Transepithelial barriers represent important mechanisms by which epithelial cells delimit tissue compartments and maintain distinct extracellular environments. Such cellular barriers are key in regulating organ and tissue homeostasis and their dysregulation leads to a wide variety of pathologies. Novel tight junctions termed tricellular junctions (TCJs) appear to provide this barrier activity at the molecular level. Despite their proposed key role in barrier function, our understanding of these junctions is limited, with only a few molecules localized to tricellular junctions having been reported. Here we add to this understanding by identifying a LIG family member, Kek6, in Drosophila that represents the first example of a molecule uniquely excluded from TCJs. LIGs represent transmembrane molecules with Leucine-rich repeats and Immunoglobulin domains whose expression is often enriched in the developing nervous system. Data on Kek6 confirms this nervous system expression. Investigation into the mechanism which controls Kek6€™s unique exclusion from TCJs has proved that it is not solely mediated by the C-terminal intracellular PDZ domain-binding site. Although PDZ domain-binding sites of various proteins have been implicated as important for protein localization, it is thought that it is the extracellular domain of Kek6 that is the part of the protein which is responsible for its unique localization pattern. Shown here, it is believed that Kek6 participates in a stabilizing homophilic interaction which may support the hypothesis that the extracellular domain is required for localization. Kek6 expression in one cell is not sufficient for expression in the bicellular junctions. Adjacent cells must both express Kek6 in order for Kek6 to be stably localized to the bicellular junction. Studies on the potential relationship between Kek6 and Gliotactin, the Drosophila protein which localizes to TCJs, revealed that there is no direct relationship between these two proteins but does not eliminate the potential of unidentified shared interactors. Further investigation of Kek6 will allow for the elucidation of the role of Kek6 in TCJs which will help further develop the junction biology field. In addition to the information provided on Kek6, this study reports the first localization and functional knowledge of Kek3. Misexpression of Kek3 leads to cross vein defects and reduction/loss of bristles revealing that Kek3 may be a modulator of BMP signaling. Although family member Kek5 has been previously identified as a modulator of BMP signaling, the mechanism of this function is still under investigation but it is believed that Kek3 is acting through a different mechanism.

La Dysplasie Epithéliale Intestinale (DEI) est une maladie intestinale rare chez l'homme qui a récemment été liée à des mutations des gènes EPCAM et SPINT2. Peu de données sont disponibles pour ces deux gènes, principalement étudiés comme marqueurs du cancer. Le but de ma Thèse était d'identifier les défauts cellulaires induits par l'inactivation de ces gènes et de comprendre les fonctions d’EpCAM et de Spint2dans les entérocytes. Matériel et méthode : Nous avons utilisé comme modèle des cultures cellulaires de cellules Caco2‐BBe stablement inactivées pour EpCAM ou pour Spint2, en utilisant la stratégie shRNA, en comparaison avec les biopsies intestinales de patients DEI, mutés pour l'un ou l'autre gène. J'ai analysé par microscopie électronique,microscopie confocale ou vidéo microscopie, différents marqueurs, et étudié ces cellules sur des modèles 3Dtrès novateurs développés dans notre laboratoire.Résultats: Notre étude a mis en évidence que EpCAM est un acteur clé dans le maintien de l'organisation entérocytaire apico‐basale. Son inactivation conduit à une hyperactivation majeure de l'actomyosine focalisée sur les sommets tricellulaires, entraînant une profonde perturbation de l'organisation cellulaire globale. Les inhibiteurs de la myosine II restaurent l'organisation cellulaire normale. L'étude de Spint2 vient de commencer,mais il semble que l'inactivation de Spint2 conduit à la perturbation de l'adhésion cellulaire en relation avec desdéfauts dans la fin de la division cellulaire à l'étape de la cytocinèse. Cette hypothèse nécessite des analyses complémentaires afin de confirmer ces résultats, les compléter et préciser le mécanisme impliqué.Conclusion: Au cours de ma thèse, j'ai dévoilé de nouvelles fonctions d'EpCAM, et en soupçonne d'autres pour Spint2. Nos résultats devraient aider la recherche de traitements pour ces malades. Les données devraient non seulement servir la cause de la DEI mais également aider les biologistes cellulaires de l'intestin et la recherche sur le cancer. / Congenital Tufting Enteropathy is a rare human intestinal disease that has recently been ascribed tomutations in EPCAM and SPINT2. Few data were available for both, mainly studied as cancer markers. Thepurpose of my thesis was to identify cell defects induced by the inactivation of these genes and to understand EpCAM and Spint2 functions in enterocytes. Materiel & Method : The model used was cell culture of Caco2‐BBe cells stably inactivated for EpCAM or for Spint2 using shRNA strategy, in comparison with intestinal biopsies from patients mutated for either one or theother gene. I analysed by electron microscopy, confocal, or video microscopy different markers. Moreover, Istudied the cells on very novative 3D patterns developped in the laboratory. Results : My studies highlighted that EpCAM is a key player in maintenance of enterocyte apico‐basalorganization. Its inactivation leads to major actomyosin hyperactivation focused in tricellular vertices resulting in tremendous perturbation of the global cell organization. Inhibitors of myosin‐II rescued the normal cellorganization. The Spint2 study has just started, but it seems that Spint2 inactivation leads to cell adhesionperturabtion in relationship with defects in finishing cell division at the cytokinesis step. This hypothesis needs further analyses to be confirmed and to precise the involved mechanism. Conclusion : During my thesis I unraveled new functions of EpCAM, suspected others for Spint2, and have progressed in the seeking of treatments. The data should not only serve the cause of CTE, but also should help members of intestinal biology and cancer research.