Tensin, paxillin and focal adhesion kinase may be involved in actin reorganization stimulated by ECM in corneal epithelia
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Purpose. Embryonic Avian corneal epithelial tissue isolated without basal lamina (-BL) responds to extracellular matrix (ECM) molecules using an actin dependent mechanism. The basal cell surface flattens and the F-actin organizes into microfilament bundles (actin cortical mat, ACM) in the presence of laminin (LM), fibronectin (FN) or collagen (COL). The ACM has different configurations in the presence of the three different ECM molecules. Epithelia required contact with ECM for 15 min. for the ACM to reform in 2hrs. In addition, ACM reorganization could be blocked by phosphotyrosine (p-tyr) inhibitors, genistein and herbimycin A. Using western blot analysis, focal adhesion kinase (FAK) and paxillin (PAX) protein levels appeared the same with or without ECM, however the level of p-tyr proteins increased after 15 min. of ECM incubation. Our current goals were: 1. Can we increase the sensitivity of the assay to determine the p-tyr changes in FAK, tensin and PAX? 2. Does tensin or paxillin have a role in the reorganization of the ACM? Methods. Proteins with p-tyr residues were immunoprecipitated (using PY20) then examined by western blot analysis (FAK, PAX and tensin). Lysates were collected from epithelia isolated -BL and cultured in the presence of control media, +50 ug/ml FN, or +100ug/ml COL from 5 min. to 2hrs. The intracellular distribution of tensin, PAX and FAK were determined with immunohistochemistry and confocal microscopy. Results. PAX, FAK, tensin and other proteins were immunoprecipitated with anti p-tyr antibodies in the presence of all ECM molecules tested. FAK was concentrated near cell-cell and cell-matrix junction areas in both periderm and basal cells. PAX had a similar distribution to FAK, however, it was not in the periderm layer. Tensin was localized to the cell-cell junctions in the periderm cells and at the cell-matrix attachment complex (CMAX) in the basal cells. Conclusions. FAK, PAX and tensin were localized in the CMAX and were phosphorylated in embryonic epithelia.
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
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Svoboda, K., & Orlow, D. L.
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