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K. H. Svoboda, C. Cowan, S. Senkayi; YFP-Rho Responded to ECM and LPA Stimulation; Whereas, ROCK Inhibitor Blocked YFP-Rho Redistribution in Embryonic Corneal Epithelial Sheets. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2713.
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Chicken corneal epithelial sheets were transfected with chicken YFP-RhoA. The purpose of these experiments was to determine if the YFP-RhoA was functional in the whole epithelial sheets in vitro. Previously, we had demonstrated that chicken YFP-RhoA was expressed in the epithelial cells with Western Blots. We had also shown that the transfected epithelial sheets incubated with actin cortical mat stimulators: lysophosphatidic acid (LPA) and collagen (COL) caused a redistribution of YFP-RhoA. We have also shown that blocking the RhoA downstream kinase, ROCK prevented actin reorganization in the presence of LPA. In these experiments we inhibited ROCK to determine if the intracellular distribution of YFP-RhoA was changed. In addition, we determined if the YFP-RhoA could bind GTP to become activated.
Chicken RhoA RNA was isolated from corneal epithelia extracts, and cloned into an eYFP-N1 plasmid vector (BD Biosciences). Primary chicken corneal epithelial sheets were isolated from E8 chicks and cultured. Epithelial sheets were stimulated with COL or LPA. Cells were counter stained with phalloidin or a nuclear stain (ToPro). ROCK activity was inhibited with Y27632 (3um, EMD Biosciences). The GTP state of YFP-RhoA was tested using a RhoA activation assay (Cytoskeleton) in combination with a primary antibody against YFP (JL-8 BD Biosciences).
LPA mediated YFP-RhoA stimulation increased YFP-RhoA co-localization with actin at the cell membranes. ROCK inhibition blocked actin polymerization and cell membrane localization of YFP-RhoA.
YFP-RhoA retained functional activity, demonstrated by movement to the membrane after LPA and COL stimulation. Blocking ROCK prevented YFP-RhoA from localizing to the cell surface.
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