Les mutations MKS1 sont impliquées dans un groupe de ciliopathies récessives létales, telles que le syndrome de Meckel-Gruber (MKS) et le syndrome de Joubert (JBT), caractérisées par une dysplasie rénale kystique, des anomalies du système nerveux central (encéphalocèle occipitale), une polydactylie, une dysgénésie biliaire et une fibrose hépatique. MKS1 a été localisée dans la zone de transition du cil dans de nombreux types cellulaires où elle joue un rôle essentiel pour la structure et la fonction des cils, en particulier la régulation de plusieurs voies de signalisation telles que Wnt et Shh.Dans le présent travail, nous avons identifié la fonction pré-ciliaire de MKS1 dans des cellules épithéliales. Nous avons montré que la localisation subcellulaire de MKS1 varie au cours de la maturation de l’épithelium, passant du cytosol où MKS1 co-localise avec le réseau de kératine, aux jonctions cellulaires, où elle co-colocalise avec les caténines. De plus, la translocation de MKS1 des jonctions au cytosol s'est avérée être mécano-sensible, suggérant que MKS1 participe à l'homéostasie épithéliale en stabilisant les jonctions cellulaires, via la transduction des signaux mécaniques liés à la compaction de l’épithelium.L’analyse fonctionnelle a démontré que le « knockdown » de MKS1 désorganise le réseau de kératine, et déstabilise les jonctions adhérentes des cellules épithéliales en culture, avec une diminution de la β-caténine jonctionnelle et une libération de l’E-cadhérine, l’α-caténine et la vinculine dans le cytosol. De plus, la déplétion de MKS1 entraîne une diminution notable du réseau apical d’actine, ainsi que la désorganisation de la structure épithéliale et une transition partielle vers un état mésenchymateux. Ces résultats illustrent une fonction indépendante du cil de MKS1 dans l’homéostasie épithéliale, et apporte de nouvelles hypothèses quant à son rôle et celui des filaments intermédiaires dans les processus d’organogenèse des épitheliums, en particulier la tubulogenèse, qui repose à la fois sur l’équilibre de la transition épithelium/mesenchyme et la mécanotransduction des sollicitations mécaniques durant l’embryogenèseDans le but de caractériser les partenaires de MKS1, des expériences de Co-IP et d’analyses protéomiques ont permis d’identifier l’epiplakine comme un partenaire possible de MKS1. L'Epiplakine est un cytolinker capable de lier la kératine à la membrane et à l'actine ; l’interaction de MKS1 avec l’epiplakine pourrait ainsi rendre compte de la stabilisation à la fois du réseau de kératine et des jonctions cellulaires. Des analyses complémentaires de protéomique et des études fonctionnelles devront compléter ces résultats préliminaires.Finalement, ces travaux ont également permis de révéler le rôle de MKS1 dans la stabilisation des jonctions communicantes ; la déplétion de MKS1 conduisant à une diminution de la CX43 jonctionnelle et à une altération de la fonction de communication intercellulaire dans les cellules épithéliales en culture. Ces travaux, qui constituent la première mention d’une altération possible des jonctions communicantes dans ce type de maladies, devront être approfondis pour caractériser leur impact dans les processus de tubulogenèse.En conclusion, ce travail qui a permis de révéler un rôle pré-ciliaire de MKS1 dans l'homéostasie épithéliale, apporte de nouvelles hypothèses pour l’étiologie de ces maladies, jusqu’alors considérées comme essentiellement consécutives à des défauts de transduction de la signalisation ciliaire. Il propose également de nouveaux mécanismes pour rendre compte des anomalies du développement hépatique, telles que la dysgénésie des voies biliaires, et plus largement des processus de tubulogenèse impliqués dans le développement de nombreux organes. / MKS1 mutations are involved in a group of lethal recessive ciliopathies, such as Meckel-Gruber syndrome (MKS) and Joubert's syndrome (JBT), characterized by cystic renal dysplasia, central nervous system abnormalities (occipital encephalocele) , polydactyly, biliary dysgenesis and hepatic fibrosis. MKS1 has been located in the transition zone of the cilia in many cell types where it plays an essential role in the cilia structure and function, in particular in the regulation of signaling pathways such as Wnt and Shh.In the present work, we have identified the preciliary function of MKS1 in epithelial cells. We have shown that the subcellular localization of MKS1 varies during the maturation of the epithelium, from the cytosol where MKS1 co-localizes with the keratin network, to the cell junctions, where it co-localizes with the catenins. In addition, the MKS1 translocation to cytosol junctions proved to be mechano-sensitive, suggesting that MKS1 participates in epithelial homeostasis by stabilizing cell junctions, via the transduction of mechanical signals related to epithelial compaction.Functional analysis has shown that the knockdown of MKS1 disrupts the keratin network, and destabilizes the adherent junctions of epithelial cells in culture, with a decrease in the junctional β-catenin and a release of E-cadherin, the α-catenin and vinculin in the cytosol. In addition, the depletion of MKS1 results in a significant decrease in the apical actin network, as well as disorganization of the epithelial structure and a partial transition to a mesenchymal state. These results illustrate a ciliary-independent function of MKS1 in epithelial homeostasis, and provides new hypotheses regarding its role and that of intermediate filaments in epithelial organogenesis processes, in particular tubulogenesis, which is based both on the equilibrium of the epithelium / mesenchyme transition and the mechanotransduction of mechanical stresses during embryogenesisIn order to characterize MKS1 partners, Co-IP experiments and proteomic analyzes have identified epiplakin as a possible MKS1 partner. Epiplakin is a cytolinker capable of binding keratin to membrane and actin; the interaction of MKS1 with epiplakin could thus account for the stabilization of both the keratin network and cell junctions. Additional proteomic analyzes and functional studies will complement these preliminary results.Finally, this work has also revealed the role of MKS1 in the stabilization of gap junctions; the depletion of MKS1 leading to a decrease in the junctional CX43 and an alteration of the intercellular communication function in the epithelial cells in culture. This work, which constitutes the first mention of a possible alteration of gap junctions in this type of disease, will have to be further developed to characterize their impact on tubulogenesis processes.In conclusion, this work which revealed a pre-ciliary role of MKS1 in epithelial homeostasis, provides new hypotheses for the etiology of ciliopathies, previously considered as essentially consecutive to signal transduction defects. It also proposes new mechanisms to account for abnormalities of hepatic development, such as bile ducts dysgenesis, and more broadly tubulogenesis processes involved in the development of many organs.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.