Cell migration is an essential physiological process required for embryogenesis, tissue repair and immune surveillance. Directional cell migration requires coordinated regulation of multiple integrin-mediated cellular processes, including dynamic regulation of the actin cytoskeleton, precise control of membrane protrusion events and the constant recycling of adhesion receptors. While it is clear that regulation of the small guanosine triphosphatases (GTPases) Rac1, Arf6 and RhoA is critical for these processes, the integrin-dependent mechanisms responsible for cyclic activation and dynamic coordination of GTPase signalling are only partially understood. Here, analysis of three published mass spectrometry (MS) studies cataloguing integrin-dependent adhesion complexes identified filamin-A and IQGAP1 as potential candidates linking β1 integrin to the regulation of Rac1 activity. Using immunoprecipitation, MS analysis, immunocytochemistry and RNAi, filamin-A and IQGAP1 were found to be recruited to integrin activation sites, where they constrained Rac1 activity via the recruitment of the GTPase-activating protein RacGAP1. The functional relevance of Rac1 deactivation, through a RacGAP1 and IQGAP1-mediated mechanism, is to permit efficient membrane protrusion and directional cell migration. Subsequently, IQGAP1 was identified as a molecule co-ordinating Rac1 and Arf6 activities downstream of β1 integrin engagement, via the recruitment of the GTPase activity modulators RacGAP1, srGAP2 and HERC1. This lead us to propose a model whereby IQGAP1, through the recruitment of multiple small GTPase activity modulators, co-ordinates the two small GTPases Rac1 and Arf6, to efficiently regulate directional cell migration. Dyregulated cell migration due to integrin over-activation is associated with tumour invasion. Increased recycling of α5β1 integrin, resulting from expression of mutant p53 or inhibition of αVβ3 integrin function, leads to random cell migration on 2D substrates and promotes tumour invasion via activation of the pro-invasive kinase Akt. Here, the RacGAP1- IQGAP1 complex was identified as a key component of this pathway. In particular, RacGAP1 was found to be phosphorylated by Akt2 on T249, a phosphorylation event that promoted RacGAP1 recruitment to IQGAP1, at the cell front, and triggered cell invasion by inducing a Rac1/RhoA activity switch. These findings demonstrated that Akt activation, downstream of α5β1 integrin recycling, promotes fibronectin-mediated cell invasion by activating a novel RacGAP1/IQGAP1/Rac1/RhoA pathway. Taken together, we identified a novel signalling nexus, downstream of integrin activation and/or recycling that co-ordinate the small GTPases Rac1, Arf6 and RhoA during cell migration and invasion.

La maladie de Parkinson (MP) est caractérisée par l'accumulation d'alpha-synucléine (aSyn) mal repliée dans la substantia nigra pars compacta (SNpc), entraînant la mort des neurones dopaminergiques (DA). Les mécanismes qui sous-tendent la toxicité de l’aSyn sont encore peu connus, mais on suppose qu'ils impliquent des défauts dans l'autophagie (ALP). Les mutations dans LRRK2 sont fréquentes dans la MP familiale et sporadique. L'inhibition pharmacologique de l'activité de la kinase LRRK2 réduit les déficits dans l’ALP et les inclusions d’aSyn phosphorylée (paSyn), ce qui suggère que ces phénotypes dépendent de l'hyperactivation de LRRK2. Nous avons observé une diminution de l’expression du gène RIT2 dans des cellules mutantes LRRK2 (G2019S). RIT2 encode la petite GTPase Rin, qui est enrichie dans les neurones DA, et moins abondante dans la SNpc des cerveaux des patients atteints de la MP. Notre objectif est d'évaluer si Rin peut moduler l'activité de LRRK2 pour contrecarrer les altérations dans l'autophagie et promouvoir la clairance de l’aSyn. Nous avons utilisé des cellules de neuroblastome exprimant LRRK2-G2019S ou LRRK2-sauvage (WT) et évalué les déficits dans l’ALP et la toxicité de l’aSyn avec ou sans surexpression de Rin. Nous avons ensuite testé notre approche in vivo en utilisant des vecteurs viraux encodant aSyn et/ou Rin afin de surexprimer ces gènes chez la souris. Nous avons évalué la déficience locomotrice et fait une analyse histopathologique de coupes de cerveau. La surexpression de Rin dans les cellules LRRK2-G2019S a permis de renverser les altérations dans l'ALP et de diminuer les inclusions d'aSyn. Dans notre modèle de souris, la surexpression de Rin a empêché les déficits moteurs induits par l'injection d'AAV-aSyn. La surexpression de Rin a également protégé contre la perte d'axones dopaminergiques dans le striatum et la dégénérescence neuronale. Nos données indiquent que Rin inhibe LRRK2 pour compenser le déficit ALP et contrecarrer l'agrégation d'aSyn et les déficits connexes. Cela suggère que de cibler la signalisation par Rin pourrait représenter une nouvelle stratégie pour combattre la MP familiale et sporadique. / Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (aSyn) in the substantia nigra (SNc), leading to the death of dopaminergic (DA) neurons. The mechanisms underlying aSyn pathology are still unclear but hypothesized to involve autophagy and endosome-lysosome pathways (ALP). LRRK2 mutations are a major cause of familial and sporadic PD. Pharmacological inhibition of LRRK2 kinase activity ameliorates ALP deficits and reduces paSyn inclusions, indicating that these phenotypes depend on LRRK2 hyperactivation. We observed selective downregulation of the novel PD risk factor RIT2 in LRRK2 mutant cells (G2019S). RIT2 encodes the small GTPase Rin, which is enriched in DA neurons and reduced in SNpc of PD brains. We aim to evaluate if Rin can modulate LRRK2 activity to rescue alterations in autophagy and promote aSyn clearance. We used neuroblastoma cells expressing LRRK2-G2019S or LRRK2-WT and evaluated ALP deficits and aSyn pathology with or without Rin overexpression. We then tested our approach in vivo using viral vectors encoding aSyn and/or Rin in mice SNpc. We evaluated the locomotor impairment and performed histopathological analysis on brain sections. Rin overexpression in LRRK2-G2019S cells rescued the alterations in ALP and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rin prevented motor deficits induced by AAV-aSyn injection. Overexpression of Rin also protected against the loss of dopaminergic axons in the striatum and neural degeneration. Our data indicate that Rin inhibits overactive LRRK2 to remove ALP impairment and counteract aSyn aggregation and related deficits. This suggests that targeting Rin signaling could represent a novel strategy to combat neuropathology in familial and idiopathic PD.

Tube formation, or tubulogenesis, is an elaborate form of epithelial morphogenesis that includes processes such as cell migration and cell shape changes. The embryonic Drosophila heart, or dorsal vessel, is an excellent model of tubulogenesis and more specifically the signaling mechanisms required for cell migration and lumen formation. Similar to vertebrate heart formation, Drosophila heart tubulogenesis begins with the collective migration of cardioblasts that meet at the midline and adhere at specialised junctions, enclosing a lumen between them. Roundabout, and its ligand Slit, are required to restrict cell-to-cell adhesions to the junctional domains of contralateral cardioblasts, as well as maintain the integrity of the lumen. The localisation patterns of Robo, and other luminal cell surface receptors important for lumen formation are significantly modified throughout heart formation. Initial receptor expression is broadly distributed over the cardioblast surface. Receptors are then relocalised to specific cell surface domains by late embryonic development. The mechanisms by which Robo and other cell surface receptors are localised have yet to be determined. Endocytosis is a promising mechanism by which cell surface receptors are targeted and trafficked to cell surface domains. Specifically, vesicular trafficking proteins, such as Rab GTPases, are molecular switches that regulate endocytic events. Here, we investigated the roles of Rab5, Rab11, and Sec6 during heart formation. Of these, only Rab5, a regulator of the early endosome, was required for lumen formation. Particularly, gain of function, loss of function, and overexpression of rab5 resulted in reduced lumen phenotype, characterised by lumen pockets rather than a continuous lumen along the anterior-posterior axis. Perturbed Rab5 function also resulted in the mislocalisation of Robo at the basal domain. Live imaging showed that expression of rab5 dominant negative, constitutively active, and overexpression constructs did not perturb apical membrane motility of migrating cardioblasts in the developing heart. / Thesis / Master of Science (MSc)

O Corpo Ciliar (CC) é uma fonte de células tronco da retina de animais adultos, mas sua ativação permanece desconhecida. GTPases Rho são proteínas que reorganizam do citoesqueleto de actina, regulam vias de sinalização e transcrição gênica, sobrevivência celular e proliferação. Neste trabalho, investigamos a expressão das GTPases Rho nas células do CC e seu efeito na regulação do ciclo celular. As GTPases RhoA, RhoB e Rac1 foram expressas nas células do CC e sua ativação pelo ácido lisofosfatidico (LPA) aumentou a expressão dos genes progenitores retinianos Pax6 e Chx10. A inibição das proteínas por Toxina A de Clostridium difficile aumentou a proliferação no CC e potencializou o efeito proliferativo dos fatores de crescimento. A inibição especifica destas proteínas diminuiu a expressão dos transcritos de p21cip, p27kip, p16INK4a e p19INK4d e aumentou de Ki67, CiclinaA e D1. O estudo da via de Wnt indicou que Rac1 regulou os genes de componentes da degradação de -catenina e Lef1. Concluímos que a inativação das GTPases Rho induziu a proliferação das células progenitoras retinianas localizadas no CC e regulou a via de Wnt. Sua ativação induziu o perfil de célula progenitora, sugerindo uma nova ferramenta para o mecanismo de reparo retiniano. / Ciliary Body (CB) is a potential source of stem cells in the adult retina, but its activation is still unknown. Rho GTPases play a role in actin-based cytoskeleton reorganization, regulate signaling pathways and gene transcription, cell survival and cell proliferation. In this study we investigated the expression of Rho GTPases in CB cells and their role on cell cycle regulation. The GTPases RhoA, RhoB and Rac1 were present in CB cells and the activation by lysophosphatidic acid (LPA) increased the expression of the progenitor genes Pax6 and Chx10. The inhibition by Clostridium difficile Toxin A increased the proliferation of CB cells and potentiated the proliferative effect of growth factors. The specific inhibition decreased the expression of p21cip, p27kip, p16INK4a and p19INK4d as well as increased Ki67, cyclinA and D1 transcripts. The Wnt pathway study indicated that Rac1 regulated -catenin degradation genes components and Lef1. Taken together, the inactivation of Rho GTPases stimulated the proliferation of progenitor cells located in CB as well as regulate the Wnt signaling pathway. The proteins activation was correlated to progenitor profile induction. These different mechanisms may provide a potential new approach on retinal repair.

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

<p>The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. </p><p>A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. </p><p>In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.</p>

The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.

La migration cellulaire collective (MCC) est un processus fondamental qui intervient au cours du développement, de la réparation tissulaire, de l'invasion tumorale et de la formation de métastases. Les cellules qui migrent collectivement possèdent deux types d'interaction avec leur environnement : i) l'un avec leur substrat et ii) l'autre avec les cellules voisines en migration. Deux grandes familles de protéines permettent ces interactions ainsi que la génération de forces mécaniques: i) la famille des intégrines (les récepteurs de la matrice extracellulaire) et ii) la famille des cadhérines (formant les jonctions intercellulaires). Les cadhérines classiques sont impliquées dans la formation des jonctions intercellulaires et sont les principaux acteurs de la MCC au cours du développement normal et tumoral. La transmission de force entre les cellules en migration est nécessaire à leur cohésion et à la communication des cellules entre elles. Des études récentes montrent que les cadhérines sont nécessaires à la transmission des forces au substrat. Néanmoins, les processus par lesquels les cadhérines agissent sur ses forces dans le contexte d'une MCC restent inexplorés. Nous avons identifié l'expression de la P-cadhérine comme étant associée à l'agressivité du rhabdomyosarcome de type alvéolaire (ARMS), sous type ayant le plus mauvais pronostic car très invasif. Nos données, ainsi que de récentes études qui démontrent que la P-cadhérine est impliquée dans l'agressivité des tumeurs du sein, nous ont conduits à étudier le rôle de cette cadhérine dans la migration cellulaire, processus majeur dans le développement tumoral. Nous nous sommes intéressés à l'impact de l'expression de la P-cadhérine sur la migration des myoblastes murins normaux C2C12. Pour ce faire nous utilisons un test de migration in vitro en 2D proche du test de blessure qui consiste à retirer une barrière physique induisant la migration des cellules vers l'espace libre ainsi créé. Nous avons pu monter que l'expression de la P-cadhérine dans les myoblastes C2C12 augmente les paramètres caractéristiques d'une MCC in vitro : la vitesse, la polarité, la persistance et la directionalité de la migration des cellules du front et au sein du feuillet. De plus, à l'aide de techniques microscopiques de mesure des forces nous avons montré une augmentation des forces intercellulaires allant du front vers le feuillet cellulaire au cours de la migration des cellules exprimant la P-cadhérine. Cela suggère une augmentation de la cohésion cellulaire. L'ensemble de ces résultats démontrent clairement que l'expression de la P-cadhérine induit une MCC. Nous avons aussi mesuré et cartographié les forces de traction au substrat connues pour être le moteur de la migration cellulaire. Nos données indiquent que l'expression de la P-cadhérine augmente l'anisotropie de ces forces de traction ainsi que leur intensité, et ce, uniquement au front de migration. L'expression de la P-cadhérine remodèle et stimule la dynamique des plaques focales d'adhérence à cet endroit.Afin de mieux comprendre comment la P-cadhérine modifie la dynamique des adhésions focales et augmente les forces de traction, nous avons étudié l'activité spatiotemporelle des petites protéines G de la famille Rho. Nous montrons que l'expression de la P-cadhérine active Rac1 et Cdc42 au front de migration, entrainant ainsi le remodelage et l'organisation des plaques focales d'adhérence à cet endroit. L'inhibition de Rac1 et Cdc42 bloque la MCC induite par la P-cadhérine. Pour conclure, en combinant la mesure des paramètres de migration cellulaire avec la mesure des forces mécaniques intercellulaires et au substrat, nous avons démontré que la P-cadhérine induit un comportement collectif des cellules et ce dépendamment de l'activité de Rac1 et de Cdc42. De plus nous mettons en avant l'existence de propriétés mécano-transductrices de cette cadhérine au cours de la MCC. / Collective cell migration (CCM), the coordinated movement of multiple cells that are connected by cell-cell adhesion, is a fundamental process in development, tissue repair and tumor invasion and metastasis. Cells part of a moving collective have two different types of interactions, i) one with the substratum, and ii) one with surrounding moving cells. Two protein families allow these interactions and also the generation of mechanical forces: i) typically integrins on the underlying extracellular matrix (ECM) and ii) cadherins at intercellular adhesion sites. Classical cadherins are involved in cell-cell adhesion and are major drivers of collective cell migration in embryonic development and tumorigenesis.Mechanical coupling between migratory cells may result in the production of force-dependent signals by which the cells influence each other. Moreover, whereas recent data showed that cadherin-dependent cell-cell adhesions are important for the force transmission to the ECM, how intercellular adhesion impacts on cell-ECM forces in the context of collective cell migration is totally unexplored. We identified P-cadherin expression to be associated with alveolar rhabdomyosarcoma (ARMS) aggressiveness, tumors with a bad prognosis due to the propensity for early and wide dissemination. Our data and recent findings showing that P-cadherin is associated with breast tumor invasiveness and aggressiveness, led us to investigate the role of P-cadherin in cell migration. We analyzed cell migration of normal mouse C2C12 myoblasts that express P-cadherin using a “wound-healing like assay” in which migration is analyzed after removal of a physical barrier. We observed that P-cadherin expression in C2C12 myoblasts increased the speed, polarity, persistence and directionality of migration toward the free space of both cells at the border and cells into the sheet. Using monolayer stress microscopy we showed that P-cadherin increases inter-cellular stresses and force transmission across the cell sheet. According to those observations we concluded that P-cadherin induces CCM.Traction forces exerted by the cells on the substrate are important for cell migration. Using traction force microscopy, we demonstrated that P-cadherin expression increases the traction forces anisotropy specifically at the multicellular leading row. To better understand how these mechanical signals induce CCM, we studied both the organization of the focal adhesions and the spatio-temporal activity of Rho GTPase. We showed that P-cadherin expression activates Rac1 and Cdc42 which induces extensive focal adhesions remodeling at the leading edge of cells at the leading row. Rac1 and Cdc42 inhibition impaired P-cadherin-induced CCM, focal adhesion remodeling and forces generation. In conclusion, combining a detailed measurement of the parameters of cell migration with physical measure of the intercellular stresses and traction forces, we have shown that P-cadherin promotes collective behavior of cells during migration through Rac1 and Cd42 activity. Also, those results provide evidence for mechano-transmission properties of P-cadherin during collective cell migration.

En plus des anomalies génétiques, plusieurs études ont rapporté des altérations des protéines chez les patients atteintes de Syndromes Myéloprolifératifs (SMP) Ph- qui pourraient participer à leurs phénotypes cliniques. Néanmoins, les altérations protéiques dans ces pathologies ne sont pas bien connues. Dans ce contexte, nous avons utilisé une approche protéomique par spectrométrie de masse pour nous aider à déchiffrer le paysage des anomalies des protéomes érythrocytaire et granulocytaire qui pourraient être liées à des altérations cellulaires fonctionnelles et à la physiopathologie des SMP. Nous avons ainsi pu identifier des dérégulations importantes de protéines qui varient selon le statut génétique des patients, [JAK2(+), JAK2(-) ou CALR(+)], selon la charge allélique de JAK2V617F, mais aussi selon le type de SMP JAK2(+) ou selon la. Ces dérégulations protéiques perturbent des voies de signalisation comme la voie IQGAP1/Rho GTPase qui pourrait être liée aux thromboses par des modifications de l'intégrité membranaire via la dérégulation de PAK1 ou des protéines du cytosquelette d'actine. Des modifications des voies de signalisation des ROS ou mTOR ont été également identifiées dans les granulocytes. En outre, nous avons montré que la protéine CALR pourrait avoir un double rôle oncogénique grâce à son expression élevée dans les SMP JAK2V617F, différent de l'altération de sa fonction décrites dans les patients CALR mutés. En conclusion, nous avons montré que les dérégulations protéomiques pourraient jouer un rôle oncogénique important dans la physiopathologie des SMP Ph- et qu’elles pourraient être impliquées dans certaines complications de ces pathologies telles que les accidents thrombotiques. / Besides genetic abnormalities, several studies have reported protein alterations in Ph-Myeloproliferative neoplasms (MPN) which could participate to the clinical phenotype of patients. Nevertheless, little is known about protein alterations in these pathologies. In this context, we used an integrative proteomic approach to decipher the landscape of the erythrocyte and granulocyte proteome abnormalities that could be related to functional cell alterations and to MPN physiopathology. We could identify significant protein deregulations that varied not only according to genetic status JAK2(+), JAK2(-) or CALR(+) but also among JAK2(+) MPNs or depending on the JAK2V617F allele burden. These protein deregulations involved pathway alterations such as the IQGAP1/Rho GTPase signaling that could be related to thrombosis via alterations on the membrane integrity by deregulation of PAK1 or the actin cytoskeleton signaling. ROS or mTOR signaling alterations were also identified in granulocytes. Finally, we stress out that CALR protein could have a dual oncogenic role through its up-regulation in JAK2V617F MPNs different from its altered function described in CALR mutated patients. Altogether, we showed that proteomic deregulations might play an important oncogenic role in MPN physiopathology and could be implicated in complication as thrombotic accidents