No description available.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II (CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher concentrations (>= 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase (FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-phosphoTyr925 to a membrane-associated compartment. This study adds further insight into the Cd2+-mediated effects on the cytoskeleton and FAs.

No description available.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 160 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Vascular smooth muscle cells (VSMC) constitute an important component of blood vessels and are primarily responsible for vessel contraction. In vascular disorders such as hypertension and atherosclerosis as well as pregnancy and exercise, VSMC demonstrate increased capacity to proliferate and migrate, resulting in vascular remodelling. The actin cytoskeleton is an important component of vascular contractility and is also essential for proliferation and migration of VSMC. Vasoactive agonists such as Endothelin-1 (ET-1) and Noradrenaline (NA), have been shown to mediate VSMC contraction through changes in actin cytoskeleton and focal adhesion (FA) remodelling, and have also been reported to cause VSMC migration in the appropriate setting. The aim of this study was to investigate the signalling mechanisms responsible for FA dependent actin cytoskeleton remodelling of VSMC in response to ET-1 and NA, with a special focus on Hydrogen peroxide-inducible clone 5 (Hic-5). The latter is a FA protein shown to regulate actin cytoskeleton dynamics in small arteries in response to Noradrenaline (NA) and the response of VSMC to arterial injury and abdominal aortic aneurysm. We have shown that Src-dependent tyrosine phosphorylation of Hic-5 regulated its subcellular localisation in mouse embryonic fibroblasts and VSMC, but was not responsible for the effects of ET-1 and NA on actin filament remodelling or Hic-5 redistribution in VSMC. ET-1 stimulation caused an increase in Hic-5 localisation at FAs concurrent with an increase in the density of actin filaments, whereas NA stimulation caused a decrease in Hic-5 localisation at FAs in VSMC concurrent with actin filament redistribution at the cell cortex. Hic-5 was the FA protein that demonstrated the most dramatic changes in subcellular localisation in response to ET-1 and NA, when compared to paxillin (Hic-5 homologue) or vinculin (classical FA marker). NA-mediated changes in Hic-5 localisation and actin filament distribution were more pronounced compared to ET-1-mediated changes. Further investigation into the NA-induced changes suggested that actin filament disassembly preceded Hic-5 relocalisation from FAs to the cytosol. These results show that vasoactive peptides cause Hic-5 relocalisation and actin filament rearrangement in VSMCs in an agonist-dependent manner. Given that VSMC FA remodelling and actin cytoskeleton reorganisation occur during contraction and arterial remodelling, our data identify Hic-5 as a key regulator of these processes in response to NA and ET-1. Furthermore, these data have implications in agonist- specific VSM function such as migration and contraction.

In cell culture, entry into mitosis of many adherent mammalian cells is accompanied by substantial changes in cellular architecture. Flat, spread-out interphase cells detach from the extracellular matrix and become more spherical. These changes in cell shape are mediated by rearrangements in the actin cytoskeleton, a dynamic network of actin filaments that are organized by actin-binding proteins. Filamin A (FLNa) is a 280 kD actin-binding protein that crosslinks actin filaments into parallel bundles or three-dimensional orthogonal networks. We previously identified FLNa as an in vitro substrate of cyclin-dependent kinase 1 (Cdk1), a kinase that regulates entry into mitosis, and hypothesized that Cdk1 phosphorylation of FLNa regulates mitotic actin remodelling. Using mass spectrometry and a p-FLNa antibody, we show that FLNa is phosphorylated in vivo in HeLa cells on multiple Cdk1 sites, including serines 1084, 1459 and 1533. All three sites match the phosphorylation consensus sequence of Cdk1. We further show that p-FLNa is almost fully dephosphorylated by anaphase, consistent with it being a cell cycle-regulated substrate. Using a phospho-specific antibody, we find that p-FLNa has decreased cortical actin localization compared to total FLNa in mitotic cells. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine and expressed this FLNa mutant (FLNa-S1084A, S1459A, S1533A, referred to as “FLNa-AAA GFP”) in FLNa-deficient human M2 melanoma cells. FLNa-AAA GFP-expressing cells have enhanced FLNa-AAA GFP localization at sites of contact between daughter cells and this correlates with defects in cell division and impaired cell migration. Therefore, mitotic delocalization of cortical FLNa is critical for successful cell division and interphase cell behaviour.

Les GTPases de la famille Rho sont les principaux régulateurs du remodelage du cytosquelette d’actine lors de la migration et l’invasion cellulaire. En particulier, deux membres de cette famille sont importants dans ce processus : les GTPases RhoA et Rac1. En effet, il existe une balance d’activité de ces GTPases, responsables respectivement de la contraction cellulaire et de la formation d’extensions cytoplasmiques, des étapes clefs de la migration. L’objectif de ce travail de thèse a été d’étudier la régulation de ces protéines dans la migration et l’invasion cellulaire. Pour cela, plusieurs stratégies ont été entreprises. Tout d’abord, une étude structure/fonction de la protéine p190RhoGAP-A (p190A), un des régulateurs majeurs de la GTPase RhoA, a été réalisée. Cette étude a permis de mettre en évidence un domaine, appelé PLS pour « protrusion localization sequence », permettant à cette protéine de se localiser au niveau des extensions membranaires appelées « replis membranaires » et « lamellipodes » où RhoA est régulée localement. D’autre part, un mutant délété de ce domaine, appelé PLSp190A, ne peux pas se localiser au niveau de ces structures et a un impact négatif sur leur formation et la migration cellulaire. De plus, l’analyse de ce mutant a révélé que le domaine PLS était impliqué dans la régulation négative de p190A. Ainsi, nous avons mis en évidence un nouveau domaine de p190A responsable de sa localisation intracellulaire et de sa fonction. La deuxième partie de ce travail de thèse a été consacrée à la mise en place d’un outil de mesure de l’activité des GTPases Rho par la technologie Alphascreen. Ce test a permis de mesurer l’activité de Rac1 in vitro et in cellulo mais a également été appliqué à un crible en vue d’identifier de nouvelles molécules régulatrices de Rac1. Ainsi, ce travail de thèse, en abordant par plusieurs angles la régulation des GTPases de la famille Rho, a permis d’apporter des informations et des outils pour la compréhension des mécanismes complexes régissant la capacité des cellules à se mouvoir dans leur environnement. / RhoGTPases are major regulators of the actin cytoskeleton during cell migration and invasion. Particularly, the two members of the RhoGTPase family, RhoA and Rac1 play important roles in these processes. Indeed, a reciprocal balance between these GTPases’activity that leads to cell contraction and cell protrusion formation, determines cell movement. The aim of this PhD thesis was to study the regulation of RhoA and Rac1 during cell migration and invasion. To this end, various strategies were undertaken.We first performed a structure/function analysis of p190RhoGAP-A (p190A), a major negative regulator of RhoA. This led to the identification of a protrusion localization sequence (“PLS”) necessary and sufficient for p190A targeting to actin-based structures. A p190A mutant deleted of the PLS domain (PLS), does not localize to ruffles and lamellipodia, where RhoA is locally regulated during cell migration. This analysis also revealed that the PLS is required for the negative regulation of p190A activity. Finally, p190APLS expression has a dominant negative effect on the formation of actin protrusions and cell migration. Thus, we identified a novel functional domain of p190A required for its proper subcellular localization and functions. The second part of this PhD thesis was focused on the design of an Alphascreen technology-based assay to study GTPases activity. This assay allowed the measurement of Rac1 activity in vitro and in cellulo. Moreover, we used this assay to screen for new regulators of Rac1 activity. In conclusion, this work provides new insights and new tools for the understanding of RhoGTPase involvement in cell migration.

Le phéochromocytome (PCC) est une tumeur neuroendocrine rare qui se développe principalement aux dépens des cellules chromaffines de la médullo-surrénale. Dans la majorité des cas, les PCCs sont caractérisés par une hypersécrétion de catécholamines responsables de divers effets délétères chez les patients dont le principal est une hypertension (phéochromocytomes symptomatiques, PS). Cependant, il existe également une forme particulière de PCCs asymptomatiques qui sécrètent des taux physiologiques de catécholamines (phéochromocytomes incidentaux, PI). Parmi les patients porteurs de PI, certains sont hypertendus (PIH) et d’autres non (PIN). Afin de mieux caractériser les différents profils sécrétoires de PCCs (PS et PI), nous avons recherché une implication potentielle des microARNs (miRNAs). Nous avons réalisé une analyse transcriptionnelle des miRNAs exprimés dans 32 échantillons de PCCs (12 PS, 12 PIN et 8 PIH). Le miRNome a été réalisé par qRT-PCR microfluidique (Taqman Low Density Array, TLDA) pour 671 miRNAs. L’analyse statistique (Limma) des données d’expression a permis d’identifier 4 miRNAs significativement sur-exprimés (hsa-miR-7-1-3p, 7-2-3p, 26a-1-3p et 550a-3p) et 3 miRNAs sous-exprimés (497-3p, 32-5p, 190b-5p) dans les tumeurs PIN par rapport aux PS. Pour identifier les cibles potentielles des miRNAs, de nombreux logiciels de prédictions bioinformatiques sont disponibles en ligne mais les résultats qu’ils génèrent sont très divergents. Afin de contourner ce problème nous avons développé miRabel, un nouvel outil de prédiction des cibles potentielles des miRNAs et des fonctions biologiques qui leurs sont associées. Le principe général consiste à agréger les résultats de 3 autres algorithmes de prédiction sélectionnés pour leur complémentarité. Au final, les analyses des courbes ROC (Receiver Operating Characteristic), de la précision et du Recall ont montré que cet outil est plus efficace i) que les algorithmes qu’il agrège et ii) que d’autres logiciels de prédictions couramment utilisés tels que miRWalk, MBSTAR et TargetScan. Une analyse d'enrichissement (Modular Enrichment Analysis ou MEA, Genecodis3) des cibles prédites pour les miRNAs différentiellement exprimés a révélé qu’ils peuvent moduler significativement l’activité de quelques dizaines de voies de signalisation dont celles du cytosquelette d’actine et des SNAREs (impliquées dans le transport vésiculaire). En se basant sur l’expression des miRNAs, leurs énergies d’hybridation avec leurs cibles ainsi que leurs effets physiologiques potentiels, les ARNm des gènes PAK3, MLCP, MLCK (cytosquelette d’actine), SNAP25 et STX1A (SNAREs) ont été retenus pour la suite de l’étude. Les essais luciférases ont mis en évidence une interaction entre la totalité de l’extrémité 3’UTR des ARNm de MLCK et miR-32, STX1A et miR-550a-3p, SNAP25 et miR-7-1-3p ainsi que miR-550a-3p. Les autres interactions testées se sont révélées négatives. Les analyses par RT-qPCR ont montré une diminution significative du niveau d’ARNm de MLCK et de STX1A suite à la transfection de miR-32-5p et miR-550a-3p respectivement. Concernant SNAP25, un effet inhibiteur de miR-550a-3p / 7-1-3p est observé. Cet effet a été confirmé au niveau protéique pour STX1A et SNAP25. / Pheochromocytomas (PCC) are rare neuroendocrine tumors which arise from chromaffin cells of the adrenal medulla. In most cases, PCCs are characterized by a hypersecretion of catecholamines, which is responsible for most of deleterious effects in the patients with hypertension being the main symptom (symptomatic pheochromocytomas, SP). However, some PCCs are asymptomatic and secrete physiological levels of catecholamines (Incidental Pheochromocytomas, IP). Among patients with an IP, some are hypertensive (HIP) and other are strictly normotensive (NIP). In order to better understand the different secretory profiles of PCCs (SP and IP), we investigated the potential role of microRNAs (miRNAs) in this process. We started by identifying differentially expressed miRNAs between 12 SP, 12 NIP and 8 SP. The miRNome was done by microfluidic qRT-PCR (Taqman Low Density Array, TLDA) for 671 miRNAs. Statistical analysis (Limma) of the expression results identified 4 miRNAs significantly over-expressed (hsa-miR-7-1-3p, 7-2-3p, 26a-1-3p et 550a-3p) and 3 under-expressed (497-3p, 32-5p, 190b-5p) in NIP tumors when compared to SP. To identify potential miRNAs’ targets, numerous bioinformatic prediction methods are available but their results are quite divergent. To circumvent this issue, we developed miRabel, a new miRNAs’ targets prediction tool and their associated biological functions. MiRabel aggregated the results of 3 other prediction algorithms selected for their features complementarity. The analysis of ROC, precision and recall curves showed that this tool is more efficient i) than the aggregated prediction methods and ii) than other recent or widely used tools such as miRWalk, MBSTAR and TargetScan. A Modular Enrichment Analysis (MEA, Genecodis3) of the miRNAs’ predicted targets revealed that they could potentially regulate the activity of a few pathways of which the actin cytoskeleton and the SNAREs (involved in vesicular transport). PAK3, MLCP, MLCK (Actin cytoskeleton), SNAP25 and STX1A (SNAREs) were selected to be experimentally validated based on miRNA’s expression, hybridization energy and potential physiological impact. Experimental validations of the selected interactions are achieved by luciferase gene reporter, RT-qPCR assays and western-blots following the transfection of studied miRNAs. Luciferase assays showed a direct interaction between the whole 3’UTR of MLCK mRNA and miR-32-5p, STX1A and miR-550a-3p, SNAP25 and miR-7-1-3p as well as miR-550a-3p. The other tested interactions came out to be negative. A significant decrease of MLCK mRNA and STX1A were observed by RT-qPCR analysis after transfecting miR-32-5p and miR-550a-3p respectively. As for SNAP25, the inhibitory effect of miR550a-3p/7-1-3p could be observed. This effect was confirmed at the protein level by western-blots for STX1A and SNAP25. We then evaluated the physiological effect of miR-550a-3p/7-1-3p on the regulated secretion of PC12 rat PCC cells. This was achieved using a nano-luciferase fused to growth hormone 1 (GH1). Once stimulated (59 mM potassium and 2 mM barium), miR-550a-3p over-expression decreased the secretory capacity of PC12 cells while miR-7-1-3p could not. This project represents the first study aiming to understand the regulation of the catecholamine secretion pathway by miRNAs in the pathophysiological context of PCC patients. Eventually, the characterization of this miRNA’s network should improve patient care in the field of hypersecreting neuroendocrine tumors.

Les cellules multiciliées (MCC) possèdent à leur surface apicale des centaines de cils mobiles générant un flux directionnel liquidien nécessaire par exemple pour le nettoyage des voies respiratoires. La fabrication de ces cils (multiciliogénèse) requiert une séquence d’évènements cellulaires dont un arrêt du cycle cellulaire, une réorganisation du réseau apical d’actine, une multiplication massive des centrioles suivie de leur migration au pôle apicale et de leur maturation en corps basal, à partir desquels les cils s’allongent.Mon laboratoire d’accueil a mis en évidence le rôle conservé de la famille de microARN miR-34/449 dans le contrôle de la multiciliogénèse en inhibant la voie de signalisation Notch ainsi qu’en induisant un arrêt du cycle. Au cours de ma thèse, j’ai mis en évidence un nouveau niveau de régulation de ces microARN par lequel ils contrôlent la réorganisation apicale du cytosquelette d’actine, en modulant l’expression et l’activité de certaines petites GTPases. Par ailleurs, j’ai identifié et caractérisé des séquences variantes des miR-34/449 canoniques, appelées isomiR. Tandis que ces isomiR partagent des fonctions semblables à celles de leurs homologues canoniques, ils apportent également une complémentarité d’action en modulant des transcrits cibles spécifiques. Enfin, le dernier axe de mon travail a permis d’identifier le rôle de la voie de signalisation BMP dans la multiciliogénèse ainsi que d’élucider certains des mécanismes moléculaires par lesquels elle contrôle ce phénomène. L’ensemble de nos découvertes offre une opportunité inédite pour développer des stratégies thérapeutiques dans le traitement de maladies associées à des désordres ciliaires / Vertebrate multiciliated cells (MCC) project hundreds of motile cilia at their apical surface which coordinately beat to generate a directional fluid flow necessary for many biological functions including airway cleansing. Biogenesis of multiple cilia (multiciliogenesis) follows different key cellular steps corresponding to a cell cycle arrest, a massive multiplication of centrioles which then migrate to the apical surface to form basal bodies, from which cilia elongate. In 2011, my host laboratory evidenced that the miR-34/449 family of microRNAs control vertebrate multiciliogenesis by inducing the cell cycle arrest and by repressing the Notch pathway. My thesis work has revealed a new role of miR-34/449 by demonstrating that they modulate expression and activity of small GTPases to drive the apical reorganization of the actin network, a prerequisite for basal body anchoring. Besides, I have identified and characterized variant sequences of canonical miR-34/449 family, named isomiRs. Whereas these isomiRs share common biological functions with canonical miR-34/449 miRNAs, they may also contribute to a complementary effect by targeting specific transcripts. Finally, the last part of my work has contributed to the identification of the conserved role of the BMP pathway in the control of multiciliogenesis. I have evidenced some molecular mechanisms by which the BMP signal controls this phenomenon. Importantly, I demonstrated that BMP inhibition promotes regeneration of tracheal MCC in vivo in an asthmatic mouse model. Overall, our findings offer an unprecedented opportunity to develop novel therapeutic strategies to treat diseases associated with ciliary disorders