In the intestinal epithelium, Wnt signaling drives the expression of the genes encoding tyrosine kinase receptors EphB2 and EphB3 and represses the expression of their membrane-tethered ligands, ephrin-Bs. Eph-ephrin interactions result in cellular repulsion and are involved in boundary formation. The project of this thesis is to understand the mechanism by which EphB−ephrin-B signals restrict cell positioning of cell types (cell sorting) in the normal intestinal epithelium and suppress colorectal cancer (CRC) progression beyond the earliest stages. We have demonstrated that at the onset of CRC EphB receptors impair the expansion of tumor cells through a mechanism dependent on E-cadherin–mediated adhesion. We show that EphB-mediated compartmentalization restricts the spreading of EphB+ tumor cells into ephrin-B1+ territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1+ intestinal cells at the onset of tumorigenesis. We have discovered that cell sorting is the outcome of two integrated mechanisms: cell contraction/repulsion and differential cell adhesion. The latter is the driving force to induce EphB/ephrin-B−mediated cell compartmentalization. We have developed in vitro models to analyze the mechanisms that induce E-cadherin remodeling upon EphB activation. We found RhoA, p120-catenin and the metalloproteinase ADAM10 as downstream effectors of EphB signaling involved in the control of cell sorting in CRC cells. / A l'epiteli intestinal, la ruta de senyalització Wnt indueix l'expressió dels gens que codifiquen per als receptors tirosina kinasa EphB2 i EphB3 i reprimeixen la dels seus lligands transmembrana, efrines de tipus B. Les interaccions Eph-efrina causen repulsió cel·lular i estan implicades en la formació de fronteres entre compartiments. La finalitat d'aquesta tesi és entendre el mecanisme pel qual la senyalització per EphB−efrina-B restringeix el posicionament dels diferents tipus cel·lulars a l'epiteli intestinal normal i suprimeix la progressió del càncer colorectal (CRC) en els primer estadis. Hem demostrat que, a l’inici del CRC, els receptors EphB restringeixen l'expansió de les cèl·lules tumorals a través d'un mecanisme depenent d'adhesió intercel·lular a través d’E-cadherina. En aquest treball es mostra in vitro i in vivo que la compartimentalització mitjançada per la senyalització dels receptors EphB restringeix l’invasió de les cèl·lules tumorals EphB+ als territoris efrina-B+. Aquests resultats indiquen que les cèl·lules de CRC han de silenciar l’expressió d'EphB per evitar les interaccions repulsives imposades per les cèl·lules intestinals normals efrina-B+ circumdants al començament del procés de tumorigènesi. Hem pogut discernir que el reordenament cel·lular per senyals EphB−efrina-B és el resultat de dos mecanismes integrats: la contracció/repulsió intercel·lular i l’adhesió diferencial entre diferents poblacions cel·lulars. Aquesta última és la força principal que condueix a la compartimentalització cel·lular mitjançada per EphB−efrina-B. Hem desenvolupat models in vitro per analitzar els mecanismes que provoquen el remodelament de la E-cadherina sota la senyalització per EphB. Presentem RhoA, p120-catenina i ADAM10 com a efectors de la senyalització de la ruta EphB implicats en el control de la compartimentalització cel·lular en el CRC.

Pathological angiogenesis, or new blood vessel formation, is involved in many pathologies, including cancer and serious eye diseases. While traditional anti-angiogenic therapies target vascular endothelial growth factor receptors to reduce or inhibit new vessel formation, this approach has several downsides, including unpleasant side effects and low efficacy over time. Therefore, identifying new targets to treat pathological angiogenesis is still needed. CMG2, one of the two identified anthrax toxin receptors, has been proposed as an alternative target to treat pathological angiogenesis. CMG2’s role as a cell surface receptor that mediates anthrax toxin internalization is very well documented. One physiological function for CMG2, not related to anthrax intoxication, is suggested by the observation that loss-of-function mutations in CMG2 cause hyaline fibromatosis syndrome (HFS), a genetic disease that results in accumulations of extra-cellular matrix (ECM) protein in different parts of the body. While the complete molecular mechanism for CMG2’s role in regulating angiogenesis has not been determined, this dissertation addresses multiple ways CMG2 regulates pathological angiogenesis. We have discovered that CMG2 plays a role in mediating ECM homeostasis via endocytosis of ECM proteins and protein fragments as a way to generate angiogenic signals from the cell. We have also demonstrated that a fragment from Col IV, S16, is endocytosed into the cells by interacting with CMG2, and S16 treatment to endothelial cells leads to a significant reduction in cell migration. Also, an endothelial cell migration assay with CMG2 knockout cells results in abolished directional migration, indicating that CMG2 is required for endothelial cell chemotaxis. Notably, we have identified that bFGF, VEGF, and PDGF are involved in CMG2 mediated chemotaxis but not insulin and sphingosine-1-phosphate (S1P). While recent literature reports show that CMG2 works closely with RhoA GTPase, which is commonly known to regulate cell migration, we have also observed that inhibition of RhoA also reduced cell chemotaxis towards VEGF but not S1P. These results could be leveraged to develop new classes of therapeutic molecules to treat pathological angiogenesis induced by multiple various growth factors via targeting CMG2.

Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19249号 / 生博第342号 / 新制||生||46(附属図書館) / 32248 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 根岸 学, 教授 渡邊 直樹, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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La cytokinèse est un processus dont le but est une séparation de deux cellules soeurs en deux entités suite à une mitose. La cytokinèse nécessite la formation d’un anneau contractile (AC) qui va conduire un sillon de clivage vers une ingression à l’équateur de la cellule. L’une des étapes critiques de ce processus est la transition d’un AC dynamique vers une structure stable surnommée l’anneau du midbody (AM), organelle qui va guider la cellule vers l’abscision. La compréhension des mécanismes moléculaires impliqués dans cette transition nous permettrait de mieux comprendre les complexes protéiques impliqués autant au niveau de l’initiation qu’à la terminaison de la cytokinèse. Des défauts ayant lieu lors de cette transition mènent à la formation de cellules binucléées tétraploïdes qui sont observées dans plusieurs pathologies comme le cancer. Afin d’approfondir nos connaissances à ce sujet j’ai utilisé un modèle d’imagerie optique en temps réel dans un modèle cellulaire de Drosophila melanogaster : les cellules S2 de Schneider. Ces études ont mis l’emphase sur un nouveau mécanisme de maturation de la transition AC/AM. Nous avons pu démontrer que la kinase Citron, Sticky, et la septine, Peanut, agissent de manière opposée sur la protéine Anillin pour retenir ou éliminer, respectivement, la membrane plasmique lors de la transition AC/AM. En effet, la diminution d’expression de Sticky par ARNi engendre une perte de contrôle de rétention membranaire de l’AM. À l’inverse, la diminution d’expression de Peanut inhibe la maturation par excrétion membranaire de l’AM. La diminution d’expression simultanée de Sticky et de Peanut conduit l’AC vers des mouvements oscillatoires typiques d’une instabilité de l’AC suite à la perte de fonction de l’Anillin. Sticky est une protéine corticale lors de la cytokinèse dont le rôle et les partenaires d’interaction restent controversés. Pour approfondie nos connaissance de ce sujet, nous avons effectué une étude structurelle et fonctionnelle de Sticky. Cette étude démontre que Sticky possède deux mécanismes de localisation corticale. Le premier dépend de l’Anillin et le deuxième dépend de la petite GTPase Rho1, le régulateur maître de la cytokinèse. Sticky est capable de se localiser à l’AC en présence de l’un ou l’autre de ces deux mécanismes, mais chacun semble être essentiel pour la réussite de la cytokinèse. Le domaine minimal d’interaction entre la Sticky et l’Anillin a été identifié. Une version d’Anillin qui manque le site de liaison à la Sticky est incapable de supporter l’achèvement de la cytokinèse, et les cellules échouent la cytokinèse d’une manière semblable aux cellules dont l’expression de Sticky est diminuée. Similairement, les cellules exprimant une protéine Sticky mutée au site d’interaction avec Rho1-GTP, sont incapables de compléter la cytokinèse lorsque les niveaux endogènes de Sticky sont diminués par ARNi. Ceci suggère que Sticky agit avec Anillin et Rho1 au niveau du cortex pour guider la transition d’un AC dynamique vers un AM stable. Par la mise en évidence et la caractérisation d’un nouveau mécanisme moléculaire essentiel à la cytokinèse, cette thèse constitue des avancements importants au niveau de la cytokinèse. / Cytokinesis is a multistep process that allows two sister cells to undergo complete separation following mitosis. Cytokinesis requires the formation of a contractile ring (CR) that will drive cleavage furrow ingression at the equator of the cell. One of the crucial steps in this process is the transition from a dynamic CR to a more stable structure named the midbody ring (MR), which directs the final separation or abscission. Our knowledge of the molecular mechanisms involved in the CR-to-MR transition would presumably improve our understanding of the molecular complexes involved throughout cytokinesis from initiation to abscission. Defects that occur during this transition can lead to the formation of bi-nucleate tetraploid cells that are often observed in pathological conditions such as cancer. I have used Drosophila melanogaster Schneider’s S2 cells to study the CR-to-MR transition. My findings have highlighted a previously uncharacterized maturation process essential for the transition. More specifically, I demonstrate that the Citron Kinase, Sticky, and the Septin, Peanut, have opposing actions on the scaffold protein Anillin to either retain or extrude, respectively, membrane-positive proteins during the CR-to-MR transition. Indeed, Sticky depletion by RNAi led to uncontrolled loss of membrane-associated Anillin at the MR. Conversely, Peanut depletion led to inhibition of MR maturation by membrane extrusion. Co-depletion of Sticky and Peanut led to oscillatory movements of the CR, typical of Anillin depletion. Sticky is a cortical protein during cytokinesis whose role and interacting partners are controversial. I have performed a structure/function analysis of Sticky to better define its role and regulation during cytokinesis. My work shows that Sticky has two mechanisms of cortical localization. The first is through an Anillin interaction and the second is through the small GTPase Rho1, a master regulator of cytokinesis. Sticky can localize to the cortex in the absence of either one of these mechanisms. However, loss of both inhibits its localization. Following the identification of the minimal interaction sites of Anillin and Sticky, I expressed an Anillin mutant that lacked part of this site and found that cells failed cytokinesis in a similar manner to cells depleted of Sticky. Mutation of the Rho1 binding site on Sticky produced similar cytokinesis failures. Altogether, the results suggest that Sticky interacts with Anillin and Rho1 at the cortex to guide the transition from dynamic CR to stable MR. This thesis advances our understanding of cytokinesis by highlighting a previously uncharacterized process of MR maturation and by defining the importance and regulation of Citron Kinase during this process.

Multiple modulating genes and environmental factors have been implicated in the pathogenesis of high-altitude pulmonary edema (HAPE). However, at the present time, there exists an incomplete understanding of the molecular mechanisms and pathways which underlie constitutional susceptibility. Genome-wide measurements of gene expression in peripheral blood mononuclear cells (PBMCs) were performed using microarray technology. Comparison of gene expression profiles of HAPE-susceptible and resistant individuals resulted in the identification of several previously undescribed candidate genes. RhoA and Rho-kinase (ROCK), regulators of vascular smooth muscle contraction, were differentially regulated in the HAPE-susceptible cohort, as compared to both HAPE-resistant patients with acute mountain sickness (AMS+) and healthy controls (p=0.0014; p=0.0020). Furthermore, biological pathways involving RhoA and Rho-kinase were strongly upregulated in subjects with HAPE. These findings represent the first description of the RhoA/Rho-kinase signaling pathway in HAPE. Currently, few pharmacologic therapies have been demonstrated to be effective in the prevention and treatment of HAPE. The results of this study provide early evidence that Fasudil, a selective Rho-kinase inhibitor, may represent a novel therapeutic intervention effective in the prevention and/or treatment of high-altitude pulmonary edema.

The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development. / Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42. / The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure. / Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.