la majorité des cellules chez les vertébrés, a un rôle primordial dans le développement embryonnaire et dans la modulation de voies de signalisation, notamment Hedgehog et Wnt. Des défauts de cette structure sont associés à des maladies héréditaires, les ciliopathies. Le gène Rpgrip1l est impliqué dans deux ciliopathies, le syndrome de Joubert de type B et le syndrome de Meckel, caractérisées par une polydactylie, des kystes rénaux et des malformations du système nerveux central. Rpgrip1l code pour une protéine localisée principalement à la zone de transition des cils, une région impliquée dans le contrôle du trafic des protéines vers et hors du compartiment ciliaire. Mon projet de thèse a porté sur les fonctions de Rpgrip1l dans les processus de différenciation et de polarité cellulaire dans le système nerveux du poisson-zèbre. Mon premier travail, basé sur la perte de fonction de rpgrip1l par injection de morpholino dans des embryons de poissons zèbre, a mis à jour une fonction de Rpgrip1l dans la mise en place de la polarité planaire via la stabilisation de dishevelled, une protéine clé de la voie Wnt/PCP. J ai ensuite étudié les fonctions plus tardives du gène rpgrip1l et de son paralogue rpgrip1 dans la rétine, via l analyse de lignées de poissons zèbre hypomorphes. Cette étude a montré l existence de multiples isoformes de Rpgrip1l et suggère de nouvelles fonctions extra-ciliaires pour Rpgrip1l dans la morphogenèse des photorécepteurs. L ensemble de ce travail devrait permettre de mieux comprendre l origine développementale des anomalies cérébrales et rétiniennes observées dans les ciliopathies.In vertebrates, primary cilia are present in virtually every cell and are involved in several signaling pathways such as the Hedgehog and Wnt pathways. Cilium dysfunctions have been causally linked to a group of pleiotropic and genetically heterogeneous human diseases, the ciliopathies. The human RPGRIP1L gene is one of the causal genes in Meckel and Joubert type B syndromes, two ciliopathies characterized by polydactyly, kidney cysts, and central nervous system malformations. The Rpgrip1l protein is enriched in the ciliary transition zone that establishes the ciliary gate controlling entry and exit of proteins in and out of the cilium. During my PhD, I studied the function of the Rpgrip1l gene in differentiation and planar cell polarity events that participate in brain morphogenesis. The first study, based on the loss of function of rpgrip1l by morpholino injection in zebrafish embryo, highlights rpgrip1l function in planar cell polarity via dishevelled stabilization, a core protein of Wnt/PCP pathway. Then, I studied the later functions of rpgrip1l and its paralogue rpgrip1 in the retina, using hypomorphic zebrafish. This work, by the discovery of rpgrip1l variants and description of photoreceptors defects, provides leads for new extra-ciliary functions of rpgrip1l in retinal morphogenesis. Ultimately, this work should result in a better understanding of the developmental origin of cerebral and retinal defects found in ciliopathies.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Functional analysis of the transition zone protein Rpgrip1l during zebrafish development

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Dishevelled stablisation at the cilium by RPGRIP1L is essential for planar cell polarity

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Dynamics of centriole amplification in centrosome-depleted brain multiciliated progenitors

International audienceReproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed

Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity.

International audienceCilia are at the core of planar polarity cellular events in many systems. However, the molecular mechanisms by which they influence the polarization process are unclear. Here, we identify the function of the ciliopathy protein Rpgrip1l in planar polarity. In the mouse cochlea and in the zebrafish floor plate, Rpgrip1l was required for positioning the basal body along the planar polarity axis. Rpgrip1l was also essential for stabilizing dishevelled at the cilium base in the zebrafish floor plate and in mammalian renal cells. In rescue experiments, we showed that in the zebrafish floor plate the function of Rpgrip1l in planar polarity was mediated by dishevelled stabilization. In cultured cells, Rpgrip1l participated in a complex with inversin and nephrocystin-4, two ciliopathy proteins known to target dishevelled to the proteasome, and, in this complex, Rpgrip1l prevented dishevelled degradation. We thus uncover a ciliopathy protein complex that finely tunes dishevelled levels, thereby modulating planar cell polarity processes

Ependymal cilia beating induces an actin network to protect centrioles against shear stress

International audienceMulticiliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability