Thesis advisor: David R. Burgess / Polarity established by the first cleavages in sea urchin embryos was investigated in this thesis revealing precocious embryonic polarity. Studies of embryonic polarity have focused on protostomes such as <italics>C. elegans</italics>, and those on deuterostomes have focused on later developmental stages. I find asymmetries in the sea urchin membrane cell cortex as early as the first division after fertilization as a result of new membrane addition in the cleavage furrow. Membrane domains and the polarity determinants Par6, aPKC, and Cdc42 are polarized to the apical, or free, cell surface, while the cell-cell contact site remains distinct. Using immunofluorescence, fluorescence recovery after photobleaching (FRAP), and specific inhibitor treatments, myosin filaments were identified as the major regulator of membrane cortex polarity. However, membrane domains and cortical polarity determinants are differentially regulated with respect to blastomere dissociation. These asymmetries are required for proper spindle alignment and cleavage plane determination and are responsible for polarized fluid phase endocytosis. The work in this thesis and future studies addressing the connection between the membrane cortex and myosin filaments has and will lead to a greater understanding of the maintenance of embryonic polarity in cleavage stage sea urchin embryos. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR / Cell polarity is a fundamental process that controls cell’s functional and physiological specificities. This process relies on membranous compartments differently composed both on proteins and on phosphatidyl-inositols (PIs). Indeed, through their asymmetric localization, polarity proteins, such as the PAR proteins, are essentials to establish and maintain polarity of the cells. During my PhD, I studied the interplay between the polarity proteins and the PIs. Using the Drosophila egg chamber, as a model, I aimed to characterized the upstream events that regulate the PI(4,5)P2 producing kinase (PIP5K), Skittles (SKTL), activity and localization. I also studied the downstream molecular process that link the PI(4,5)P2, SKTL and the PAR proteins in cell polarity. I contributed to the characterization of the importance of PI(4,5)P2, mainly produced by SKTL in maintaining the apical-basal polarity and during the morphogenesis of the follicle cells. The PI(4,5)P2 is ensuring PAR3 and adherens junctions but not PAR1 proper localizations. Next, through a precise quantification method, I showed that SKTL and the PI(4,5)P2, probably via vesicular traffic, were also ensuring PAR3 proper localizations (anterior accumulation and stage 9B posterior exclusion) in the oocyte. PAR3 accumulation also relies on a Dynein mediated transport and the IKKε kinase while its posterior exclusion relies on PAR1 phosphorylation. Finally, I studied SKTL post translational modifications and their relevance on cell polarity. I identified palmitoylation and phosphorylations that are regulated by the kinase PAR1 and the phosphatase PP1. SKTL phosphorylations seem to be related to its role on the vesicular traffic. Altogether these results clarify some mechanisms involving both PIs and PAR proteins in cell polarity maintaining and establishment

O cristalino se origina de um epitélio simples e cuboidal que recobre a vesícula óptica. Neste estádio, os filamentos de actina são distribuídos ao longo do eixo apicobasal. As células do ectoderma pré- placodal, em contato com a vesícula óptica, formam um epitélio pseudoestratificado, chamado de placóide do cristalino, com acúmulo de actina no domínio apical. Nós propusemos estudar o papel da proteína PAR3 e sua fosforilação no estabelecimento de actina apical. A superexpressão de PAR3 no placóide forma pontos ectópicos de PAR3 na membrana baso-lateral e induz o recrutamento de actina ectópica para esses pontos. O recrutamento de actina e aPKC ectópicos é independente do estado de fosforilação da treonina 833, resíduo localizado no domínio de ligação do PAR3 ao aPKC. Além disso, no ectoderma peri-placoidal, onde a actina localiza-se baso-lateralmente, PAR3 induz o recrutamento ectópico de actina apical e esse recrutamento é independente da fosforilação da treonina 833. Esses dados nos sugerem que PAR3 é suficiente para recrutar actina no placóide do cristalino. / The lens originates from a simple cuboidal epithelium that overlies the optic vesicle. At this stage, the actin filaments are distributed along its apical-basal sides. The pre-placodal ectoderm, in contact with the optic vesicle, forms a pseudostratified tissue, the lens placode, with accumulation of actin network at the apical domain. Here, we focused on the role of the polarity protein PAR3 and its phosphorylation in the establishment of this apical actin network. Overexpression of PAR3 in the lens placode, induced formation of ectopic actin clusters in the basolateral membrane of the lens placode. The formation of these actin clusters, as well as recruitment of aPKC was independent of Threonine 833 phosphorylation at the PAR3 aPKC-binding site. In addition, PAR3 induced ectopic actin networks in the apical membrane of the periplacodal ectoderm independent of the Threonine 833 phosphorylation. Taken together, these data suggest that PAR3 is sufficient for actin recruitment in the lens placode.

O cristalino se origina de um epitélio simples e cuboidal que recobre a vesícula óptica. Neste estádio, os filamentos de actina são distribuídos ao longo do eixo apicobasal. As células do ectoderma pré- placodal, em contato com a vesícula óptica, formam um epitélio pseudoestratificado, chamado de placóide do cristalino, com acúmulo de actina no domínio apical. Nós propusemos estudar o papel da proteína PAR3 e sua fosforilação no estabelecimento de actina apical. A superexpressão de PAR3 no placóide forma pontos ectópicos de PAR3 na membrana baso-lateral e induz o recrutamento de actina ectópica para esses pontos. O recrutamento de actina e aPKC ectópicos é independente do estado de fosforilação da treonina 833, resíduo localizado no domínio de ligação do PAR3 ao aPKC. Além disso, no ectoderma peri-placoidal, onde a actina localiza-se baso-lateralmente, PAR3 induz o recrutamento ectópico de actina apical e esse recrutamento é independente da fosforilação da treonina 833. Esses dados nos sugerem que PAR3 é suficiente para recrutar actina no placóide do cristalino. / The lens originates from a simple cuboidal epithelium that overlies the optic vesicle. At this stage, the actin filaments are distributed along its apical-basal sides. The pre-placodal ectoderm, in contact with the optic vesicle, forms a pseudostratified tissue, the lens placode, with accumulation of actin network at the apical domain. Here, we focused on the role of the polarity protein PAR3 and its phosphorylation in the establishment of this apical actin network. Overexpression of PAR3 in the lens placode, induced formation of ectopic actin clusters in the basolateral membrane of the lens placode. The formation of these actin clusters, as well as recruitment of aPKC was independent of Threonine 833 phosphorylation at the PAR3 aPKC-binding site. In addition, PAR3 induced ectopic actin networks in the apical membrane of the periplacodal ectoderm independent of the Threonine 833 phosphorylation. Taken together, these data suggest that PAR3 is sufficient for actin recruitment in the lens placode.

No description available.