Zonula Occludens (ZO)-1 and -2 Regulate Apical Cell Structure and the Zonula Adherens Cytoskeleton in Polarized Epithelia

The structure and function of both adherens (AJ) and tight junctions (TJ) are dependent on the cortical actin cytoskeleton. The Zonula Occludens (ZO)-1 and -2 proteins have contextdependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting they might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable MDCK cell lines depleted of both ZO-1 and ZO-2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2 depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of pMLC and ROCK1, contraction of the actomyosin ring and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of adherens junction proteins or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are not just required for tight junction assembly, but that they also regulate the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and speculate these activities are relevant both to cellular organization and epithelial morphogenesis.