OCCLUDIN IS A FUNCTIONAL COMPONENT OF THE TIGHT JUNCTION

Occludin's role in mammalian tight junction activity was examined by ' labeling' the occludin pool with immunologically detectable chick occl udin, This was accomplished by first transfecting MDCK cells with the Lac repressor gene. Hyg(R) clones were then transfected with chick occ ludin cDNA inserted into a Lac operator construct. The resulting Hyg(R )/Neo(R) clones were plated on porous inserts and allowed to form tigh t junctions. Once steady state transepithelial electrical resistance w as achieved, isopropyl-beta-D-thiogalactoside was added to induce chic k occludin expression, Confocal laser scanning microscopy of monolayer s immunolabeled with Oc-2 monoclonal antibody revealed that chick occl udin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canin e ZO-1 co-localized to punctate sites in the cytoplasm suggesting thei r association within the same vesicular structures. In low calcium med ium both proteins also colocalized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of addi ng isopropyl-beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coin cided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electr ical resistance, above control steady state values, began five hours a fter addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isop ropyl-beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; tran sepithelial electrical resistance also returned to baseline values dur ing this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cad herin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected t he process of tight junction assembly, monolayers in low Ca2+ medium w ere treated with isopropyl-beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly, Chick occl udin did not alter the rate at which transepithelial electrical resist ance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent, By freeze fractu re analysis, the number of parallel tight junction strands shifted fro m a mode of three in controls to four strands in cells expressing chic k occludin and the mean width of the tight junction network increased from 175+/-11 nm to 248+/-16 nm. Two days after plating confluent mono layers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With cont inued incubation with the inducing agent, mannitol flux increased on d ay 11 to 50%, and TER rose to 45% above controls. Both of these change s were reversible upon removal of isopropyl-beta-D-thiogalactoside. Th ese data are consistent with the notion that occludin contributes to t he electrical barrier function of the tight junction and possibly to t he formation of aqueous pores within tight junction strands.