The experiments outlined in the proposal will continue the molecular characterization of the zonula occludens (ZO) or tight junction. This intercellular junction is responsible for maintaining and regulating the transepithelial permeability barrier between the two extracellular compartments defined by an epithelium. The tight junction also participates in maintenance of polarized membrane domains characteristic of an epithelial cell. The majority of the studies outlined here focus on the regulation and assembly of the tight junctions in the intestinal epithelial cell. One objective is to define the molecular basis for the """"""""loosening"""""""" of intestinal tight junctions by cells such as polymorphonuclear leukocytes as they invade the intestinal mucosa during an inflammatory response. This process can be modeled in vitro by addition of the cytokine, interferon-gamma (IFN-g) to cultured monolayers of the intestinal cell line T-84. Studies are proposed to test several hypotheses for regulation of tight junction permeability by IFN-g in cultured intestinal cell monolayers. Direct effects on the tight junction protein, ZO-1, including changes in its biosynthetic profile (transcription; stability), isoform expression, and phosphorylation state will be examined, in comparison to a number of other 'differentiation' specific proteins of the enterocyte brush border (BB) membrane and cytoskeleton. Using video-enhanced light microscopy, we will monitor IFN-g effects on junctional contractility in vivo to test the hypothesis that junctional permeability is regulated by the actomyosin ring associated with the adherens junction. A second objective is to define early molecular events in the assembly of the tight junction and polarized membrane skeletal assembly in cultured intestinal cells. This will be done by a series of paired time-lapse video and immunocytochemical studies, using immuno-probes for tight junction (ZO-1, cingulin), and BB proteins. Finally, pilot studies are proposed to assess genetic and molecular genetic approaches for the dissection of the tight junction. A series of experiments will further characterize a ZO-1 immunogen expressed in the CNS of the Drosophila where glial cell tight junctions form the equivalent of a blood-brain barrier. Studies to verify the expression of a ZO-1 homolog, together with initial genetic characterization are proposed.
