Rôle de la protéine G Cdc42p et de son facteur d'échange Cdc24p dans la fusion cellulaire, au cours de la reproduction sexuée de la levure Saccharomyces cerevisiae

La croissance polarisée est un processus fondamental chez les eucaryotes, qui fait intervenir des protéines conservées de la levure à l'homme, notamment les protéines G de type Rho et leurs régulateurs. Cdc42 joue un rôle essentiel dans le contrôle de l'établissement et du maintien de la polarité cellulaire. Chez la levure Saccharomyces cerevisiae, Cdc42p possède un seul facteur d'échange, Cdc24p. Cdc42p et Cdc24p sont nécessaires pour l'orientation de la croissance pendant la conjugaison. L'objectif de ce travail de thèse est d'identifier d'autres fonctions de Cdc24p et de comprendre comment Cdc24p contrôle l'activation de Cdc42p, au cours de ce processus. A cette fin, nous avons identifié et caractérisé des mutants de cdc24 et de cdc42 spécifiquement déficients dans la conjugaison, en particulier cdc24-m6 et cdc42[V36M]. Ces mutants répondent normalement aux phéromones et orientent leur croissance en direction du partenaire, mais ont un défaut dans l'étape de fusion, ils accumulent les prézygotes. cdc24-m6 et cdc42[V36M] sont incapables de localiser une protéine requise pour la fusion, au niveau de la zone de contact entre les deux cellules, en dépit d'une exocytose normale. Nous avons pu établir que la fonction de Cdc42p dans la fusion ne fait pas intervenir la cascade des MAPKs et que Cdc42[V36M]p est capable d'interagir avec les effecteurs à domaine CRIB. La surexpression de Cdc24p dans les cellules cdc42[V36M], et de Cdc42[C18A]p (mutant " fast-cycling ") dans cdc24-m6, permet de compenser partiellement les défauts de fusion de ces mutants. Nos résultats indiquent que l'oscillation de Cdc42p entre les formes liées au GDP et au GTP est nécessaire à la fusion cellulaire, peut-être en activant ou en localisant la machinerie de fusion au site de contact.Polarized growth is a fundamental process in eukaryotic cells, which requires proteins conserved between yeast and mammals such as Rho GTPases and their regulators. Cdc42 plays a central role in controlling the establishment and maintenance of cell polarity. In the yeast, Saccharomyces cerevisiae, Cdc42p is activated by the guanine nucleotide exchange factor, Cdc24p. Cdc42p and Cdc24p are required for oriented growth during yeast mating. The goal of this study is to identify additional functions of Cdc24p and to understand how Cdc24p controls Cdc42p activation during this process. We identified and characterized two mating specific cdc24 and cdc42 mutants, cdc24-m6 and cdc42[V36M]. These mutants respond normally to pheromone and orient their growth towards a mating partner, yet are defective in cell fusion and accumulate prezygotes. cdc24-m6 and cdc42[V36M] cells do not correctly localize a protein required for cell fusion, to the cell contact region, despite normal exocytosis. This function of Cdc42p in cell fusion does not require the MAPK cascade, and furthermore Cdc42[V36M]p is able to bind CRIB domain containing effectors. Overexpression of Cdc24p in cdc42[V36M] cells, and overexpression of fast cycling Cdc42[C18A]p in cdc24-m6 cells, partially suppressed their fusion defects. Together, our results show that Cdc42p GDP/GTP cycling is necessary for cell fusion, perhaps in activating or localizing the fusion machinery at the site of cell contact. We have identified a new function of the Cdc42p GTPase module in polarization of the fusion machinery during yeast mating and we suggest that Cdc42p cycling might be regulated during cell fusion.NICE-BU Sciences (060882101) / SudocSudocFranceF

The exchange factor Cdc24 is required for cell fusion during yeast mating.

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating

Choroidal and peripapillary changes in high myopic eyes with Stickler syndrome

International audienceBackground: To compare different clinical and Spectral-Domain Optical Coherence Tomography (SD-OCT) features of high myopic eyes with Stickler syndrome (STL) with matched controls.Methods: Patients with genetically confirmed STL with axial length ≥ 26 mm and controls matched for axial length were included. The following data were obtained from SD-OCT scans and fundus photography: choroidal and retinal thickness (respectively, CT and RT), peripapillary atrophy area (PAA), presence of posterior staphyloma (PS).Results: Twenty-six eyes of 17 patients with STL and 25 eyes of 19 controls were evaluated. Compared with controls, patients with STL showed a greater CT subfoveally, at 1000 μm from the fovea at both nasal and temporal location, and at 2000 and 3000 μm from the fovea in nasal location (respectively, 188.7±72.8 vs 126.0±88.7 μm, 172.5±77.7 vs 119.3±80.6 μm, 190.1±71.9 vs 134.9±79.7 μm, 141.3±56.0 vs 98.1±68.5 μm, and 110.9±51.0 vs 67.6±50.7 μm, always P< 0.05). Furthermore, patients with STL showed a lower prevalence of PS (11.5% vs 68%, P< 0.001) and a lower PAA (2.2±2.1 vs 5.4±5.8 mm2, P=0.03), compared with controls.Conclusions: This study shows that high myopic patients with STL show a greater CT, a lower PAA and a lower prevalence of PS, compared with controls matched for axial length. These findings could be relevant for the development and progression of myopic maculopathy in patients with STL

Autophagy plays a critical role in the degradation of active RHOA, the control of cell cytokinesis, and genomic stability.

International audienceDegradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance

Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia

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Cdc42p GDP/GTP Cycling Is Necessary for Efficient Cell Fusion during Yeast Mating

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP–GTP cycling is critical for efficient cell fusion