<p>Bud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.</p><p>Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.</p> / Dissertation

La polarité apico-basolatérale, qui est essentielle pour le maintien de l'architecture tissulaire et pour des fonctions de l'épithelium sain, est fréquemment fragilisée, voire perdue, dans des lésions prénéoplasiques et au cours de la tumorigénèse. Les patients atteints de l'hépatite C chronique ont un risque accru de développer des carcinomes hépatocellulaires. Le contexte inflammatoire et cirrhotique, caractéristique de l'infection chronique par le virus de l'hépatite C (VHC), constitue un facteur de risque important de la tumorigenèse. De plus, le virus exerce des effets directs sur la physiopathologie de la cellule hôte, qui semblent favoriser la transformation oncogénique. Mon travail de thèse a permit de développer et de caractériser un modèle de culture tridimensionnelle qui reproduit la morphologie complexe des hépatocytes pour ensuite étudier l'effet d'une proteine virale, NS5A sur la physiopathologie de cellules hépatiques dans un contexte tridimensionnelle. Des cellules hépatiques, immortalisées ou primaires, s'organisent en organoides polarisés et acquièrent l'expression de marqueurs hépatocytaires matures. L'organisation tridimensionnelle et la polarité des organoides influent sur des voies de signalisation intracellulaires, dont la voie PI3K/Akt, un acteur crucial de la physiologie de la cellule et de sa transformation. Dans notre modèle, l'activation constitutive de la kinase Akt perturbe l'organisation tridimensionnelle des hépatocytes. NS5A, une des protéines du VHC, active la voie PI3K/Akt et interfère avec l'intégrité des organoides. Dans des précurseurs hépatiques primaires, les BMELs l'expression de NS5A conduit à la perte de marqueurs épithéliaux et à l'acquisition de marqueurs mésenchymateux et au phénotype migratoire et invasif. Ces effets de NS5A, qui suggèrent une transition épithelio-mésenchymateuse (TEM), sont additifs à l'action du TGF-β, un inducteur connu de la TEM. / Apico-basolateral polarity is essential to maintain tissue architecture and function of healthy epithelium. It is weakened or lost in preneoplastic lesions and in the course of carcinogenesis. Patients suffering from chronic hepatitis C are at risk for hepatocellular carcinoma. In addition to the necroinflammatory liver microenvironment, which favours tumorigenesis, direct effects of the virus on its host cell physiopathology also participate in the initiation of oncogenic transformation.To study the effects of viral proteins on cellular polarity and function, we developped an in vitro threedimensional (3D) culture model that reproduces complex hepatocyte morphology. Both immortalized cells and primary hepatocyte precursors organise themselves into intricate 3D organoids and acquire markers of mature hepatocytes. Their organisation and polarity impacts on several intracellular signal transduction pathways, including the PI3K/Akt axis, a major actor of physiology and of oncogenic transformation. Interestingly, constitutive Akt signalling perturbs hepatocyte 3D organization.NS5A, an HCV viral protein with pleiotropic activities, is an upstream activator of Akt. NS5A expression interferes with organoid formation and integrity. In primary hepatocyte precursors it leads to downregulation of epithelial and to acquisition of mesenchymal markers, suggesting an induction of epithelial-to-mesenchymal transition (EMT). Moreover, the effects of NS5A are additive to that of TGF-beta, a bona fide EMT inducer relevant to HCV-related pathologies. NS5A is also inducing pro-migratory and invasive phenotypes in BMEL cells as well as the hepatic cell line AML12. The signaling pahways underlying this results might involve Twist factors, well known actors of EMT, as NS5A is capable of their transcriptional regulation.The molecular mechanisms linking viral proteins to alterations of hepatocyte morphology are under investigation. Alterations of cell shape and function are of major interest in the context of virus-induced phenotype, relevant to tumour initiation and progression.

La polarité planaire ou PCP est un processus dans lequel les cellules et des structures apicales sedéveloppent et s’orientent dans le plan de l’épithélium. Cette polarité est régulée par un ensemble deprotéines incluant Vangl2. Un mutant murin de Vangl2 nommé looptail présente une forme sévèred’absence de fermeture du tube neural. Des pertes de fonctions de Vangl2 sont également retrouvées chezdes enfants atteints de défaut de fermeture du tube neural. Vangl2 possède un motif d’interaction pour desdomaines PDZ et s’organise en protéine à quatre domaines transmembranaires. Malgré de nombreusesdonnées génétiques disponibles concernant la perte de fonction de Vangl2, beaucoup reste à faire pourélucider les mécanismes moléculaires impliqués. Le locus du gène vangl2 humain est localisé dans unerégion fréquemment réarrangée dans différents cancers incluant les cancers du sein. Nous nous sommesintéressés à l’expression de Vangl2 dans les cancers du sein. Nous montrons que Vangl2 est surexprimédans des cancers du sein de sous-type basal (cancers du sein agressifs et de mauvais pronostic) danslesquels le locus génétique de Vangl2 est significativement amplifié et où le niveau d’expression del’ARNm et de la protéine est également élevé. De plus, par des études in vitro et in vivo, nous démontronsle rôle de Vangl2 dans la croissance tumorale et dans les processus de migration cellulaire. Ces effets sontdépendants de son motif d’interaction aux domaines PDZ et sont dépendants de Scrib, une autre protéinede la PCP, étudiée dans l’équipe et impliquée dans les processus de migration cellulaire. Par desexpériences de gain et de perte de fonction dans des cellules de cancers du sein, nous démontrons quel’expression de Vangl2 régule la signalisation JNK via l’activité des GTPases Rac1 et Cdc42 importantespour les processus de réorganisation du cytosquelette d’actine. L’ensemble de nos données indique un rôlerégulateur de Vangl2 dans les processus de tumorigenèse l’impliquant dans la croissance tumorale, laprolifération et migration cellulaires. / Planar cell polarity (PCP) is a process by which cells and apical structures grow in a uniformorientation within the plane of the epithelium. This type of polarity is regulated by a set of “core” proteinsincluding Vangl2. A vangl2 mutant, known as looptail exhibits a severe form of neural tube defect.Mutations of vangl2 associated to neural tube defects were also described in human. Vangl2 possesses aPDZ binding motif and is potentially organized in a four transmembrane domain structure. While geneticdata has very well described the importance of Vangl2 in embryonic development, its molecular functionsare still unknown. The human vangl2 gene is localized in a region with frequent rearrangements involvedin multiple cancers, including breast cancer. We decided to explore the expression of Vangl2 in breastcancer and defined Vangl2 as a PCP “core” protein associated with a signature of poor prognosis basaltypebreast cancer. The Vangl2 genetic locus is amplified in breast cancers, and this amplificationcorrelates with high mRNA and protein levels. Moreover, in in vitro and in vivo studies, we demonstratethe role of Vangl2 in tumor growth and cell migration. These functions are dependent on its PDZ bindingmotif and implicates Scrib, a PCP protein containing PDZ domains and involved in cell migration. Finally,using gain-of-function and loss-of-function approaches in breast cancer cell lines, we demonstrate thatVangl2 modulates the JNK pathway activation via the Rac1 and Cdc42 GTPases which are important forcytoskeleton reorganization. Together, our data reveal that Vangl2 has a role in tumor growth, cellproliferation and cell migration.

The development of organs such as the lung and kidney occurs by branching morphogenesis. Changes in the cytoskeletal architecture, cell-cell adhesion and cell polarity are necessary for the formation of new branches. Interactions and reciprocal signalling between epithelial and mesenchymal cells mediate these organised cell movements that give rise to a complex system of tubes suitable for the transport of gas or fluids. Mutations that disrupt formation of either the correct number, or shape of epithelial branches, affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signaling pathways that drive epithelial tube formation will likely shed light on both congenital and adult lung disease. This study shows that mutations in the planar cell polarity (PCP) genes: Celsr1; Vangl2 and Scribble, lead to disrupted lung development and defects in lung architecture. Examination of Vangl2 mutant kidneys reveals similar impairment of branching morphogenesis. Detailed histological and immunocytochemical analysis reveals that lungs from Celsr1Crsh/Crsh, Vangl2Lp/Lp and ScribbleCrc/Crc mice are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. Moreover, epithelial integrity is disrupted, cytoskeletal remodeling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. In ex-vivo culture, inhibition of Rho kinase, an important downstream effector of the PCP signaling pathway, can mimic the branching defects observed in these three mouse mutants. Furthermore, all three proteins are present in restricted spatial domains within lung epithelium. ScribbleCrc/Crc lungs, the most severely affected line, exhibit additional defects in components of the tight and adherens junctions; this in turn affects lumen diameter. These findings show that components of the PCP pathway: Celsr1; Vangl2 and Scribble are required for normal foetal lung development, thereby revealing a novel signalling pathway critical for this process. Examination of postnatal mice was not possible as homozygous mutations result in embryonic lethality. However, an assessment of Vangl2Lp/+ mice reveals that loss of a single copy of Vangl2 is enough to cause defects in embryonic lung development that persist into adult life, affecting lung function. Similarly, Vangl2Lp/+ mice show a small but significant reduction in kidney glomeruli.

ASPP2 has been identified as a haploinsufficient tumour suppressor in mice, and an activator of the apoptotic function of the p53 family. Yeast two-hybrid experiments have also shown that ASPP2 interacts with a large number of proteins involved in other major signalling pathways. The mechanism(s) of action of ASPP2 are therefore complex, and likely to involve more than just the stimulation of the apoptotic programme. Since a study previously conducted in our laboratory revealed that the deletion of ASPP2 in vivo leads to severe hydrocephalus in the J129/C57BL6 background (Vives et al., 2006), it can be hypothesised that ASPP2 safeguards the normal development of the mammalian central nervous system (CNS), in addition to its role as a tumour suppressor. Deletion of ASPP2 leads to the development of hydrocephalus, most probably by affecting tight junctions (TJs) in the choroid plexus, thereby impairing its blood-cerebrospinal fluid (CSF) barrier function. TJ defects are likely to be the underlying cause of the loss of cell polarity observed in the neuroepithelium of several areas of the CNS. As cell polarity plays a key role in multiple aspects of CNS development, ASPP2 appears to be required for the proper lamination of the cerebral cortex and retina.

Planar cell polarity (PCP) is a developmental signaling mechanism that establishes a polarity within the plane of an epithelium. PCP has been shown to play a role in guiding numerous neurodevelopmental processes such as convergent extension, neuron migration, and axon pathfinding. Certain commissural neurons in the dorsal spinal cord make a series of guidance decisions en route to the brain: first, a ventral projection along the D-V axis, followed by a midline crossing, and after exiting the floorplate, a dorso-anterior turn along the A-P axis. Here, we provide in vivo evidence that the axons of the Commissural Primary Ascending (CoPAs) neurons in zebrafish require the PCP genes fzd3a, vangl2, and scribble for rostral pathfinding both before and after crossing the midline. Dorsoventral guidance of CoPA axons is unaltered in fzd3a, vangl2, and scribble mutants, suggesting that the PCP signaling pathway only controls A-P guidance of CoPAs. Our results have provided evidence for two potential non- mutually exclusive models: (i) A-P axon guidance is achieved by cell-autonomous Wnt-Frizzled signaling or that (ii) A-P axon guidance is achieved by non-cell-autonomous PCP signaling in the neuroepithelial environment. The single-cell nature of the CoPA axon system allows for simple genetic manipulation and visualization, which will potentially elucidate the validity of either model. Scribble (Scrib), a member of the LAP family, plays a critical role in establishing and regulating cell polarization in epithelia and during cell migration. In zebrafish, Scrib mutants have defects in convergent extension (CE) cell movements and facial branchiomotor neuron (FBMN) migration. Despite our understanding of Scrib’s genetic role in neurodevelopment, little is known about the subcellular localization of endogenous Scrib in vivo during CE and FBMN migration. We have generated a monoclonal antibody against the C-terminus of zebrafish Scrib and have shown that this antibody is specific against endogenous Scrib in both western blot and immunocytochemical applications. Confocal microscopy of Scrib immunocytochemistry shows that at various developmental stages, Scrib distinctly localizes to basolateral membranes of non polarized epithelium, to the membrane in mesodermal cells undergoing CE, and to the membrane of migrating FBMNs. Furthermore, the distribution of Scrib puncta along membranes of FBMN- FBMN contact is significantly altered in the PCP mutant pk1b. Further application of our newly generated Scrib antibody will potentially lead to new insight on Scrib’s role in neurodevelopment.

Extracellular signal-regulated kinases (ERKs) modulate cellular activities in response to extracellular stimuli and play important biological roles. Thus, perturbed kinase pathways induce pathological conditions, such as tumor development. Rit, a novel member of the Ras family GTPases, activase ERK6, and its over-expression confers tumorigenicity. We hypothesized the presence of scaffolding molecules specific to ERK6, similar to other known MAP kinases. We performed yeast two-hybrid assays using ERK6 as bait, and Scribble was identified as a binding partner. Scribble contains 16 LRR domains and four PDZ domains. We performed immunoprecipitation (IP) assays and discovered ERK2 as another binding partner. Surprisingly, no interaction was observed with the highly homologous MAP kinase, ERK1. No other representative kinases showed binding capabilities with Scribble. IP data confirmed that both ERK2 and ERK6 bind to Scribble through its LRR and PDZ domains. Deletion of ten aminoi acids from the C-terminus of ERK2 and ERK6 abolished these interactions. In vitro kinase assays indicated the kinase suppressing ability of Scribble. Focus formation assays were performed with RitQ79L and H-RasV12 as constitutive activators of ERK6 and ERK2, respectively, in the presence of Scribble. Results confirmed the role of Scribble as a tumor suppressor.

In zebrafish, CoPA (Commissural Primary Ascending) is the first among ascending commissural axons to pathfind anteriorly and form the spinal commissure. One pathway that guides their anterior growth is the planar cell polarity (PCP) signaling pathway, but it is not fully known how PCP signaling regulates anterior guidance. We examined CoPA pathfinding in various PCP mutants to determine if anterior-posterior (A-P) guidance of CoPAs is dependent on PCP signaling. We found that certain PCP mutants exhibited anterior pathfinding defects, with approximately half of all affected CoPAs migrating incorrectly posteriorly. By using a translation-blocking DCC (Deleted in Colorectal Cancer) morpholino to prevent CoPA midline crossing, we discovered that CoPA axons in Fzd3a and Scribble mutants show severe defects in A-P guidance, which suggest that PCP influences A-P guidance of CoPAs prior to and after midline crossing.

Le cycle cellulaire eucaryote est caractérisé par des changements abrupts et dynamiques de la polarité cellulaire lorsque les chromosomes sont dupliqués et ségrégés. Ces évènements nécessitent une coordination entre la machinerie du cycle cellulaire et les régulateurs de la polarité. Les mécanismes qui contrôlent cette coordination ne sont pas totalement compris. Dans la levure S. cerevisiae, comme dans d’autres organismes eucaryotes, la GTPase Cdc42 joue un rôle important dans la régulation de la polarité cellulaire. En effet ses régulateurs constituent un module GTPase qui subit une phosphorylation dynamique, au cours du cycle cellulaire, par des kinases évolutivement conservées dont la Cycline-Dependent Kinase 1 (Cdk1) et la p21-Activated Kinase (PAK). Ces kinases et substrats pourraient relier la polarité et la progression dans le cycle cellulaire. En utilisant une approche in vitro, nous avons reconstitué la phospho-régulation du Guanine nucléotide Exchange Factor (GEF) de Cdc42, la protéine Cdc24. Nous avons identifié un possible mécanisme de régulation de la phosphorylation impliquant une protéine d’échafaudage qui augmente la phosphorylation de Cdc24 par la PAK et Cdk1. Cette phosphorylation accroit modérément l’affinité de Cdc24 pour cette même protéine d’échafaudage, Bem1. De plus, en testant les effets d’autres composants du module GTPase sur la phosphorylation de Cdc24, nous avons identifié un effet antagoniste pour une GTPase Activating Protein (GAP), Rga2. Cette protéine est présente dans le même complexe que Cdc24 et Bem1, les membres de ce complexe sont tous phosphorylés par Cdk1. Des mutants rga2 suggèrent que la phosphorylation que subie Rga2 inhibe son activité GAP. Nous proposons un modèle provisoire pour expliquer la présence de Rga2 dans ce complexe et l’inhibition qu’elle oppose à la phosphorylation de Cdc24. La présence de la protéine GAP dans le complexe pourrait être un mécanisme de contrôle de la phosphorylation de Cdc24 dans le but de déstabiliser son intéraction avec la protéine Bem1 en cas de mauvaise localisation du complexe. Par ailleurs, la PAK est activée par l’activité de Cdc42, nos résultats sont consistants avec un modèle dans lequel des signaux du cycle cellulaire engendreraient une auto-amplification de l’activation du module GTPase. Chez S. pombe, la croissance polarisée nécessite un gradient d’activation de Cdc42 dû à une ségrégation de GEF et de GAP. Dans ces travaux nous montrons que toutes les protéines GAPs de Cdc42 localisent aux sites de croissance au cours du cycle cellulaire. Ces localisations sont consistantes avec le besoin de cyclage de Cdc42 pour maintenir sa polarisation. Ces résultats suggèrent que la localisation des protéines GAP régulant Cdc42 chez S. cerevisiae semble différente de ce qui est connu chez S. pombe. / The eukaryotic cell cycle is characterized by abrupt and dynamic changes in cellular polarity as chromosomes are duplicated and segregated. Those dramatic cellular events require coordination between the cell cycle machinery and polarity regulators. The mechanisms underlying this coordination are not well understood. In the yeast S. cerevisiae, as in other eukaryotes, the GTPase Cdc42 plays an important role in the regulation of cell polarity. Cdc42 regulators constitute a GTPase module that undergoes dynamic phosphorylation during the cell cycle by conserved kinases including Cyclin-Dependent Kinase 1 (Cdk1) and p21-activated kinase (PAK). These kinases and substrates may link cell polarity to the cell cycle progression. Using in vitro approaches, we have reconstituted the phospho-regulation of the Cdc42 Guanine Nucleotide Exchange Factor (GEF), Cdc24. We have identified a possible mechanism of Cdc24 regulation involving a scaffold-dependent increase in Cdc24 phosphorylation by Pak and Cdk1. This phosphorylation moderately increases the affinity of Cdc24 for another GTPase module component, the scaffold Bem1. Moreover, by testing the effect of other GTPase module components on the phosphorylation of Cdc24, and thus on its interaction with the scaffold, we identified an antagonistic function for the GTPase Activating Protein (GAP) Rga2. Our in vivo data of rga2 mutants suggest that Rga2 phosphorylation by Cdk1 inhibits its GAP activity. We propose a tentative model to explain the inhibition of Cla4 by Rga2 and its presence in a complex containing Cdc24 and Bem1. The presence of the GAP protein in the complex may be a mechanism that reduces Cdc24 phosphorylation in case of a mistargetting of the complex in order to downregulate the GEF/Scaffold dimer. Since the PAK component of the GTPase module is itself activated by Cdc42 activity, our results are consistent with a model in which inputs from the cell cycle lead to auto-amplification of the Cdc42 GTPase module. In S. pombe, polarised growth requires a gradient of activation of Cdc42 due to GEF and GAP segregation. Here we show that all Cdc42 GAPs localise to the polarised site during the cell cycle. Those localisations are consistent with a requirement of Cdc42 cycling to maintain a polarity cap. Our results may suggest that Cdc42 GAPs localisations in S. cerevisiae are different from current knowledge in S. pombe.

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.