ZO/afadin KD disrupts barrier function and resistance to external mechanical stress. (A–D) Occludin and Ecad. Junctional occludin changes from continuous in control or afadin single KD to punctate after ZO KD. ZO/afadin KD dramatically disrupts junctional occludin. Ecad still localizes to regions where occludin is lost but no longer concentrates at ZA (D–D″, asterisks; projection, apical 6 µm). (E and F) Barrier function. TER (E) or dextran flux (F) across monolayer. ZO/afadin KD dramatically reduced barrier function, whereas afadin single KD had no effect. Error bars are SDs of three independent experiments. (G–J) Monolayers detached by dispase and mechanically disrupted by pipetting. (G′–J′) 5× zoomed images. ZO KD did not affect tissue integrity. Afadin single KD slightly reduced sheet integrity. ZO/afadin KD dramatically reduced this (I). (K) Model, ZA actomyosin organization in cells with increased contractility. Contractile actomyosin arrays run parallel to BCJs and make end-on contacts at TCJs, generating a “zig-zag” membrane topology at TCJs. (L) Germband extending wild-type or canoe mutant fly embryos—wild-type myosin localizes to planar polarized cables tightly localized to the ZA at anterior-posterior boundaries (arrows). Canoe loss results in myosin broadening.