means by which an actin filament network can be re- cruited to and organized in a functionally relevant manner at the TJ. The actin cytoskeleton is also a major structural and functional element of the AJ (Farquhar and Palade, 1963; Rimm et al., 1995; Yonemura et al., 1995), and is present in a bundled actin belt around the apical periphery cell at the level of the AJ. Interestingly, in MDCK/NZO-3 cells, there is a delay in actin recruitment and formation of this perijunctional apical actin ring (Fig. 6). Because the amino-terminal half of ZO-3 is responsible for binding F-actin, this may represent one mechanism whereby ex- pression of this construct affects TJ and AJ assembly. Not only did expression of the amino terminus of ZO-3 alter the distribution of b-catenin, but there was also an in- crease in the TX-100–soluble pool of signaling-active b-catenin. Presumably the presence of an increased level of the NZO-3 construct at early time points after calcium switch results in a downstream alteration of the E-cad- herin/catenins complex at the adherens junction, releasing b-catenin from a cytoskeletal linkage into the TX-100–sol- uble pool. A corresponding change in the levels of b-cate- nin in the insoluble pool is not observed, although any possible change may be masked by the overall high levels of b-catenin present in these samples. Normally cytoplas- mic b-catenin levels are strictly regulated via a ubiquitin- mediated proteolysis pathway requiring b-catenin interac- tion with the cytoplasmic tumor suppressor APC (Aberle et al., 1997). Soluble b-catenin that escapes this targeted proteolysis is capable of translocating to the nucleus where it acts as a transcriptional transactivator in a complex with TCF/LEF family of transcription factors to direct tran- scription of a variety of genes that promote a proliferative phenotype (Behrens et al., 1996; Huber et al., 1996; Tetsu and McCormick, 1999). Recently, Stewart et al. (2000), showed that expression of a mutant signaling-active (solu- ble) form of b-catenin in MDCK cells caused a delay in the establishment of tight confluent cell monolayers com- pared with control cells, and the cells appeared more mo- tile and formed less compact colonies when plated at a low density. These results, taken together with our data show- ing that NZO-3 expression delays TER formation after a calcium switch and results in an increased level of signal- ing-active, soluble b-catenin in these cells, suggests that NZO-3 might act through b-catenin to exert its effects on epithelial junctional complex formation. At present we do not know if this action is direct or indirect. In summary, through these studies we attempted to fur- ther elucidate the role of ZO-3 in epithelial cell physi- ology, including the role it plays in junctional complex assembly and its protein binding interactions. Exoge- nous expression of partial constructs is the experimental method we chose to approach the dissection of ZO-3 pro- tein function and TJ physiology. This study demonstrates that exogenous expression of the PDZ domain-containing amino-terminal half of ZO-3 perturbs both TJ and AJ as- sembly. Moreover, expression of NZO-3 alters actin dy- namics and increases the amount of soluble signaling- active b-catenin. We are currently testing the hypothesis that NZO-3 exerts its effect on junctional assembly via a mechanism that involves its F-actin and ZO-1 binding abil- ity and/or the increase in soluble b-catenin. This paper is dedicated to the memory of Bernie Gilula. His contributions to both our understanding of the tight junction and the career of B.R. Stevenson were considerable. Cell biology has lost a friend. We thank Alan Fanning, Warren Gallin, Barry Gumbiner, Lijie Gu, Tom Hobman, Brigitte Keon, Carolyn Machamer, Manijeh Pasdar, San- dra Citi, and Alpha Yap for providing reagents and/or advice. We also thank Naveen Basappa for his expert assistance in this project. This work was supported by grants from the Medical Research Council of Canada, the Kidney Foundation of Canada, and the Canadian Associa- tion of Gastroenterology/Janssen-Ortho Inc. E.S. Wittchen holds a Medi- cal Research Council studentship. Submitted: 18 July 2000 Revised: 14 September 2000 Accepted: 4 October 2000 References Aberle, H., A. Bauer, J. Stappert, A. Kispert, and R. Kemler. 1997. Beta-cate- nin is a target for the ubiquitin-proteasome pathway. EMBO (Eur. Mol. Biol. Organ.) J. 16:3797–3804. Anderson, J.M., B.R. Stevenson, L.A. Jesaitis, D.A. Goodenough, and M.S. Mooseker. 1988. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell Biol. 106:1141–1149. Ando-Akatsuka, Y., S. Yonemura, M. Itoh, M. Furuse, and S. Tsukita. 1999. Dif- ferential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity. J. Cell Physiol. 179:115–125. Bazzoni, G., O.M. Martinez-Estrada, F. Orsenigo, M. Cordenonsi, S. Citi, and E. Dejana. 2000. Interaction of Junctional Adhesion Molecule with the tight junction components ZO-1, cingulin, and occludin. J. Biol. Chem. 275: 20520–20526. Beatch, M., L.A. Jesaitis, W.J. Gallin, D.A. Goodenough, and B.R. Stevenson. 1996. The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs- large/ZO-1) domains and an alternatively spliced region. J. Biol. Chem. 271: 25723–25726. Behrens, J., J.P. von Kries, M. Kuhl, L. Bruhn, D. Wedlich, R. Grosschedl, and W. Birchmeier. 1996. Functional interaction of beta-catenin with the tran- scription factor LEF-1. Nature. 382:638–642. Cereijido, M., E.S. Robbins, W.J. Dolan, C.A. Rotunno, and D.D. Sabatini. 1978. Polarized monolayers formed by epithelial cells on permeable and translucent support. J. Cell Biol. 77:853–880. Cordenonsi, M., F. D’Atri, E. Hammar, D.A. Parry, J. Kendrick-Jones, D. Shore, and S. Citi. 1999. Cingulin contains globular and coiled-coil domains and inter- acts with ZO-1, ZO-2, ZO-3, and myosin. J. Cell Biol. 147:1569–1582. Fagotto, F., N. Funayama, U. Gluck, and B.M. Gumbiner. 1996. Binding to cad- herins antagonizes the signaling activity of beta-catenin during axis forma- tion in Xenopus. J. Cell Biol. 132:1105–1114. Fanning, A.S., B.J. Jameson, L.A. Jesaitis, and J.M. Anderson. 1998. The tight junction protein ZO-1 establishes a link between the transmembrane pro- tein occludin and the actin cytoskeleton. J. Biol. Chem. 273:29745–29753. Farquhar, M.G., and G.E. Palade. 1963. Junctional complexes in various epi- thelia. J. Cell Biol. 17:375–412. Funayama, N., F. Fagotto, P. McCrea, and B.M. Gumbiner. 1995. Embryonic axis induction by the armadillo repeat domain of beta-catenin: evidence for intracellular signaling. J. Cell Biol. 128:959–968. Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol. 123:1777–1788. Furuse, M., M. Itoh, T. Hirase, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1994. Direct association of occludin with ZO-1 and its possible in- volvement in the localization of occludin at tight junctions. J. Cell Biol. 127: 1617–1626. Gumbiner, B., and K. Simons. 1986. A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of an uvomorulin- like polypeptide. J. Cell Biol. 102:457–468. Gumbiner, B., B.R. Stevenson, and A. Grimaldi. 1988. The role of the cell ad- hesion molecule uvomorulin in the formation and maintenance of the epi- thelial junctional complex. J. Cell Biol. 107:1575–1587. Haskins, J., L. Gu, E.S. Wittchen, J. Hibbard, and B.R. Stevenson. 1998. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J. Cell Biol. 141:199–208. Hirokawa, N., and L.G. Tilney. 1982. Interactions between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear. J. Cell Biol. 95:249–261. Howarth, A.G., M.R. Hughes, and B.R. Stevenson. 1992. Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am. J. Physiol. 262:C461–C469. Huber, O., R. Korn, J. McLaughlin, M. Ohsugi, B.G. Herrmann, and R. Kemler. 1996. Nuclear localization of beta-catenin by interaction with tran- scription factor LEF-1. Mech. Dev. 59:3–10. Itoh, M., A. Nagafuchi, S. Moroi, and S. Tsukita. 1997. Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J. Cell Biol. 138:181–192. Wittchen et al. Delayed Junctional Complex Assembly 835