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Vol. 19, Issue 9, 3801-3811, September 2008

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ZO-1- and ZO-2-Dependent Integration of Myosin-2 to Epithelial Zonula Adherens

Yuji Yamazaki^*,, Kazuaki Umeda, Masami Wada^*, Shigeyuki Nada, Masato Okada, Shoichiro Tsukita, and Sachiko Tsukita^*

*Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Kyoto 606-8501, Japan; Department of Molecular Pharmacology, Graduate School of Medical Science, Kumamoto University, Honjo, Kumamoto 860-8556, Japan; and Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan

Submitted April 7, 2008; Revised June 9, 2008; Accepted June 25, 2008
Monitoring Editor: Keith E. Mostov

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 (zonula occludens-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 (PSD-95/discs large/zonula occludens-1)-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 or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, 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. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2–dependent way to establish ZA from primordial forms in epithelial cells.

Address correspondence to: Sachiko Tsukita (atsukita{at}biosci.med.osaka-u.ac.jp).