A hallmark of cell migration is the reorganization of the actin based cytoskeleton. The Rho-Rho Kinase (ROCK) pathway has been implicated in the process of actin remodeling because of its many downstream targets all being effectors of cell migration. The Rho/ROCK pathway negatively regulates the actin-severing protein cofilin via Ser3 phosphorylation. We have previously shown an increase of parietal endoderm outgrowth upon ROCK inhibition by use of Y-26732, a highly specific and potent chemical inhibitor of ROCK. In addition, ROCK inhibited cells subjected to western analysis exhibited increased levels of dephosphorylated cofilin.
To further investigate the role of cofilin in actin-mediated migration, we used indirect immunofluorescence to verify localization of coflin in Parietal Endoderm. Our data suggest uniform distribution of cofilin throughout the cytoplasm. We have also observed areas of increased intensity which may be indicative of areas of increased localization.
To this aim, we plan to investigate the sub-cellular localization of phosphorylated and dephosphorylated cofilin in both wild type and ROCK-inhibited Parietal Endoderm outgrowth.