A dominant mutant of occludin disrupts tight junction structure and function

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is a n integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular s eal. In order to elucidate the role of occludin at the tight junction, a fu ll length and an N-terminally truncated murine occludin construct, both FLA G-tagged at the N terminus, were stably introduced into the murine epitheli al cell line CSG 120/7, Both constructs were correctly targeted to the tigh t junction, as defined by colocalization with another tight junction protei n, ZO-1, The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as dem onstrated by low transcellular electrical resistance values and an increase d paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands , as visualized by freeze-fracture electron microscopy. These findings demo nstrate an important role for the N-terminal half of occludin in tight junc tion assembly and maintaining the barrier function of the tight junction.