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© The Rockefeller University Press, 0021-9525/1997//181 $5.00
The Journal of Cell Biology, Volume 138, Number 1, , 1997 181-192

Article

Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to {alpha} Catenin and Actin Filaments



Masahiko Itoh*, Akira Nagafuchi*, Seiji Moroi*,{ddagger}, and Shoichiro Tsukita*

* Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan; and {ddagger} Department of Urology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/{alpha}, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with {alpha} catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and {alpha} catenin was ~0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ~10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with {alpha} catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

1. Abbreviations used in this paper: aa, amino acid(s); AJ, adherens junctions; C-ZO-1 and N-ZO-1, carboxyl-terminal half ZO-1 and amino-terminal half dlg-like ZO-1; dlg, discs large; GST, glutathione-S-transferase; MAGUK, membrane-associated guanylate kinase homologues; TJ, tight junctions.

Address all correspondence to Shoichiro Tsukita, M.D., Department of Cell Biology, Kyoto University, Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4372. Fax: 81-75-753-4660. E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp


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