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Identification of PKA-dependent phosphoproteins promoting cytoskeleton reorganization during cell fusion and tumor progression

proposé par Guillaume PIDOUX, UMR-S-1139 : PhysioPathologie et PharmacoToxicologie Placentaire

Projet de stage : Cell fusion processes are complex biological phenomena essential in fertilization, fetal development, skeletal muscle formation and bone homeostasis. Moreover, cell fusion has recently been shown to play a role in the metastatic process. Cell fusion and syncytial formation result in the mixing of plasma membrane components and merging of cell content between two or more cells. Although occurring in different biological contexts, cell fusion processes share many of the same steps. Important cytoskeleton reorganization is needed to commit cell fusion. However, the precise role of cytoskeleton in cell fusion remains mysterious. Specifically, how could organization of the intracellular cytoskeleton and its spatiotemporal regulation affect plasma membrane dynamics and fusion. Despite the paucity of studies on cell fusion in human cancer, accumulating experimental evidence in recent years has suggested a possible broad involvement of cell fusion during the initiation, progression, and phenotypic diversification of cancer. Our recent data on carcinoma cell line (BeWo) suggested that the fusion process is associated with a degradation of total actin protein as well as modification in F/G actin ratio. The aim of this project is to identify by the use of state of the art proteomic analysis PKA-dependent phosphoproteins involve in the cell fusion process. First, a time course of PKA activity will be performed on stimulated BeWo cells in order to determine the time point corresponding to the maximum of PKA activity. Then at this determined time point, cAMP/PKA signaling will be activated on BeWo cells and subsequently phosphoproteins contained in cytoskeleton fraction will be enriched on TiO2 column prior to mass spectrometry analysis. A better understanding of these mechanisms could putatively propose new targets in order to block tumor progression by inhibiting cell fusion and cytoskeleton reorganization.

Techniques mises en œuvre par le stagiaire : Culture cellulaire : culture de lignée BeWo (carcinome) Spectrométrie de masse Biochimie : immunoblot, immunocytochimie, pharmacocinétique

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