The long-term objective of this grant is to test the hypothesis that corneal epithelial cells have specialized cell-cell and cell-matrix domains that are responsible for stability, differentiation and communication. A specialized basal-lateral domain in the intact embryonic corneal epithelial tissue that contains many actin associated and signaling proteins has been defined. The intracellular protein distribution in this domain is filamentous-actin dependent. The next logical set of experiments involves further characterization of this cell matrix attachment complex and the signal transduction pathways initiated by extracellular matrix that lead to tissue viability.
These specific aims will be addressed with whole avian embryonic epithelia isolated without basal lamina and cultured with extracellular matrix (ECM) molecules in the absence or presence of specific inhibitors or oligonucleotides that block synthesis of specific proteins.
Specific aims : I. Test the hypothesis that zyxin is a nucleating site for ECM stimulated actin bundle assembly. The actin associated protein, zyxin is homologous to an actin nucleating protein and it shuttles from the nucleus to fibroblast focal contacts. Zyxin has a polarized distribution in corneal epithelial sheets that is actin and ECM dependent. The sequence of zyxin and actin reassembly will be determined, then endogenous zyxin will be decreased with antisense oligonucleotides to establish if zyxin acts as a nucleation site for ECM stimulated actin bundle assembly. Zyxin binding proteins will be identified with co-immunoprecipitation, followed by western blot analysis. Zyxin s turnover rate and stability will be determined. II. Test the hypothesis that RhoGTP regulates actin reorganization and cytoplasmic retraction. Rho is a small signaling protein that shuffles between an inactive form (GDP) to an active form (GTP). One of the proteins that participates in changing the active state of Rho has been isolated from the embryonic corneal epithelium (p190RhoGAP). The current project will determine the role of this protein (p190RhoGAP) and other related downstream signaling proteins (ROCK, Dia, MAP kinase and P13 kinase) in extracellular matrix stimulated actin bundle formation. III. Test the hypothesis that epithelial cell-cell and cell-matrix interactions suppresses apoptosis. This model maintains cell-cell attachments while separating the cell-matrix attachment complex. The current project will determine if the stress-inducing signal transduction pathways become active in epithelia isolated without the basal lamina. Further experiments will determine if exogenous ECM can rescue epithelia isolated without the basal lamina or treated with signal transduction inhibitors. In summary, these experiments in our well defined corneal epithelial model will focus on how ECM mediated signal transduction stimulates actin bundle formation and also save the cells from programmed cell death.
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