ZO-1/2-dependent integration of myosin-2 to epithelial zonula adherens

For the zonula adherens (ZA) to be established by linear-arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA, as well as administration of LPA or Y27632 which activates RhoA or inhibits ROCK (Rho-kinase), respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. FRET analyses revealed that spatio-temporal Rho-activation occurred in a ZO-1/2-dependent way to establish ZA from primordial forms in epithelial cells.