Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen. Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt. In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet. In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine. Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden. Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam. Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen. / Evidence exists that cAMP stabilizes the endothelial barrier in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2´-O-methyladenosine-3´,5´-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (Epac/Rap1) signaling enhanced transendothelial electrical resistance (TER) and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin5. Ultrastructural analysis of the intercellular contact zone morphology documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contacts displaying complex interdigitations of cell borders in which membranes of neighboring cells were closely apposed over comparatively long distances and which were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP whereas F/R-mediated barrier enhancement was not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement and contact zone reorganization. These results indicate that in microvascular endothelium Rac1-mediated alterations in contact zone architecture contributes to cAMP-induced barrier stabilization.

Most patients undergoing androgen deprivation therapy relapse eventually and progress to androgen-independent (AI) prostate cancer. Although the mechanisms underlying progression to AI prostate cancer are not well understood, studies suggest that androgen receptor (AR) is still required for AI prostate cancer. Our lab found that Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is up-regulated during the progression of androgen-dependent human prostate cancer cells to androgen-independence in vivo and in cell-based experiments. Since Vav3 significantly increases ligand-dependent AR transcriptional activity and this action requires the Vav3 pleckstrin homology (PH) domain but not Vav3 GEF activity, we explored the role of the Vav3 PH domain in ligand-dependent AR coactivation by Vav3. We found that targeting the Vav3 PH mutant into nuclei but not the plasma membrane restored Vav3 PH mutant in AR coactivation. Targeting Vav3 to the plasma membrane eliminated the capacity of Vav3 to coactivate AR. In agreement with nuclear targeting of Vav3 via its PH domain, chromatin immunoprecipitation assays showed that Vav3 enhancement of AR transcriptional activity was accompanied by Vav3 recruitment to AR transcriptional complexes at an AR target gene enhancer. Further, Vav3 increased AR occupancy at the target gene enhancer upon androgen treatment and this may underlie the capacity of Vav3 to enhance AR transcriptional activity. Because Vav3 can also be activated by growth factors (GFs) and GFs activate AR in the absence of androgen (ligand-independent), we investigated the crosstalk between Vav3 and GF activation of AR and found Vav3 strongly enhanced AR transcriptional activity induced by GFs. GEF function and the downstream Rho GTPase, Rac1 were required for constitutively active (Ca) Vav3 activation of AR, which differs from Vav3 activation of AR in the presence of androgen. We also investigated the possible signal pathways contributing to AR activation by Ca Rac1. Ca Rac1 caused ligand-independent activation of AR in part through MAPK/ERK signaling and conferred prostate cancer growth in the absence of androgen in cell culture, soft agar and mouse tumor xenografts. Thus, our findings indicate that Vav3 activates AR in the presence or absence of ligand through two distinct mechanisms, which supports a versatile regulatory effect of Vav3 in AR signaling and prostate cancer progression.

La dendritogenèse et la spinogenèse sont des étapes clés du développement neuronal. Elles impliquent de nombreuses protéines jouant un rôle essentiel dans la réorganisation du cytosquelette d'actine via les RhoGTPases. Des défauts dans ces processus peuvent mener à des maladies neurodéveloppementales comme l'autisme ou la schizophrénie. Les récepteurs BAI appartiennent à la famille des RCPG d'Adhésion et ont été identifiés dans des préparations biochimiques de densités postsynaptiques. BAI1 module la RhoGTPase Rac1 via son interaction avec la protéine ELMO1. De plus, les protéines sécrétées C1q-like ont récemment été identifiées comme ligands du récepteur BAI3 in vitro et cette interaction régulerait la synaptogenèse. Nous avons émis l'hypothèse que le récepteur BAI3 pourrait réguler le développement neuronal, en particulier la dendritogenèse et la spinogenèse, en interagissant avec ELMO1. Nos travaux ont montré que BAI3 est localisé dans les dendrites, et chez les neurones matures dans les épines dendritiques. Des études morphométriques nous ont permis de montrer son rôle dans la croissance et la complexification de l'arbre dendritique des neurones in vitro. Nos données in vivo sont en accord avec un rôle du récepteur BAI3 dans la morphogenèse des cellules de Purkinje du cervelet et la mise en place de leur innervation excitatrice. Le rôle de BAI3 dans la morphogenèse dendritique semble dépendre en partie de son interaction avec ELMO1. Par ailleurs, BAI3 module l'étalement cellulaire, suggérant son implication dans la régulation des RhoGTPases. L'ensemble de nos résultats met en lumière un nouveau rôle des récepteurs BAI comme régulateurs de la dendritogenèse et de la formation des synapses, en partie via la voie de signalisation ELMO1/Rac1. Nos résultats identifient les récepteurs BAI comme de nouveaux acteurs de la morphogenèse neuronale et, au vu du lien génétique existant entre BAI3 et certains symptômes de la schizophrénie, offrent de nouvelles perspectives dans l'étude des maladies neurodéveloppementales.

Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21815号 / 農博第2328号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5187(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 矢﨑 一史, 教授 植田 充美 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

L'adhérence cellulaire à la matrice extracellulaire joue un rôle clé dans leur prolifération,leur différenciation ou l'apoptose. Par conséquent ce processus est critique pour undéveloppement normal et pour l'homéostasie tissulaire. La dérégulation de ce mécanismecontribue souvent à des situations pathologiques. Ainsi, la dérégulation de nombreux gènesimpliqués dans les adhérences cellule-cellule ou cellule-matrice extracellulaire sont liés à despathologies conduisant à un défaut de développement, la progression tumorale, oul'inflammation.Les intégrines sont des récepteurs transmembranaires hétéro dimériques jouant un rôlemajeur dans les interactions cellule-matrice extracellulaire. Ce rôle n'est pas limité à unesimple interaction mécanique puisqu'elles permettent également la transduction dessignaux de la matrice extracellulaire à la cellule afin de permettre à cette dernière des'adapter à son micro environnement. Dans le but d’étudier le rôle des intégrines à chaîneβ1 dans le développement osseux, le laboratoire a mis en place un modèle murind'inactivation conditionnelle du gène Itgb1 basée sur l'expression de la recombinase Cre austade pré-ostéoblastique. Les souris mutées présentent un défaut de développementosseux, dû à une faible prolifération des ostéoblastes.Contrairement à ce qui était généralement admis, cette faible prolifération desostéoblastes est indépendante de la voie classique mettant en jeu la voie classique des MAPkinases. En revanche, elle est contrôlée par la voie Hippo: cette signalisation a étérécemment identifiée chez la Drosophile et les Mammifères comme un mécanismeinhibiteur majeur de la prolifération cellulaire. Le cofacteur de transcription YAP, effecteurfinal de cette voie, est une navette nucléo-cytoplasmique. Son expression est amplifiée dansdivers cancers dont l'ostéosarcome où cette surexpression associée à celle de l’Itgb1 est unfacteur de mauvais pronostique.Mes travaux consistent à comprendre comment les intégrines à chaîne β1 contrôlent lavoie Hippo, et donc la prolifération. Nous avons confirmé que la délétion des intégrines β16active la phosphorylation de YAP et sa séquestration dans le cytoplasme. En utilisant destechniques de Biologie Cellulaire et de Biochimie, nous avons montré que suite à la délétionde l’Itgb1, les cellules présentent un défaut de trafic vésiculaire réduisant la translocationmembranaire de Rac1. La séquestration cytoplasmique de Rac1 diminue l’activation de soneffecteur majeur la kinase PAK responsable de la dissociation d'un complexe membranaired'inactivation composé de la protéine adaptatrice NF2, la kinase LATS et de son effecteurprincipal YAP. Les intégrines en provocant la perte de ce complexe induisent ladéphosphorylation de YAP, sa translocation nucléaire et donc stimulent la proliférationcellulaire. / Cell adhesion to the extracellular matrix plays a key role in their proliferation,differentiation or apoptosis. Therefore, this process is critical for normal development andtissue homeostasis. The deregulation of this mechanism often contributes to pathologicalsituations. Thus, the deregulation of many genes involved in cell-cell or cell-extracellularmatrix adhesions are linked to pathologies leading to developmental defects, tumorprogression, or inflammation.Integrins are heterodimeric transmembrane receptors that play a major role in cellextracellularmatrix interactions. This role is not limited to a simple mechanical interactionsince integrins also allow the transduction of the signals from the extracellular matrix to thecell in order to permit the latter to adapt to its microenvironment. In order to study the roleof β1 integrins in bone development, the laboratory has implemented a mouse model withconditional inactivation of the Itgb1 gene based on the expression of recombinase Cre at thepre-osteoblastic stage. The mutated mice show a defect in bone development due to a lowproliferation rate of osteoblasts.Contrary to what was generally accepted, this reduced proliferation is independent of theclassical pathway involving the classical pathway of MAP kinases. On the other hand, it iscontrolled by Hippo: this signaling pathway has recently been identified in Drosophila andMammals as a major inhibitory mechanism of cell proliferation. The transcription cofactorYAP, the end effector of this pathway, is a nucleo-cytoplasmic shuttle. Its expression isamplified in various cancers including osteosarcoma where this overexpression associatedwith that of Itgb1 is a factor of poor prognosis.My work involves understanding how β1 integrins control the Hippo pathway, and thusproliferation. We confirmed that deletion of β1 integrins activates the phosphorylation ofYAP and its sequestration in the cytoplasm. Using Cell Biology and Biochemistry techniques,we showed that following the deletion of Itgb1, the cells exhibit a defect in vesicular trafficthat reduces the membrane translocation of Rac1. The cytoplasmic sequestration of Rac18decreases the activation of its major effector, the PAK kinase. PAK is responsible for thedissociation of an inactivating membrane complex composed of the adaptor protein NF2,the LATS kinase, and its main effector YAP. The integrins by provoking the loss of thiscomplex induce the dephosphorylation of YAP, its nuclear translocation, and thus stimulatecell proliferation.

As espécies reativas de oxigênio são capazes de influenciar a secreção de insulina, porém ainda não está clara a influência da glicose, principal secretagogo deste hormônio, sobre a homeostase redox das células beta pancreáticas. Incubações por 1 e 48 horas com diferentes concentrações de glicose (2,8; 5,6; 8,3; 11,1; 16,7 e 20 mM) demonstraram que esta é capaz de alterar não só o conteúdo de superóxido, produzido pela mitocôndria e NADPH oxidase, mas também o sistema antioxidante, alterando a concentração de GSH e a expressão das enzimas antioxidantes. Além disso, aumenta a interação Rac1/Sod1, que mantém a NADPH oxidase ativa. Porém, não apresenta endossomas de sinalização redox, os redoxossomas, em resposta a glicose. Estas alterações podem afetar eventos chave para este tecido endócrino, como a secreção de insulina e a morte celular. / ROS production in pancreatic beta cells has been associated with the insulin secretion process but the mechanism by which glucose affects the redox state in these cells remains unknown. In order to address this issue, we evaluated the effect of 1 or 48 hours incubation of pancreatic beta cells with various glucose concentrations (2.8, 5.6, 8.3, 11.1, 16.7 and 20 mM). Glucose loading induced superoxide production by mitochondria and NADPH oxidase complex, and enhanced the antioxidant capacity by increasing GSH content and modulate expression of antioxidant enzymes. Glucose also promoted Rac1/Sod1 interaction that maintains NADPH oxidase activated. These cells however did not present redox endosomes, the redoxosomes, in response to glucose loading. These effects might be associated with the process of insulin secretion and pancreatic beta cell death.

As espécies reativas de oxigênio são capazes de influenciar a secreção de insulina, porém ainda não está clara a influência da glicose, principal secretagogo deste hormônio, sobre a homeostase redox das células beta pancreáticas. Incubações por 1 e 48 horas com diferentes concentrações de glicose (2,8; 5,6; 8,3; 11,1; 16,7 e 20 mM) demonstraram que esta é capaz de alterar não só o conteúdo de superóxido, produzido pela mitocôndria e NADPH oxidase, mas também o sistema antioxidante, alterando a concentração de GSH e a expressão das enzimas antioxidantes. Além disso, aumenta a interação Rac1/Sod1, que mantém a NADPH oxidase ativa. Porém, não apresenta endossomas de sinalização redox, os redoxossomas, em resposta a glicose. Estas alterações podem afetar eventos chave para este tecido endócrino, como a secreção de insulina e a morte celular. / ROS production in pancreatic beta cells has been associated with the insulin secretion process but the mechanism by which glucose affects the redox state in these cells remains unknown. In order to address this issue, we evaluated the effect of 1 or 48 hours incubation of pancreatic beta cells with various glucose concentrations (2.8, 5.6, 8.3, 11.1, 16.7 and 20 mM). Glucose loading induced superoxide production by mitochondria and NADPH oxidase complex, and enhanced the antioxidant capacity by increasing GSH content and modulate expression of antioxidant enzymes. Glucose also promoted Rac1/Sod1 interaction that maintains NADPH oxidase activated. These cells however did not present redox endosomes, the redoxosomes, in response to glucose loading. These effects might be associated with the process of insulin secretion and pancreatic beta cell death.

La motilité cellulaire est un processus intégré essentiel à de nombreux phénomènes physiologiques tels que la formation du cône de croissance et la plasticité synaptique. Des dérégulations de la motilité cellulaire peuvent être à l’origine de la formation de métastases ou de pathologies neuropsychiatriques comme la schizophrénie et l'autisme. La compréhension des mécanismes régulant la migration cellulaire est donc un enjeu majeur. La motilité cellulaire repose sur la formation de diverses structures constituées de réseaux d’actine branchés telles que le lamellipode. La formation du lamellipode nécessite l’intervention de protéines régulatrices de l’actine telles que Rac1 et les complexes Wave et Arp2/3. Grâce à l’utilisation de suivi de protéine unique, nous avons pu comprendre comment la coordination spatio-temporelle de ces régulateurs contrôle la formation et la morphologie des lamellipodes de cellules migrantes. Nous avons ainsi découvert que l’activation et la localisation du complexe Wave étaient régulées de manière enzymatique mais également mécanique. Dans une première étude, nous avons montré que la RhoGTPase Rac1 active le complexe Wave spécifiquement à l’extrémité du lamellipode. Dans une seconde étude, nous avons révélé que la localisation du complexe Wave est régulée par la dynamique des filaments des réseaux branchés d’actine. Ces données soulignent l’importance du complexe Wave dans la formation du lamellipode et révèlent l’existence d’une régulation mécanique de la localisation du complexe Wave. / Cell motility is an integrated process involved in critical phenomena such as axonal pathfinding and synaptic plasticity. Dysregulation of cell motility can induce metastasis and abnormal spine shapes observed in neuropsychiatric disorders like autism and schizophrenia. Therefore it is essential to understand how cell motility is regulated. Cell motility requires the formation of branched actin networks propelled by actin polymerization that lead to the formation of membrane protrusions such as the lamellipodium. Several actin regulatory proteins are involved in this process, such as Rac1 and the WAVE and ARP2/3 complexes. Using single protein tracking, we revealed key phenomena concerning the spatio-temporal regulation of lamellipodium formation by actin regulatory proteins. We found that the localization and activation of the WAVE complex was enzymatically regulated, but also mechanically. First, we showed that the Rac1 RhoGTPase activates the WAVE complex specifically at the tip of the lamellipodium. We also showed that WAVE complex localization is regulated by the dynamics of branched-network actin filaments. This study confirms the crucial role of the WAVE complex in lamellipodium formation and reveals the existence of a mechanical regulation of the localization of this complex in the cell.

The metanephric kidney consists of two types of epithelia; the Wolffian duct-derived ureteric bud and the nephrogenic components that originate from mesenchymal-toepithelial transitions in the metanephric mesenchyme. The ureteric bud forms when inductive signals from the metanephric mesenchyme stimulates the evagination of an epithelial tube from the Wolffian duct into the mesenchyme. Reciprocal signalling between the ureteric bud and the metanephric mesenchyme regulates the branching of the ureteric bud and the induction of nephron formation. Inductive and inhibitory signalling of ureteric bud growth and branching has been shown by several protein families, however, the mechanical aspects of ureteric bud branching and nephrogenesis are largely unknown. I investigated the roles of Rac1-GTPase and Rho-kinase during kidney development. These proteins are important regulators of the cytoskeleton where Rac1 is a promoter of actin filament polymerisation and Rho-kinase directly stimulates the formation and contraction of actin-myosin stress fibres. Using a cell-permeable inhibitor, Rac1 was inhibited with no effects on nephron formation or subsequent segmentation and patterning. Inhibition of active Rac1 significantly reduced the level of ureteric bud branching and also resulted in lower proliferation rates. Rho-kinase was similarly targeted using two inhibitors. Rho-kinase inhibition had important effects on nephron formation and nephron maturation. Inhibition of Rhokinase resulted in decreased levels of nephron formation and severely morphologically abnormal nephrons. The formation of apical-basal polarity was disturbed as was the development of the visceral and parietal epithelia; precursors of the renal corpuscle. Inhibition of Rho-kinase led to abnormal formation of the proximal-distal axis and abnormal segmentation of the nephron. The effects of Rho-kinase inhibition were partially mimicked by direct targeting of actin-myosin contractions using a myosin-ATPase inhibitor. This demonstrated that Rho-kinase is necessary during multiple stages of nephrogenesis and maturation, at least in part, as a result of its ability to regulate actin-myosin contraction. These results show that Rac1 and Rho-kinase play important roles during several aspects of kidney development and highlights the significance of further investigating the mechanisms involved during kidney organogenesis.