Células de músculo liso vascular são capazes de perceber estímulos mecânicos do sistema cardiovascular, coordenando pressão sanguínea e perfusão tecidual por meio da modulação do tônus e do diâmetro vascular via resposta contrátil. O gatilho inicial à contração é um aumento na concentração intracelular de cálcio e diversas vias de sinalização têm sido descritas na sustentação deste sinal inicial. Evidências atuais indicam que adesões focais desempenham papel crucial na contração através da organização do citoesqueleto de actina e engajamento com o aparato contrátil. Nosso grupo demonstrou que a proteína rica em cisteína e glicina 3 (CRP3) interage com a quinase de adesão focal (FAK) em resposta a um aumento do estiramento mecânico e existem evidências de que CRP3 modula a dinâmica do citoesqueleto de actina. Neste trabalho testamos a hipótese de que a proteína CRP3 atua como uma proteína de adesão focal que regula a contração de células musculares lisas aórticas. Por meio de ensaios de imunoprecipitação e colocalização, verificou-se a presença de CRP3 nas adesões focais de células selvagens. Evidenciou-se que a ausência de CRP3 está associada a aumento no tamanho médio de adesões focais em células musculares lisas aórticas de forma independente do substrato. Entretanto, em resposta à angiotensina II, células nocaute para CRP3 apresentam incapacidade de maturação das adesões focais, um evento que está associado ao reduzido conteúdo proteico de FAK, paxilina e MLC2 (plataformas moleculares envolvidas na maturação de adesões focais) observada em células nocaute. Consistente com o maior tamanho médio das adesões focais, células nocaute são mais rígidas e, portanto, menos elásticas que células selvagens, sendo que a rigidez avaliada por citometria magnético-óptica de oscilação se reflete na reduzida capacidade contrátil, seja em condições basais, em resposta à angiotensina II ou ao inibidor de ROCK, como evidenciado no ensaio de contração em gel de colágeno. Em síntese, os dados deste trabalho mostram que CRP3 está presente nas adesões focais, regulando tamanho e sinalização, com reflexos na rigidez (viscoelasticidade) e capacidade contrátil, variáveis fundamentais ao correto funcionamento de células musculares lisas aórticas. Em conjunto, as evidências deste trabalho suportam a hipótese de que CRP3 é um modulador de contratilidade e mecanotransdução em células musculares lisas aórticas / Smooth muscle cells act also as mecanosensors of the cardiovascular system, coordinating blood pressure and tissue perfusion by means of vascular tone and diameter modulation via the contractile response. The trigger for contraction is a rise in the intracellular calcium concentration and several signaling pathways have been described to sustain the initial calcium signal. Recent evidences highlight the crucial role of focal adhesions to the contractile response, given its role in actin cytoskeleton assembly and engagement with the actomyosin contractile apparatus. We have demonstrated that the cysteine and glycine-rich protein-3 (CRP3) interacts with focal adhesion kinase (FAK) in response to increased hemodynamic stress. Additionally, it has also been shown that CRP3 modulates actin cytoskeleton dynamics. Here, we tested the hypothesis that CRP3 acts as a focal adhesion protein that regulates the contraction of aortic smooth muscle cells. Through colocalization and immunoprecipitation studies we found that CRP3 is a focal adhesion protein in aortic smooth muscle cells. Focal adhesion mean size evaluation showed that in the baseline, CRP3 KO smooth muscle cells display greater focal adhesion size. However, upon angiotensin II (a contraction-triggering molecule) stimulation, CRP3 KO cells fail to maturate focal adhesions, an event that might be related to the reduced protein levels of FAK, paxillin, and MLC2 (key signaling molecules involved in focal adhesion maturation) observed in KO cells. Consistent with the greater mean focal adhesion size, CRP3 KO cells exhibited increased stiffness and therefore, reduced viscoelasticity when compared to wild type cells. The reduced viscoelasticity of KO cells seems to influence cell contractility, as CRP3 KO cells also displayed reduced contractile response in the baseline and in response to angiotensin II. In summary, these data showed that CRP3 is present at focal adhesions, regulating their size and signaling. Thus, CRP3 at focal adhesions influences cell stiffness and contractile capacity, which are key features of smooth muscle cell physiology. Altogether, our findings support the idea that CRP3 is a key modifier of contractility and mechanotransduction in aortic smooth muscle cells

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L'évasion immunitaire tumorale joue un rôle central dans la progression tumorale et représente un obstacle majeur au succès des immunothérapies. Dans cette Thèse nous avons étudié le rôle du cytosquelette d’actine dans la résistance des cellules de cancer du sein à la lyse induite par les cellules "natural killers" (NKs). Nous avons trouvé que les cellules de cancer du sein résistantes échappent à l’attaques des cellules NKs par une accumulation importante et rapide d’actine près de la synapse immunologique, un processus que nous avons nommé "réponse actine". Nos analyses mécanistiques suggèrent que la réponse actine induit la polarisation d’autophagosomes vers la synapse immunologique et facilite ainsi la dégradation des molécules cytotoxiques sécrétées par les cellules NKs, tel que le ganzyme B, par autophagie. De plus, la réponse actine est associée au regroupement de ligands inhibiteurs à la synapse, suggérant qu’elle est au centre de plusieurs mécanismes de résistance. Dans leur ensemble, nos résultats constituent une base pour le développement d’approches thérapeutiques visant à interférer avec la réponse actine et à restaurer une réponse immunitaire anti tumorale efficace. / Tumor immune evasion plays a central role in cancer progression and is a major hurdle to effective immunotherapy. In this Thesis, we examine the role of the actin cytoskeleton in breast cancer cell resistance to natural killer (NK) cell-mediated cell lysis. We found that resistant breast cancer cells escape from NK-cell attack through a rapid and prominent accumulation of actin near the immunological synapse, a process we termed the “actin response”. Our mechanistic investigations suggest that the actin response drives autophagosome polarization toward the immunological synapse and thereby facilitates the autophagy-mediated degradation of NK cell-derived cytotoxic molecules such as granzyme B. In addition, the actin response was associated with inhibitory ligand clustering at the immunological synapse, suggesting that it is a common driver of different immune evasion mechanisms. Taken together, our data lays the groundwork for therapeutic approaches aimed at interfering with the actin response and restoring an effective anti-tumor immune response.

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LFA-1 (Lymphocyte Function Associated antigen-1) est une intégrine centrale dans la fonction cytotoxique des lymphocytes T CD8+ car elle permet la formation de la synapse immunologique avec les cellules cibles. La régulation de cette interaction cellulaire est contrôlée par la qualité de l'engagement de LFA-1 avec son ligand ICAM-1 (Intracellular Adhesion Molecule-1). Un support clef au contrôle spatio-temporel de l'activation de LFA-1 est le cytosquelette d'actine corticale dans lequel est ancré LFA-1 par son domaine intracellulaire. Comment LFA-1 est organisée à la synapse immunologique et comment la coordination entre LFA-1 et cytosquelette d'actine s'opère de manière précise au sein des lymphocytes T CD8+ cytotoxiques sont des questions non résolues. Le but de ce projet de thèse a été d'étudier l'organisation précise de la distribution de LFA-1 à la synapse immunologique en relation avec l'actine corticale sous-jacente au contact entre lymphocytes T cytotoxiques et les cellules présentatrices d'antigènes. Pour ce faire, des approches de microscopies de super-résolution SIM (Structured Illumination Microscopy), dSTORM (direct STochastic Optical Reconstruction Microscopy) et TIRF (Total Internal Reflexion Fluorescence microscopy) ont été développées. Elles ont été appliquées à des lymphocytes T humains non transformés dérivés de contrôles sains et de patients atteints d'une immunodéficience congénitale, le Syndrome de Wiskott-Aldrich (WAS), caractérisé par un défaut de remodelage du cytosquelette d'actine à la synapse immunologique. L'emploi de l'approche de dSTORM en mode TIRF nous a permis de révéler que dans sa conformation activée, LFA-1 forme à la synapse une ceinture radiale composée de centaines de nano-clusters. L'intégrité du cytosquelette d'actine et notamment la protéine WASP s'avèrent importantes pour la formation de la ceinture de nano-clusters de LFA-1, comme le montre le défaut de formation de cette ceinture dans les lymphocytes de patients WAS. L'approche de SIM multi-couleur nous a permis de révéler le rôle de la ceinture de LFA-1 dans le confinement des granules lytiques. Par comparaison de marquages avec des anticorps spécifiques de différentes conformations de LFA-1, notre travail montre également que l'activation de LFA-1 s'opère de manière digitale, dans le sens où les nano-clusters fonctionnent comme des unités au sein desquelles l'activation de LFA-1 suit une loi du tout ou rien. En conclusion, ce travail de thèse démontre l'intérêt des approches de microscopie de super-résolution pour révéler des mécanismes clefs de l'activation des lymphocytes T et pour appréhender la nature des défauts à l'origine de dérèglements pathologiques de la fonction de ces cellules. / LFA-1 (Lymphocyte Function Associated antigen-1) is a central integrin in the function of cytotoxic CD8+ T lymphocytes since it allows the formation of the immunological synapse with target cells. The regulation of this cellular interaction is controlled by the quality of the engagement of LFA-1 with its ligand, ICAM-1 (Intracellular Adhesion Molecule- 1). A key support for the spatio-temporal control of LFA-1 activation is the cortical actin cytoskeleton in which LFA-1 is anchored by its intracellular domain. How LFA-1 is organized at the immunological synapse and how the coordination between LFA-1 and actin cytoskeleton operates accurately within cytotoxic CD8+ T lymphocytes are unresolved issues. The aim of this thesis project was to study the precise organization of the LFA-1 distribution at the immunological synapse in relation to the cortical actin underlying the contact between cytotoxic T lymphocytes and target cells. For this purpose, super-resolution microscopy approaches, including SIM (Structured Illumination Microscopy), dSTORM (direct STochastic Optical Reconstruction Microscopy) and TIRF (Total Internal Reflection Fluorescence microscopy) were developed. They were applied to untransformed human T lymphocytes derived from healthy donors and patients with a congenital immunodeficiency, the Wiskott-Aldrich Syndrome (WAS), characterized by actin cytoskeleton remodeling defects at the synapse. The use of the dSTORM approach revealed that activated LFA-1 forms a radial belt composed of hundreds of nanoclusters. The assembly of this belt depends on the integrity of the actin cytoskeleton, as shown by the impairment of this structure in the T lymphocytes derived from the WAS patients. The multi-color SIM approach allowed us to investigate the role of the LFA-1 belt in the confinement of lytic granules. Furthermore, the combination of staining with antibodies specific of LFA-1 conformation states shows that LFA-1 activation is a digital process, whereby nanoclusters operate as units in which LFA-1 activation follows an on / off rule. In conclusion, this PhD work exemplifies the great asset of super-resolution microscopy approaches to reveal key activation mechanisms in T lymphocytes and explore the nature of the defects causing pathological dysregulation of the function of these cells.

O endotélio reveste a parede vascular e possui função essencial na manutenção da homeostase. A célula endotelial é capaz de perceber estímulos extracelulares, como fatores químicos e mecânicos, transmitir a informação para dentro da célula e regular sua função e fenótipo. Neste sentido, investigamos os mecanismos moleculares associados as células endoteliais em dois contextos importantes de intervenções vasculares 1) nos stents farmacológicos, onde a rapamicina exerce funções antiproliferativas e pró-trombogênicas, e 2) na revascularização cardíaca por ponte de safena, onde o alto estiramento mecânico exerce grande impacto no remodelamento vascular e no fenótipo da célula endotelial. A rapamicina pertence à classe de drogas limus, bastante utilizadas nos stents farmacológicos usados no procedimento de desobstrução vascular. Além de sua função antiproliferativa, exploramos os efeitos deletérios associados a pró-trombogênese. Os dados demonstraram que a rapamicina ativa o receptor de TGF independentemente de seu ligante TGFbeta, promovendo aumento na expressão da PAI-1 (pró-trombogênica), alteração no fenótipo endotelial (Transição endotélio-mesenquimal - EndMT) e na formação de fibras de estresse. Os efeitos observados são dependentes da ativação de Smad2 e independentes da via clássica antiproliferativa por mTOR. Experimentos in vivo mostraram que o tratamento com inibidor do receptor de TGF diminui os efeitos pró-trombogênicos e a expressão de PAI-1 induzidos pela rapamicina em artérias carótidas de camundongos. A ponte de safena é um procedimento bastante utilizado na cirurgia de revascularização cardíaca e a arterialização do segmento venoso submetido ao estresse hemodinâmico arterial resulta em remodelamento vascular, que influencia o sucesso do procedimento. Nossos dados demonstram que a célula endotelial humana de veia safena humana (hSVEC), susceptível as modificações do tipo EndMT induzido quimicamente (estímulo pró-fibrótico e pró-inflamatório), não expressou o mesmo comportamento em resposta ao aumento de estiramento mecânico que ocorre durante a arterialização venosa. Entretanto, detectamos uma pronunciada redução dos filamentos de actina, modulação no padrão de ativação da cofilina e na proporção de actina glomerular (G-actina) entre citoplasma e núcleo, com redução da biodisponibilidade de NO. De modo interessante, demonstramos que a redução no filamento de actina é específica para a célula endotelial venosa, não sendo observado em células endoteliais de origem arterial de aorta e coronária. Em conjunto, os dados mostram que 1) efeitos pró-trombogênicos associados a rapamicina são mediados por ativação do receptor de TGF independente do seu ligante e da atividade antiproliferativa da droga e 2) a adaptação da célula endotelial venosa ao estiramento mecânico envolve modulação da síntese/degradação de filamentos de actina e redução na biodisponibilidade de NO. Estes novos elementos sobre o mecanismo de transdução de estímulos químicos e físicos pelo endotélio poderão ser explorados terapeuticamente para modular a plasticidade endotelial em disfunções cardiovasculares / Endothelium is the inner layer in vascular wall and displays an essential role in the maintenance of cardiovascular homeostasis. Endothelial cell senses the extracellular stimuli, such as chemical and mechanical factors, transduce and process these signals to regulate cell function and phenotype. Here, we investigated molecular underpinning of the endothelial cells under two important scenarios: 1) in drug-eluting stents, where rapamycin exerts antiproliferative and undesirable prothrombogenic functions, and 2) in vein graft bypass surgery, where increased stretch modulates vascular remodeling and endothelial cell phenotype. Rapamycin belongs to the class of limus drugs and is widely used in drug eluting stents (DES) to vascular restenosis. In addition to its antiproliferative function, we explore the deleterious effects associated with prothrombogenesis. Our data demonstrated that rapamycin activates TGF receptor independent of its ligand TGFbeta, in concert with promotion of PAI-1 expression (prothrombogenic), changes in endothelial phenotype (Endothelial to Mesenchymal Transition - EndMT) and stress fibers induction. These effects are Smad2 dependent and independent of the classical antiproliferative mTOR pathway of rapamycin. Our in vivo experiments showed that TGF receptor inhibitor treatment decreases prothrombogenic effects and PAI-1 expression induced by rapamycin in mice carotid arteries. Saphenous vein is widely used in coronary artery bypass surgery (CABG) and the vein arterialization remodeling in response to the increased stress influences graft patency. Our data demonstrated that human saphenous vein endothelial cell (hSVEC) is susceptible to chemically induced endothelial-to-mesenchymal transition (EndMT) by pro-fibrotic and pro-inflammatory stimuli. On the other hand, physical stimulus associated with high stretch failed to induce EndMT. However, we detected a pronounced decrease of actin filaments, modulation of the cofilin activation, changes in the proportion of glomerular actin (G-actin) between cytoplasm and nucleus, and reduction of NO bioavailability. Interestingly, the reduction of actin fibers by high stretch is specific to venous endothelial cell since arterial endothelial cells from aorta, and coronary artery failed to display the response. Altogether, our data show that 1) the thrombogenic effects of rapamycin are mediated by TGF receptor activation independent of its ligand and independent of the antiproliferative pathway of the drug, and 2) the adaptation of venous endothelial cell to mechanical stretch involves synthesis/degradation of actin filaments and reduced NO bioavailability. These new elements on signal transduction of endothelial cells in response to chemical and physical stimuli may be therapeutically explored to modulate endothelial plasticity in cardiovascular disorders

Les infections bactériennes touchant la circulation sanguine conduisent à un vaste éventail de graves pathologies, comme les chocs septiques ou les infections locales (endocardites et méningites). Neisseria meningitidis colonise avec succès l’endothélium vasculaire et cause des sepsis sévères. Ces infections résultent de la colonisation des cellules endothéliales de l’hôte, étape clef de la pathophysiologie à laquelle les travaux présentés dans ce manuscrit se sont intéressés. La colonisation de l’endothélium par N. meningitidis est un processus complexe qui implique l’adhésion et la multiplication des bactéries à la surface des cellules endothéliales dans le contexte particulier de la circulation sanguine, où des forces mécaniques sont générées par le flux sanguin sur les objets circulants. Bien que de nombreuses études se soient intéressées à l’interaction entre les cellules endothéliales et N. meningitidis, plusieurs aspects demeurent incertains comme par exemple l’impact des contraintes générées par le flux sanguin et la participation relative des deux partenaires de l’interaction dans la colonisation de l’endothélium par N. meningitidis.L’adhésion de la bactérie à la surface des cellules endothéliales est dépendante de facteurs bactériens (les pili de type IV, PT4) et induit une réponse de la part de la cellule hôte, qui se traduit par un remodelage de la membrane plasmique et une réorganisation du cytosquelette d’actine sous les microcolonies. Dans un premier temps, ces travaux de thèse montrent que la réponse cellulaire induite par N. meningitidis participe activement à la colonisation. En effet, la formation de projections membranaires permet à chaque bactérie de la microcolonie d’établir des contacts avec la cellule hôte, nécessaires à la résistance des microcolonies face aux forces mécaniques générées par le flux sanguin. De plus, nous montrons que la protéine PilV, composant des PT4, est impliquée dans le remaniement de la membrane plasmique et la réorganisation du cytosquelette. Nous avons développé une méthode combinant vidéo-microscopie et analyse de fluorescence pour décrypter les événements précoces prenant place lors du contact entre les bactéries et la surface des cellules hôtes. Nous avons alors montré que le remodelage de la membrane induit par N. meningitidis ne dépend pas de la réorganisation du cytosquelette d’actine au site d’infection mais plutôt des propriétés intrinsèques de la bicouche lipidique.Dans un second temps, nous nous sommes intéressés aux étapes tardives de l’infection, c'est-à-dire à l’initiation d’un nouveau cycle de colonisation. Bien que solidement ancrées à la surface des cellules par l’intermédiaire des projections membranaires, quelques bactéries se détachent des microcolonies pour coloniser des nouveaux sites au sein de l’hôte. Nous avons démontré l’importance de modifications post-traductionnelles de la piline majeure dans cette étape de l’infection et caractérisé les mécanismes impliqués.Cette étude a permis d’affiner les mécanismes impliqués dans l’induction de la réponse cellulaire induite par N. meningitidis et son impact sur la colonisation efficace de l’endothélium par ce pathogène. / Bacterial infections targeting the bloodstream lead to a wide array of severe clinical manifestations, such as septic shock or focal infections (endocarditis and meningitis). Neisseria meningitidis colonizes successfully the vascular wall and causes severe sepsis. Such infections result from an efficient colonization of host endothelial cells, a key step in meningococcal diseases which has been the subject of the work presented here. Endothelium colonization by N. meningitidis is a complex process implying bacterial adhesion and multiplication on the endothelial cell surface in the specific context of the bloodstream, where mechanical forces generated by the blood flow are applied on circulating bacteria. Even though many studies focused on the interaction between N. meningitidis and the endothelial cell, many aspects remain elusive, such as the impact of shear stress generated drag forces and the relative contribution of the two partners involved in this interaction.Adhesion to the endothelial cell surface is dependent on bacterial factors called type IV pili (Tfp) and leads to induction of a host cell response, characterized by a local remodeling of the plasma membrane and reorganization of actin cytoskeleton underneath bacterial microcolonies. First, we have shown that the cellular response induced by N. meningitidis actively participate in the colonization process. Indeed, membrane deformation allows contact with every bacterium inside the microcolony, which is necessary for microcolony resistance to mechanical forces. Additionally, we have demonstrated that the PilV protein, a Tfp component, is involved in plasma membrane remodeling and actin cytoskeleton reorganization. We designed a method combining high resolution live-cell fluorescence video-microscopy and fluorescence quantification to decipher the early events induced on contact of bacterial aggregates with the host cell surface. Using this technique we have shown that membrane remodeling does not rely on actin cytoskeleton reorganization but rather on intrinsic properties of the lipid bilayer. Second, we focused on latter steps of the infection process when initiation of a new colonization cycle is initiated. While firmly attached to the host cell surface through the membranous projections, some bacteria can detach from the microcolony to disseminate throughout the host. We have demonstrated the importance of post-translational modification of the major piline in this step and characterized the underlying mechanisms.This work allows refinement of the molecular mechanisms involved in the induction of the cellular response induced by N. meningitidis and its impact on successful endothelium colonization by this pathogen.

La mucoviscidose est causée par des mutations du gène CFTR (p.Phe508del étant la plus fréquente). Celui-ci code pour la protéine CFTR qui constitue un canal chlorure exprimé à la face apicale des cellules épithéliales. Au niveau du reticulum endoplasmique (RE), le contrôle de qualité conformationnelle oriente la majorité du CFTR en cours de repliement vers une voie de dégradation. Une fraction limitée du WT-CFTR parvient cependant à se replier correctement et peut ensuite progresservers la surface cellulaire, contrairement au Phe508del-CFTR (qui est néanmoins fonctionnel). Lorsque des formes mutées sont exportées à partir du RE, grâce à des traitements correcteurs, elles sont alors instables à la membrane plasmique. Par ailleurs, il a été montré que l'organisation des microfilaments d'actine participe à l'ancrage du canal au cytosquelette et à sa stabilité. Or, la petite GTPase Cdc42 influence la dynamique de nucléation de l'actine fibrillaire. Au cours de nos travaux, nous avons testé l'implication de Cdc42 et de certains de ses effecteurs dans la régulation de WT-CFTR dans des cellules épithéliales bronchiques. Dans ce cadre, la fonction de la voie Cdc42 a été perturbée par des traitements pharmacologiques et par ARN interférence. Les résultats obtenus, principalement par biotinylation de surface, ont permis de proposer que (1) la protéine Cdc42 participe à ladégradation de formes mal repliées de CFTR dans les étapes précoces et tardives de la voie de sécrétion et (2) la voie Cdc42, par son implication dans l'organisation de l'actine F corticale, affecte l’ancrage du canal chlorure au cytosquelette et régule ainsi son recrutement dans des vésicules d'internalisation. / Cystic Fibrosis is caused by CFTR gene mutations (p.Phe508del being the most frequently encountered). The CFTR protein functions as a chloride channel expressed at the plasma membrane of epithelial cells. Its productive folding in the endoplasmicreticulum (ER) is poorly efficient and unfolded proteins are therefore targeted to degradation. Nevertheless, a limited fraction of WT-CFTR acquires a native conformation and then progesses into the secretory pathway. In the case of Phe508del-CFTR, virtually all channels are degraded at this step except through corrector treatments. Under these conditions the mutant remains unstable at the plasma membrane (although it is functionnaly competent). Furthermore, it has been shown that fibrillar actin organization is involved in CFTR tethering to the cytoskeleton and channel stability. Moreover, the small GTPase Cdc42 promotes F actin nucleation. In the present study, we aimed at testing the involvement of Cdc42, and of some of its effectors, in WT-CFTR regulation in epithelial airway cells. In this context, Cdc42 pathway function was altered through pharmacological treatments or siRNAmediated depletions. Our results, mainly obtained via cell surface biotinylation assays, led us to propose that (1) Cdc42 is involved in misfolded CFTR degradation at early and late steps of the secretory pathway, and (2) Cdc42 pathway, through its F actin organization function, affects CFTR anchoring to the cytoskeleton and thus regulates its endocytosis.