The experiments outlined in this proposal are designed to study the cell and molecular biology of the zonula occludens, or tight junction. This intercellular junction is responsible for forming a semi-permeable barrier in the paracellular pathway in most vertebrate epithelia. The junction forms the basis of blood-tissue barriers in brain, thymus and testis, and constitutes the normal seal permitting epithelial cells to boundary separate physiological compartments. ZO-1, the first protein to be described as part of the structure of the zonula occludens has been identified and characterized. Based on these studies, the present proposal will focus on two specific aims. In the first, ZO-1 will be used as a specific probe to identify additional molecules associated with the tight junction, with the long-term aim of providing a complete molecular description of this intercellular junction. Particular emphasis will be placed on identifying integral membrane proteins which specifically associate with ZO-1. Two strategies for identifying these proteins are planned. Isolated, purified ZO-1, which has been demonstrated to be competent to rebind to tight junctions at the morphologically correct sites, will be sepharose- coupled for affinity chromatography. ZO-1-depleted tight junction membranes, solubilized with detergents known not to interfere with Z0-1/plasma membrane binding, will be reacted with these ZO-1- coupled beads, and specific binding of membrane proteins assayed. The second strategy will be to directly solubilize ZO-1/membrane protein assemblies under conditions which do not extract ZO-1 from native binding sites, and to gradient isolate and immune precipitate these assemblies and study their protein composition. In the second specific aim of this proposal, the cell biology of ZO-1 will be studied in bead-loaded MDCK cells, wherein cells in monolayer culture may be intracellularly loaded with anti-ZO-I antibodies, and the effects of these antibodies on the biogenesis and stabilization of transepithelial permeability and epithelial cell polarity directly demonstrated.
