UNIVERSITE D’AIX-MARSEILLE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE / ED 62 LABORATOIRE DE LA POLARITE CELLULAIRE ET MORPHOGENESE DES EPITHELIUMS/INSTITUT DE BIOLOGIE DU DEVELOPPEMENT DE MARSEILLE (UMR 7288) Thèse présentée pour obtenir le grade universitaire de docteur Spécialité : Biologie du développement Veronika AKSENOVA Role of Crumbs and Bazooka in the organization and distribution of DE-cadherin in Drosophila embryo Soutenue le 18/12/2017 devant le jury : Alexandre DJIANE Rapporteur Grégoire MICHAUX Rapporteur Julien ROYET Président du jury André Le BIVIC Directeur de thèse Thomas LECUIT Invité (Co-Directeur de thèse) Résumé Les tissus épithéliaux sont des couches de cellules adhérentes qui servent de barrières entre différents compartiments morphologiques et procurent un transport directionnel de molécules. Pour remplir leurs fonctions, ces cellules épithéliales possèdent une architecture hautement polarisée en trois domaines distincts - apical, latéral et basal. Le domaine apical fait face à l’environnement extérieur ou lumen tandis que le domaine latéral assure l’adhésion entre les cellules voisines et le domaine basal est en contact avec la membrane basale. L’action coopérative de plusieurs déterminants de la polarité gouverne l’identité et la morphogenèse spécifiques de ces domaines : 1) le cytosquelette d’actomyosine, 2) les jonctions adhérentes (AJs) basées sur la E-cadhérine et 3) les complexes de polarité conservés au cours de l’évolution. Ainsi, l’intégrité dynamique de ces acteurs donnent lieu à l’établissement et au maintien de la polarité épithéliale. Une perte de l’adhérence via la DE-cadhérine (DE-Cad) conduit à des défauts de polarité apico-basale, tandis que la localisation apicale de DE-Cad nécessite les protéines de polarité Crumbs (Crb) et Bazooka (Baz) (L’homologue de Par3 chez la mouche). Notablement, DE- Cad forme des amas qui co-localisent partiellement avec les amas de Baz, génèrent l’adhésion intercellulaire et transmettent la tension. Cependant, les mécanismes impliqués dans le contrôle de la taille, le nombre, la répartition et la dynamique des amas de DE-Cad restent peu connus. Pendant ma thèse, je me suis concentrée sur l’interaction complexe entre les AJs formées par DE-Cad, la protéine de polarité Baz et un déterminant majeur de la polarité apicale, le complexe transmembranaire Crumbs, dans l’embryon de Drosophila. En particulier, j’ai étudié le rôle de Crumbs et Baz dans la régulation de la distribution fine de DE-Cad à la membrane. J’ai montré que Crb contrôle la distribution macroscopique de DE-Cad, au moins, partiellement via Baz. En générant des mutations de Baz sur des sites régulateurs variés grâce à de la transgenèse spécifique de site et en utilisant de la microscopie en temps réel quantitative, j’ai montré que Crb agit via le domaine d’oligomérisation CR1 et le site Ser980 de Baz afin d’ajuster les niveaux de DE- Cad aux jonctions. Remarquablement, j’ai aussi révélé que le domaine d’oligomérisation de Baz est inutile à la formation d’amas Baz-DE-Cad et j’ai caractérisé la réciprocité de l’interaction DE- Cad-Baz. En conclusion, ma thèse fournit une preuve expérimentale forte d’une interaction génétique entre les protéines de polarité Crb et Baz qui est impliquée dans les niveaux et le contrôle de la distribution de DE-Cad à la membrane. Ainsi, les protéines de polarité ne jouent pas seulement leur rôle canonique requis pour l’établissement et le maintien de la polarité apico- basale mais aussi régulent la stabilité et l’intégrité des AJs pendant l’extension de la bande germinale (GBE) lors de la gastrulation de l’embryon de Drosophila. 2 Abstract Epithelia are sheets of adherent cells that serve as barriers between distinct morphological compartments and provide directed transport of molecules. To fulfil their functions, epithelial cells possess a highly polarized architecture with three distinct domains – apical, lateral and basal. Apical domain faces external environment or lumen while lateral domain ensures adhesion between neighboring cells and basal domain contacts basement membrane. The cooperative action of several polarity determinants governs the proper identity and morphogenesis of these domains: 1) actomyosin cytoskeleton; 2) E-Cadherin-based adherens junctions (AJs) and 3) evolutionarily conserved polarity complexes. Thus, the dynamic integrity of these players results in the epithelial polarity establishment and maintenance. A loss of DE- cadherin (DE-Cad) adhesion leads to apico-basal polarity defects, while the apical localization of DE-Cad requires the polarity proteins Crumbs (Crb) and Bazooka (Baz) (Par3 homolog in fly). Notably, DE-Cad builds clusters that display a certain degree of colocalization with the clusters of Baz, provide intercellular adhesion and transmit tension. However, the mechanisms involved in the control of DE-Cad cluster size, number, repartition and dynamics remain poorly understood. In my thesis, I have focused on the complex interplay between DE-Cad-mediated AJs, Baz polarity protein and a major apical polarity determinant, transmembrane Crumbs complex in Drosophila embryo. In particular, I have addressed the role of Crumbs and Baz in the regulation of DE-Cad fine distribution on the membrane. I demonstrated that Crb controls DE-cad macroscopic distribution, at least, partially via Baz. By generating Baz mutants on various regulatory sites using site-specific transgenesis and quantitative live-imaging microscopy, I showed that Crb acts via CR1 oligomerization domain and Ser980 site of Baz to adjust DE-Cad levels at the junctions. Strikingly, I also revealed that Baz oligomerization domain is dispensable for Baz-DE-Cad clusters formation and characterized the reciprocity of DE-Cad-Baz crosstalk. In conclusion, my thesis provides a strong experimental evidence of a genetic interplay between polarity proteins Crb and Baz that is implicated in the DE-Cad levels and distribution control on the membrane. Hence, polarity proteins do not only play their canonical role required for the establishment and maintenance of apico-basal polarity but also regulate AJs stability and integrity during germ band elongation (GBE) in gastrulating Drosophila embryo. 3 Acknowledgements First of all, I would like to thank Dr. Alexandre Djiane, Dr. Grégoire Michaux and Prof. Julien Royet for their agreement on reading my manuscript and to be members of my PhD Jury. I look forward to receiving their constructive criticism that, I believe, would significantly enrich my work in theoretical and experimental prospects and to initiating fruitful discussions during my Defense. I would like to express my deep gratitude to my scientific advisors – André Le Bivic and Thomas Lecuit. Being very different by their nature, they both contributed a lot into my personal growth from different aspects. The weekly meetings with both of them strongly developed my analytical and presentation skills, as well as conflict-solving skills sometimes. André has always been very supportive and kind to me, consistently helped to overcome tough moments during my PhD. It was a real pleasure for me to work under André’s guidance and in the André’s lab. I would like to acknowledge Thomas for investing into my potential and inviting me to work under the Labex Inform funding. Every meeting with Thomas was scientifically demanding and I am thankful to him for setting ambitious goals and challenging me every week. In addition, I appreciate their mutual contribution into my scientific mobility and expanding my professional network by sending me to numerous international conferences and workshops. I would like to acknowledge André Le Bivic team members. From the very first day in the lab, they have been very supportive and helpful with me. I thank Benoit for being my teacher in Drosophila genetics and for helping me with imaging analysis. I would like to thank Christopher Toret for his sense of humor, for intruding me to the American culture, for educating me in general and just not letting down during tough periods. I thank Pierre Mangeol for introducing me to the world of Physics and to spend hours trying to explain me the physics laws and the coding language. I also appreciate the support of Elsa and Magali and for sharing their knowledge of the mammalian cell culture. I would like to thank Dominique for her attentive reading of my manuscript. We spent great time doing experiments together with our collaborators in CRCM of Institut Paoli-Calmettes. Discussions with all of them have significantly broadened my fundamental expertise in the 4 polarity proteins. In addition, I would express my deep gratitude to Stéphane Audebert, Head of Proteomics and Mass Spectrometry platform at the CRCM, for allowing me to conduct my experiments in his lab. I am much obliged to Pauline Salis for her constant emotional and professional support, she has been my navigator in my life in France. I appreciate Pauline for accepting me to follow her project since our first meeting and for taking me to painting classes. Thanks to Pauline, I can draw a Drosophila embryo now. Thomas Lecuit team has been also truly supportive to me. I thank Jean-Marc Phillipe for generating Baz constructs for me and for helping me with molecular biology techniques. I also appreciate Alain Garcia de las Bayonas, Anaïs Bailles and Girish Kale for their comprehensive help and always positive attitude. Finally, I would like to acknowledge my first scientific advisor at the Institute of Carcinogenesis in Moscow, Natalya Gloushankova, for introducing me to the world of Cell, for sharing her knowledge on the molecular oncology and for always supporting me in my ambitious scientific plans. In addition, I would have never achieved any progress without a constant support from my family who often visited me on my long way to get a PhD degree to ensure my well-being in France. This work would have never been possible without a help of my husband Dmitry Ilyushin who has been providing not only emotional support but, in fact, studying all these years with me.