Implication des Rho GTPases dans la survie cellulaire et l'adhérence : caractérisation d'une nouvelle voie d'activation de la Kinase Akt par Rac

Les RhoGTPases régulent une multitude de fonctions cellulaires. Les travaux présentés dans cette thèse mettent en évidence les interrelations entre deux phénomènes majeurs contrôlés par cette classe de molécules que sont la régulation de l’organisation du cytosquelette d’actine et la survie cellulaire. L’adhérence à la matrice extracellulaire joue un rôle actif dans la survie cellulaire essentiellement grâce à la kinase Akt qui assure la protection contre l’apoptose. La 1ère partie de ce travail a permis de caractériser une nouvelle voie d’activation d’Akt par la GTPase Rac, sollicitée dans des situations où l’interaction avec la matrice est réduite. Mise en évidence dans des cellules non adhérentes, nos travaux montrent que cette voie est également opérationnelle dans des cellules adhérentes mises en suspension. Ces observations sont supportées par la mise en évidence, in vitro, d’un complexe contenant Akt et Rac activées dans les cellules en suspension. Dans la 2ème partie, nous avons exploré le rôle des RhoGTPases dans la morphologie de cellules leucémiques atypiques, qui présentent des prolongements cytoplasmiques caractéristiques, les Tricholeucocytes. Nous démontrons une relation entre cette morphologie et un fort taux d’activation des GTPases Rac et Cdc42. Le traitement de ces cellules à l’IFN normalise ces activités par un mécanisme faisant intervenir RhoA et p53. Dans la 3ème partie, nous nous sommes intéressés à des structures d’adhérence appelés podosomes dont la formation est dépendante des GTPases Cdc42 et RhoA. Nous avons montré pour la première fois, l’existence de ces structures in vivo.RhoGTPases control a multitude of cellular functions. The work presented in this thesis highlights the relationships between two major phenomena controlled by this class of molecules which are the regulation of the organization of the actin cytoskeleton and cellular survival. Adhesion to the extracellular matrix plays an essential role in cellular survival thanks to the Akt kinase which ensures protection against the apoptose. The first part of this work led us to characterize a new pathway of activation of Akt by the GTPase Rac, requested when the interactions with the matrix are reduced. Highlighted in nonadherent cells, our work shows that this way is also operational in adherent cells grown in suspension. These observations are supported by the description, in vitro, of a complex containing Akt and Rac activated in the cells in suspension. In the 2nd part, we explored the role of RhoGTPases in the morphology of atypical leukemic cells, the Tricholeucocytes which present characteristic cytoplasmic protrusions. We show a relation between this morphology and a strong rate of activation of GTPases Rac and Cdc42. Treatment of these cells by IFN normalize the activities by a mechanism RhoA and p53 dependant. In the 3rd part, we were interested in structures of adherence called podosomes on which formation is dependent on GTPases Cdc42 and RhoA. We showed for the first time, the existence of these structures in vivo

CytoBinning: Immunological insights from multi-dimensional data

New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses

Human Immunodeficiency Virus Type 1 Vif causes dysfunction of Cdk1 and CyclinB1: implications for cell cycle arrest

The two major cytopathic factors in human immunodeficiency virus type 1 (HIV-1), the accessory proteins viral infectivity factor (Vif) and viral protein R (Vpr), inhibit cell-cycle progression at the G2 phase of the cell cycle. Although Vpr-induced blockade and the associated T-cell death have been well studied, the molecular mechanism of G2 arrest by Vif remains undefined. To elucidate how Vif induces arrest, we infected synchronized Jurkat T-cells and examined the effect of Vif on the activation of Cdk1 and CyclinB1, the chief cell-cycle factors for the G2 to M phase transition. We found that the characteristic dephosphorylation of an inhibitory phosphate on Cdk1 did not occur in infected cells expressing Vif. In addition, the nuclear translocation of Cdk1 and CyclinB1 was disregulated. Finally, Vif-induced cell cycle arrest was correlated with proviral expression of Vif. Taken together, our results suggest that Vif impairs mitotic entry by interfering with Cdk1-CyclinB1 activation

CytoBinning:Immunological insights from multi-dimensional data

<div><p>New cytometric techniques continue to push the boundaries of multi-parameter quantitative data acquisition at the single-cell level particularly in immunology and medicine. Sophisticated analysis methods for such ever higher dimensional datasets are rapidly emerging, with advanced data representations and dimensional reduction approaches. However, these are not yet standardized and clinical scientists and cell biologists are not yet experienced in their interpretation. More fundamentally their range of statistical validity is not yet fully established. We therefore propose a new method for the automated and unbiased analysis of high-dimensional single cell datasets that is simple and robust, with the goal of reducing this complex information into a familiar 2D scatter plot representation that is of immediate utility to a range of biomedical and clinical settings. Using publicly available flow cytometry and mass cytometry datasets we demonstrate that this method (termed CytoBinning), recapitulates the results of traditional manual cytometric analyses and leads to new and testable hypotheses.</p></div

The Necrotic Signal Induced by Mycophenolic Acid Overcomes Apoptosis-Resistance in Tumor Cells

The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42.Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers.These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells

Wild-Type p53 Enhances Endothelial Barrier Function by Mediating RAC1 Signalling and RhoA Inhibition

Inflammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 orchestrates the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho-cofilin. 17AAG treatment resulted in reduced levels of active cofilin. Silencing of cofilin pyridoxal phosphate phosphatase (PDXP) blocked the LPS-induced hyper-permeability, and P53 inhibition reversed the 17AAG-induced PDXP down-regulation. P190RHOGAP suppression enhanced the LPS-triggered barrier dysfunction in endothelial monolayers. 17AAG treatment resulted in P190RHOGAP induction and blocked the LPS-induced pMLC2 up-regulation in wild-type mice. Pulmonary endothelial cells from super p53 mice, which carry additional p53-tg alleles, exhibited a lower response to LPS than the controls. Collectively, our findings help elucidate the mechanisms by which p53 operates to enhance barrier function

Importance of the difference in surface pressures of the cell membrane in doxorubicin resistant cells that do not express Pgp and ABCG2

P-glycoprotein (Pgp) represents the archetypal mechanism of drug resistance. But Pgp alone cannot expel drugs. A small but growing body of works has demonstrated that the membrane biophysical properties are central to Pgp-mediated drug resistance. For example, a change in the membrane surface pressure is expected to support drug–Pgp interaction. An interesting aspect from these models is that under specific conditions, the membrane is predicted to take over Pgp concerning the mechanism of drug resistance especially when the surface pressure is high enough, at which point drugs remain physically blocked at the membrane level. However it remains to be determined experimentally whether the membrane itself could, on its own, affect drug entry into cells that have been selected by a low concentration of drug and that do not express transporters. We demonstrate here that in the case of the drug doxorubicin, alteration of the surface pressure of membrane leaflets drive drug resistance

Exposed Hydrophobic Residues in Human Immunodeficiency Virus Type 1 Vpr Helix-1 Are Important for Cell Cycle Arrest and Cell Death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity