Our long-term interest continues to be understanding adhesive interactions between epithelial cells and basement membranes, which regulate epithelial tissue formation and maintenance. Laminin-5 (Ln-5) is a unique basement membrane ligand for epithelial cells. We and others determined that no other extracellular matrix was as effective as Ln-5 in promoting efficient attachment and spreading of epithelial cells. However, behavior of cells in contact with Ln-5 may vary from static adhesion and hemidesmosome formation, to vigorous migration. We hypothsize that these apparently opposing functions of Ln-5 in vitro are the reflection of regulatory mechanisms underlying epithelial morphogenesis in vivo. The goal of this proposal is to elucidate these mechanisms. We hypothesize tht regultion of spreading, static adhesion and migration occurs at several levels, including structure/ function modulation of Ln-5 and of its receptors and modifications in the components of the focal complexes in contact with Ln-5. We are in an excellent position to study these mechanisms, since we have identified a source for purified rat Ln-5 protein, we are advanced in the characterization of integrins that bind Ln-5, and we have defined a system of paried cell types with either static or migratory behavior on ln-5. We will: 1) complete the structural and functional characterization of Ln-5, by a combination of recombinant DNA techniques, purification of intact or fragmented protein and functional epitope mapping; 2) thoroughly characterize its receptors, especially integrins, by binding and competition assays; 3) define and test a mechanistic model for the regulation of adhesion and migration on Ln- 5, using a series of assays measuring strength of adhesion, spreading, migration and motility in a system of paried cell lines; 4) define the regulatory role of molecular components of focal complexes by cDNA cloning, mutagenesis, transfection and testing in the cell behavior assays. By these approaches, we hope to better our understanding of molecular interactions occuring in the focal complexes of epithelial cells in contact with Ln-5, and to help elucidate the molecular mechanisms of cell spreading, static adhesion and migration on Ln-5, and their hierarchy in the context of epithelial tissue morphogenetic events. Clarifying mechanical and biochemical aspects of epithelial adhesion to basement membranes may engender a better understanding of human diseases caused by epithelial tissue degeneration, including cancer metastasis, and lay the foundations for rational approaches to tissue healing and regeneration.
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