ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent

Previous studies demonstrated that corneal epithelial cells isolated withou t basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorgani zes. We hypothesize that the actin reorganization is initiated by intracell ular signaling mechanisms that includes tyrosine phoshporylation and activa tion of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. O ur goals were to develop a morphological assay to. test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have: the same configuration in response to different ECM mol ecules? 2) What is the minimum time ECM molecules need to be in contact wit h the tissue for the actin to reorganize? 3) Will blocking tyrosine phospho rylation inhibit reorganization of the actin? 4) Are known signal transduct ion proteins phosphorylated in response to soluble matrix molecules? The ac tin cortical mat demonstrated distinct bundle configurations in the presenc e of different ECM molecules. Soluble fibronectin accumulated at the basal cell:surfaces 75-fold over 30 min in a clustered pattern, The cells need co ntact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In. contrast, two substances that bind to heptahelical receptors to stimul ate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the ac tin bundles in 15-30 min. Focal adhesion: kinase; p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorg anization induced by ECM molecules. In conclusion, we have developed two mo rphological assays to examine the response of corneal epithelial cells to E CM molecules. In addition, actin bundle reorganization involved tyrosine ph osphorylation, MAP kinase, and PI3 kinase activation. Anat Rec 254:348-359, 1999. (C) 1999 Wiley-Liss, Inc.