Exogenous Expression of the Amino-terminal Half of the Tight Junction Protein ZO-3 Perturbs Junctional Complex Assembly Erika S. Wittchen, Julie Haskins, and Bruce R. Stevenson Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Abstract. The functional characteristics of the tight junction protein ZO-3 were explored through exoge- nous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junc- tions induced by calcium switch treatment or brief ex- posure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction- speciﬁc proteins by immunoﬂuorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junc- tion proteins E-cadherin and b-catenin were also de- layed in their recruitment to the cell membrane, and Introduction Regulated transport of substances across a polarized epi- thelial cell sheet occurs via two distinct routes: the trans- cellular pathway, where substances are vectorially trans- ported through the cytoplasm (transcytosis); and the paracellular pathway, where molecules selectively pass through the spaces between adjoining cells. The tight junc- tion (TJ)1 is the structural element that forms a physical barrier to the movement of molecules through the paracel- lular space (gate function) and also maintains the polariza- tion of apical and basolateral membrane domains by re- stricting the movement of lipids and membrane proteins within the plane of the membrane (fence function). Since the identification and characterization of the membrane-associated guanylate kinase (MAGUK) pro- tein ZO-1 (Stevenson et al., 1986), other TJ-associated proteins have been discovered, now totaling at least 15 pe- Address correspondence to Dr. Bruce R. Stevenson, Department of Cell Biology, University of Alberta, Med. Sci. 5-19, Edmonton, Alberta, Can- ada T6G 2H7. Tel.: (780) 492-1841; Fax: (780) 492-0450; E-mail: bruce.stevenson@ualberta.ca 1Abbreviations used in this paper: AJ, adherens junction; cD, cytochala- sin D; CZO-3, carboxy-terminal half of ZO-3; FLZO-3, full-length ZO-3; GUK, guanylate kinase; MAGUK, membrane-associated guanylate ki- nase; NZO-3, amino-terminal half of ZO-3; PDZ, PSD95/dlg/ZO-1; SH3, Src homology 3; TER, transepithelial resistance; TJ, tight junction; TX- 100, Triton X-100; VSVG, vesicular stomatitis virus glycoprotein. NZO-3 expression had striking effects on actin cyto- skeleton dynamics. NZO-3 expression did not alter ex- pression levels of ZO-1, ZO-2, endogenous ZO-3, oc- cludin, or E-cadherin; however, the amount of Triton X-100–soluble, signaling-active b-catenin was increased in NZO-3–expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hy- pothesize that NZO-3 exerts its dominant-negative ef- fects via a mechanism involving the actin cytoskeleton, ZO-1, and/or b-catenin. Key words: zonula occludens-3 protein • tight junc- tion • cadherins • actin cytoskeleton • b-catenin ripheral and 3 integral membrane proteins including the multiple members of the claudin family (Morita et al., 1999). However, our understanding of the functions of these proteins, particularly those peripherally associated with the TJ membrane, is minimal. As a first step towards this understanding, determining how junctional proteins interact will help decipher TJ physiology at the molecular level. Two additional MAGUK family proteins, ZO-2 (Je- saitis and Goodenough, 1994; Beatch et al., 1996) and ZO-3 (Haskins et al., 1998) have been shown to interact directly with ZO-1 (Haskins et al., 1998; Wittchen et al., 1999) but not with each other (Haskins et al., 1998). ZO-1/ZO-2 and ZO-1/ZO-3 likely exist as two distinct heterodimers in vivo and not as a trimeric complex as previously thought (Wittchen et al., 1999). All three ZO proteins interact di- rectly with the carboxy-terminal 150 amino acids of occlu- din (Furuse et al., 1994; Haskins et al., 1998; Wittchen et al., 1999) and claudin 1–8 (Itoh et al., 1999a). ZO-1, ZO-2, and ZO-3 also bind actin filaments (Itoh et al., 1997; Fan- ning et al., 1998; Wittchen et al., 1999), providing multiple linkage sites to the actin cytoskeleton. Furthermore, ZO-1, ZO-2, and ZO-3 all interact with another TJ plaque pro- tein, cingulin (Cordenonsi et al., 1999), and ZO-1 can asso- ciate with the Ras-effector molecule AF-6 (Yamamoto et al., 1997) and the integral membrane protein JAM (Baz- zoni et al., 2000). ª The Rockefeller University Press, 0021-9525/2000/11/825/12 $5.00 The Journal of Cell Biology, Volume 151, Number 4, November 13, 2000 825–836 http://www.jcb.org/cgi/content/full/151/4/825 825