Shroom3 is recruited to the ZA in response to ZO KD and stimulates actomyosin contraction. (A and B) Control versus ZO KD cells. ZO-1 and Shroom3 are shown. (C) Control cells or cells expressing murine Shroom3L cocultured. Shroom3 and ROCK1 are shown. (D) mShroom3L overexpression in MDCK cells led to ZA actomyosin assembly and border straightening. (E) Afadin KD in mShroom3L-overexpressing cells. ZA actomyosin assembled, but defects were seen at MCJs (arrows). (F and G) ZO KD cells transiently transfected with siRNA targeting canine Shroom3. Shroom3, afadin, and F-actin are shown. Cells with reduced Shroom3 (F′ and G′) have reduced F-actin and afadin and larger apical areas. (H) ZO KD and ZO/afadin KD cells cocultured. ZO/afadin KD cells have less uniform Shroom3 at the ZA.