Chemotaxis, the ability of a cell to change its motility as a response to environmental stimuli, belongs to the most important signal transduction mechanisms in life. Evolution has created a plethora of different molecular mechanisms to enable cells to react appropriately to extracellular changes. In this thesis, chemotactic signal transduction pathways in bacteria, in particular two component signalling in R. sphaeroides, and eukaryotes, in particular human axon guidance, are studied by means of X-ray crystallography complemented with biophysical, biochemical and cellular studies. Two-component signal transduction in bacteria is one of the most abundant signalling pathways in biology. Here the determinants for specificity for a crucial sensor kinase-response regulator interaction are presented and used to design novel, synthetic two-component pairs. The activation mechanism of response regulators has been extensively studied and a model for activation that crucially involves a Thr and a Tyr residue has been widely accepted in the field. In this thesis, two structures of a response regulator from R. sphaeroides are presented and together with biophysical and cellular assays suggest a novel paradigm for response regulator activation. Axon guidance is an essential process in human development and relies crucially on chemotaxis. Two signalling pathways, the plexin-semaphorin and the Rgm-Neogenin pathway are studied extensively in this work. Structures of the intracellular region of Plexin-B1 provide an elegant mechanism explaining how ligand binding events on the extracellular and intracellular side can be integrated into a single signalling output. The study of RgmB in complex with its receptor Neogenin provides the first structural insight into the important family of repulsive guidance molecules and explains their role in the disease juvenile hemachromatosis. In summary, this work provides insights into a plethora of chemotactic pathways and extends our current knowledge of these important mechanisms significantly.

Many signaling pathways have been shown to be involved in the formation of the vascular system. Among them are the endothelial specific receptor families such as VEGF, Ang/Tie, as well as other signaling pathways such as semaphorins, which are also involved, in axonal guidance. It is known that the interaction between receptor tyrosine kinase, Tie2, VE-Cadherin, and VE-PTP mediate endothelial cell quiescence and adhesion. However, the structural basis of these interactions is not well understood. The aim of our study is to characterize the binding interactions between these players. Another important part of our study is describing the cross-talk between vasculature and nervous system by characterizing the Neuropilin/Plexin/Semaphorin system. VE-Cadherin along with neuropilins plays an essential role by directing VEGF signals to the appropriate location and coordinating the activation of downstream molecules. We characterize the interaction between Tie2, VE-PTP and VE-Cadherin by (FRET)-based proximity assay, fluorescence lifetime imaging, and co-immunoprecipitation assays. Our data showed a consistent localization of the protein and FRET signal for Tie2 and VE-PTP prior to ligand recognition. We showed the association between Tie2 and VE-Cadherin complex by co-immunoprecipatation. However, our FRET data was not consistent. The examination of VE-PTP and VE-Cadherin for association and localization of the protein showed a very unique, mutually exclusive localization of the protein. Our study of Neuropilin/Plexin/Semaphorin system showed changes in the protein localization, FRET signal and morphology upon stimulation of HEK293 cells expressing Nrp/plexin with Sema3D. In this system VE-Cadherin along with neuropilins plays an essential role by directing VEGF signals to the appropriate location and coordinating the activation of downstream molecules. The characterization of extracellular binding between Tie2, VE-PTP, and VE-Cadherin, will help to better understand the molecular mechanisms of normal and tumor angiogenesis to develop new anti-angiogeneic therapies.

Les Sémaphorines constituent une grande famille de protéines sécrétées et membranaires, qui ont été initialement impliquées dans le guidage axonal au cours du développement du système nerveux, mais qui jouent aussi un rôle important dans la cancérogenèse. Ainsi, les Sémaphorines agissent via leurs récepteurs, qui font partie de la famille des Neuropilines et des Plexines, pour réguler des fonctions multiples qui contribuent au développement des cancers et à la progression métastatique, telles que l'adhésion cellulaire, la motilité, la survie, l'angiogenèse et la réponse immunitaire. Mon travail de thèse a visé à mieux comprendre le rôle de la signalisation induite par la Sémaphorine-3E (Sema3E) et son récepteur Plexine-D1 dans le cancer du sein. Il a été proposé récemment, que la signalisation autocrine induite par le couple Sema3E/Pleixne-D1 dans les cellules tumorales, favorise l’apparition de métastases via l’augmentation de leurs capacités de migration et d’intravasation/extravasation.Dans ce travail de recherche, nous avons trouvé qu’une nouvelle signalisation autocrine induite par Sema3E/Plexine-D1 régit à la fois la tumorigenèse et la formation de métastases. Ainsi, la sécrétion de Sema3E par les cellules tumorales favorise leur survie grâce à l’inhibition d’une voie de mort induite par le récepteur à dépendance Plexine-D1. Bloquer l’interaction entre Sema3E et son récepteur Plexine-D1, via l’utilisation d’un ligand TRAP dans des modèles murins de cancer du sein, permet de tuer spécifiquement les cellules tumorales et exerce une action anti-tumorale et anti-métastatique. Des données préliminaires sur la voie de mort induite par le récepteur Plexine-D1, suggèrent un rôle important du clivage de son domaine intracellulaire par les Caspases et/ou une translocation nucléaire. Ensemble, ces résultats indiquent que l’expression de Sema3E dans les cancers du sein chez la femme, permet un avantage sélectif pour la survie des cellules tumorales. Bloquer l’interaction entre Sema3E/Plexine-D1 apparaît donc comme une stratégie thérapeutique prometteuse pour traiter ces cancers. De plus, ces données permettent une nouvelle approche dans l’étude des fonctions du couple Sema3E/Plexine-D1 dans d’autres contextes physiologiques et pathologiques. / The Semaphorins constitute a large family of secreted and membrane-bound proteins, which have initially been implicated in axon guidance during development of the nervous system, but also have an important role in cancer. Semaphorins signal through Neuropilin and Plexin receptors to regulate multiple functions contributing to cancer development and progression, including cell adhesion and motility, cell survival, angiogenesis and immune response. My work is aimed at better understanding the role of the secreted glycoprotein Semaphorin 3E (Sema3E) and its binding receptor PlexinD1 in breast cancer. It has been recently proposed that Sema3E/PlexinD1 autocrine signaling in tumor cells promotes metastasis by enhancing cancer cell migration and intravasation/extravasion steps. Here we found another mechanism by which Sema3E/PlexinD1 autocrine signaling regulates both tumorigenesis and metastasis. We show that release of Sema3E by tumor cells promoted their survival through inhibition of an endogenous cell death pathway triggered by the unbound PlexinD1 “dependence receptor”. Interrupting Sema3E/PlexinD1 binding using a ligand-TRAP for PlexinD1 receptor specifically increased tumor cell death and exerted anti-tumoral and anti-metastatic activities in animal models of breast cancers. Preliminary investigations of the underlying cell death pathway suggested a crucial role of PlexinD1 intracellular domain via its cleavage by caspases or its possible translocation to the nucleus. Together, the results indicate that up-regulation of Sema3E in human breast cancers provides a selective advantage for tumor cell survival and that antagonizing Sema3E binding to PlexinD1 may represent a promising therapeutic strategy. Moreover, these data may provide new insight into the functions of Sema3E/PlexinD1 in other physiological and pathological contexts.

During embryonic development, cells respond to extracellular signals to establish proper tissue organization. Semaphorins (Semas) are a large class of secreted and transmembrane proteins that signal through Plexin (Plxn) receptors to guide migrating cells to their correct position and thus play critical roles in the development of various tissues including the nervous and cardiovascular systems. We have previously shown that Sema6A-PlxnA2 signaling is essential for visual system development, as decreasing endogenous Sema6A or PlxnA2 in zebrafish results in decreased cohesion of the early eye field, impaired retinal lamination, and smaller eye size. However, the molecular mechanisms governing these phenotypes are unknown. This dissertation describes the elucidation of functionally-relevant mechanisms of Sema6A-PlxnA signaling during eye development using biochemical and proteomic approaches in cell culture systems and the zebrafish as an in vivo vertebrate model of eye development. We first describe our investigations on the receptor-proximal mechanisms of Sema6A-PlxnA signaling. The Src-family tyrosine kinase Fyn was known to bind to and phosphorylate PlxnA receptors. However, the specific sites of phosphorylation and their function were unknown. Using mass spectrometry, we identified highly-conserved, Fyn-induced PlxnA tyrosine phosphorylation sites. Mutation of these tyrosines to phenylalanine nearly eliminated Fyn-dependent PlxnA phosphorylation. Furthermore, unlike mRNA encoding wild type human PlxnA2, mRNA encoding the tyrosine-to-phenylalanine mutant PlxnA2 could not rescue the smaller eye size phenotype caused by endogenous PlxnA2 knockdown in zebrafish. This suggests that Fyn-dependent PlxnA2 phosphorylation is critical for proper vertebrate eye development. Next, we report the discovery and functional characterization of a naturally-released soluble ectodomain of Sema6A (sSema6A). We show that sSema6A production is increased by PKC activity. The identification of several PKC-dependent phosphorylation sites in the intracellular region of Sema6A suggests a mechanism for PKC-dependent release of sSema6A. Importantly, we show that sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This is the first report of a soluble ectodomain of the Sema6 class and suggests that Sema6A can have regulated, long-range signaling capacity in addition to its canonical contact-mediated functions. Finally, we present our findings characterizing the role in eye development of CRMP2, a downstream effector of Sema-Plxn signaling. CRMP2 is known to be critical for lamination of the cerebral cortex, leading us to hypothesize that CRMP2 could also be involved in the lamination of the retina. Using morpholino-based knockdown of endogenous zebrafish Crmp2, we show that Crmp2 has a critical function in visual system development. Crmp2 knockdown results in smaller eye size, impaired retinal lamination and a weakened optic tract. Together, this dissertation describes important novel Sema6A-PlxnA signaling mechanisms and places them in the context of vertebrate eye development.

Background: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. Methods: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. Results: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. Conclusion: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

The role that signaling molecules play during morphogenesis and their interactions is a field of intense study and the sea urchin represents a facile system to study these aspects of development in the early embryo. In many instances, the S. purpuratus genome contains relatively simple receptor-ligand signaling systems compared to vertebrate counterparts and this provides interesting opportunities to study their diversity of function during the morphogenetic events that shape the embryo. The Eph-Ephrin signaling components are an excellent example of this and they are represented by dozens of members in the vertebrate system with developmental functions that include axon guidance, cell migration and tissue segregation. In contrast, the sea urchin genome contains a single Eph receptor and a single Ephrin ligand and by interacting with different effectors of signal transduction, this simple, bipartite system can fulfill a variety of functional roles during morphogenesis. Studying the function of Eph-Ephrin signaling in the sea urchin embryo, I have revealed two distinct morphogenetic movements in which Eph-Ephrin signaling is necessary; apical constriction of ciliary band cells and pigment cell migration. In both examples, a functionally relevant Ephrin gradient establishes spatial information in the developing tissues, producing a reaction from cells expressing the Eph receptor. In the case of pigment cells, the distribution of migrating cells is affected and in the case of ciliary band cells, apical constriction occurs. The different outcomes of Eph-Ephrin signaling in these two tissues exemplifies signaling components communicating spatial information and initiating morphogenetic programs with outcomes dependent on cellular context. Furthermore, I have identified downstream components of Eph-Ephrin signaling that have necessary functions in both models, illustrating how different cellular programs can be induced by the same signaling iii iv components. My research contributes to understanding fundamental aspects of how complex 3 dimensional tissues arise from the genes and regulatory elements encoded in metazoan genomes. / Graduate

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Durant le développement, l’innervation d’une zone précise du cerveau par certaines branches axonales est un mécanisme encore mal compris. Afin d’aborder cette question, je me suis intéressée aux axones rétiniens qui innervent deux cibles principales du système visuel : le corps genouillé latéral dorsal (CGLd) et le colliculus supérieur. J’ai étudié le rôle de la protéine de guidage Sémaphorine 6D et de son récepteur Plexine-A1 dans l’innervation spécifique du CGLd par les axones rétiniens. J’ai ainsi découvert que chez les souris Sema6D-/- et Plexine-A1-/-, le tractus optique (formé par les axones rétiniens) entre dans le CGLd au lieu de le contourner et certains axones rétiniens innervent des régions ectopiques de l’autre côté du tractus optique. De plus, l’analyse des souris simple ou double hétérozygotes indique que ces deux protéines interagissent avec un mécanisme dose-dépendant. Grâce à des expériences de perte et de gain de fonction par électroporation rétinienne in utero, j’ai pu montrer la nécessité de Sema6D et de Plexine-A1 dans la rétine pour l’innervation des axones rétiniens et ce via des effets non cellulaire autonomes. Ces résultats révèlent un mécanisme dose-dépendant dans lequel Sema6D et PlexineA1 interagissent et assurent une communication axone-axone permettant l’innervation précise du CGLd par une sous-population d’axones rétiniens. / During development, axons branch at precise points to innervate a specific brain target, yet the mechanisms at hand are still unclear. To address this question, I used retinal axons forming the optic tract that innervate two principal targets of the visual system: the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus. I investigated the role of the guidance receptor Plexin-A1and its ligand Semaphorin-6D (Sema6D) in this targeting process. Here I highlight a new type of phenotype in Plexin-A1-/- or Sema6D-/- mice. In these mice, the optic tract enters in the dLGN instead of circumscribing it and some retinal axons innervate ectopic regions at the other side of the optic tract. Furthermore, the analysis of simple or double heterozygotes mice reveals that Plexin-A1 and Sema6D interact together with a dose-dependent effect. Using loss and gain of function experiments (via retinal in utero electroporation), I showed that both are necessary in the retina for proper retinal innervation through non-cell autonomous effects. All these results reveal for the first time a dose-dependent mechanism, in which Sema6D and Plexin-A1 interact together. They monitor axon-axon communication to allow the correct innervation of the dLGN by a subpopulation of retinal axons.

Ce travail poursuit l’exploration du potentiel thérapeutique de peptides antagonistes des domaines transmembranaires (TM) de récepteurs impliqués dans la croissance tumorale. J’ai montré l’effet anti-angiogénique de MTP-NRP1, un peptide ciblant le récepteur Neuropline-1 et confirmé sa capacité d’inhibition de prolifération, migration et de croissance d’une lignée de glioblastome (GBM) humain. J’ai ensuite démontré que le récepteur Plexine-A1 est corrélé à l’agressivité des gliomes et semble être un marqueur pronostique négatif de la survie des patients atteints de GBM. J’ai démontré le rôle du segment TM de PlexA1 dans ses interactions. Le peptide MTP-PlexA1, inhibe la signalisation et la formation du complexe NRP1-PlexA1, réduit la prolifération et la migration des cellules de GBM, impacte la croissance tumorale in vivo y compris de cellules souches tumorales. J’ai décrit le rôle pro-angiogénique de PlexA1 par des tests d’angiogenèse et de CAM où MTP-PlexA1 bloque cette fonction. / This thesis work continues the exploration of the therapeutic potential using peptides targeting transmembrane (TM) domains of receptors involved in tumor growth. I showed the anti-angiogenic effect of MTP-NRP1, a peptide targeting Neuropilin-1 and confirmed its capability to impact proliferation, migration and in vivo growth of a human glioblastoma (GBM) cell line. Then, I demonstrated that the expression of Plexin-A1 is correlated with glioma aggressiveness and seems to be a bad prognosis marker for GBM patients. We described the importance of PlexA1 TM domain in the control of their interactions. The peptide MTP-PlexA1 inhibits complex formation and signaling of NRP1-PlexA1, impacts tumor growth in vivo and cancer stem cells engrafting and development. I demonstrated the pro-angiogenic role of PlexA1 with in vitro angiogenesis assays and CAM assay in which MTP-PlexA1 is able to block this function.