We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.The formation and maintenance of these barriers is dependent on a series of cellcell contacts that circumscribe the apicallateral margin of each cell, known collectively as the apical junction complex. Both AJ and TJ are intimately associated with the cortical actin cytoskeleton and are functionally regulated by circumferential actomyosin filaments; the dynamic interaction between cell junctions and the cytoskeleton is critical for the morphogenesis of epithelial tissues during development or tissue repair. The zonula occludens are multidomain scaffolds that bind directly to the barrierforming claudin proteins and also interact with other signaling and structural proteins implicated in TJ structure and function. Targeted deletion of either ZO or in mice results in relatively late embryonic lethality. Both observations suggest that there is considerable functional redundancy among ZO proteins, which has made experimental elucidation of ZO protein function technically difficult.Depletion of ZO and in cultured epithelial cells is associated with a delay in the assembly of the beltlike AJ and associated perijunctional actomyosin ring. These observations strongly implicate ZO proteins in some aspect of AJ function, although the precise mechanistic role is not understood.Studies to date support the hypothesis that ZO proteins regulate the recruitment of actin and myosin II into the circumferential AJ during the early events of junction assembly in cultured cells. We confirmed the requirement for ZO proteins in TJ assembly and barrier formation.This expansion is associated with changes in cell shape, the organization of cells within the epithelium, and the structure of the apical plasma membrane.In three independently isolated clones, ZO expression was reduced by over, while ZO expression was almost undetectable. In addition, although ZO expression was not directly targeted, expression in all three doubleknockdown expression could be effectively restored by expression of a recombinant myctagged ZO rescue transgene. Restoration of either ZO or was also sufficient to restore ZO expression to normal levels. This decrease in expression of the three ZO proteins was confirmed by immunocytochemistry. For example, the localization of both the transmembrane protein occludin and the cytoplasmic scaffolding protein cingulin were reduced at the TJ. In contrast, the distributions of the occludin paralogue tricellulin and the cell adhesion molecule JAMA were Naftifine hydrochloride unaltered. Similarly, while the accumulation of claudin and at the AJC was reduced relative to control cells, that of claudin and appeared normal. However, as noted above, the distribution of all proteins in the AJC was much more linear in dKD cells than in control MDCK cells, which characteristically have wavy cell junctions.These shape changes were most obvious in an mthick section that contained the AJC, and were not noticeable in more basal Albamycinsodium aspects of the epithelium. In spite of clear alterations in distribution, the expression levels of these proteins in dKD and control cells were not different. Changes in the distribution of occludin and claudin in dKD cells could be rescued by tetracycline, as could the changes in junction contour and cell shape.