ZO-1, ZO-2, and ZO-3 contain a conserved series of well-known protein–protein interaction motifs. All three proteins have three amino-terminal PSD95/dlg/ZO-1 (PDZ) domains, followed by a Src homology 3 (SH3) do- main and a guanylate kinase (GUK) domain. The car- boxy-terminal portion of ZO-1 and ZO-2 consists of a pro- line-rich region; however, in ZO-3 this proline-rich region is located in the amino-terminal half of the protein be- tween PDZ2 and PDZ3. The specific domains responsible for the various binding interactions of ZO-1, ZO-2, and ZO-3 have, in part been defined. ZO-1 and ZO-2 form a complex through reciprocal interactions between their PDZ2 domains (Fanning et al., 1998; Itoh et al., 1999b), and ZO-3 likely interacts with ZO-1 in the same manner (Itoh et al., 1999a). ZO-1 interaction with occludin likely occurs through the GUK domain, and actin binding via a region that encompasses the proline-rich domain of ZO-1 (Fanning et al., 1998). ZO-1, ZO-2, and ZO-3 binding to the carboxy-terminal YV sequence of claudin 1–8 requires the first PDZ domain of each protein (Itoh et al., 1999a). Finally, the amino-terminal region of ZO-1 and ZO-2 binds to a-catenin, and ZO-1 and ZO-2 are components of the adherens junction (AJ) in certain nonepithelial cells (Itoh et al., 1997, 1999b). No further information on indi- vidual domain binding characteristics or overall protein function(s) is available for ZO-3. One current model for TJ assembly involves the coordi- nate assembly of the AJ and TJ. This model proposes that the first step in de novo junction formation involves E-cad- herin–mediated cell adhesion (Gumbiner et al., 1988), fol- lowed by ZO-1 recruitment to the lateral surface of the cell via a transient interaction with the a-/b-catenin complex (Rajasekaran et al., 1996). Indeed, it was subsequently found that ZO-1 binds a-catenin in vitro (Itoh et al., 1997), providing the means by which ZO-1 is associated with the catenin complex during this early stage in junction forma- tion. As epithelial polarization proceeds, ZO-1 is sorted out of the AJ and forms part of a separate TJ complex (Ando-Akatsuka et al., 1999). In vitro binding experiments have shown that ZO-2 can also bind a-catenin (Itoh et al., 1999b), indicating the possibility that ZO-2 may follow a similar path during initial stages of junctional complex as- sembly, either directly via interaction with a-catenin or in- directly via its association with ZO-1 (Wittchen et al., 1999). Recent data suggest that this hierarchy of junctional complex assembly is not absolute. Troxell et al. (2000) have shown that TJ assembly was extensive in MDCK cells expressing a mutant E-cadherin protein lacking the extra- cellular domain required for cell–cell adhesion, suggesting the possibility that TJs can assemble in the absence of an initial E-cadherin–mediated cell adhesion event. Here we pursue the role of ZO-3 in epithelial cell physi- ology by expressing mutant ZO-3 constructs in MDCK cells. Through studies on TJ assembly and the domains re- sponsible for protein–protein interactions, we have dem- onstrated that exogenous expression of the PDZ domain- containing, amino-terminal half of ZO-3 perturbs both TJ and AJ assembly. Moreover, such expression also affects actin dynamics and the biochemical properties of at least one adherens junction protein. Through in vitro binding assays we show that this amino-terminal half of ZO-3 pref- erentially binds ZO-1 and actin filaments. These studies provide the first functional information on ZO-3 and help to define how the molecular constituents of the junctional complex may interact and assemble in epithelial cells. Materials and Methods Plasmid Construction and MDCK Transfections The ZO-3 constructs used in this study are shown in Fig. 1. A vesicular stomatitis virus glycoprotein (VSVG) epitope-tagged version of full- length ZO-3 (FLZO-3) in the expression vector pBK-CMV used for trans- fection of MDCK cells has been described (Haskins et al., 1998). The stable cell line MDCK/FLZO-3 was generated by transfection using Lipo- fectin (GIBCO BRL) and G418 selection as described (Haskins et al., 1998). A construct composed of the amino-terminal half of ZO-3 (NZO-3) contains the three PDZ domains, arginine-rich region, and proline-rich domain (amino acids 1–454). For stable transfection in MDCK cells, this construct was subcloned into pBK-CMV with a VSVG epitope tag in- serted at the carboxy terminus. NZO-3 was also subcloned into the pFast- Bac HT vector series of the Baculovirus eukaryotic expression system (GIBCO BRL) to generate a 6-histidine–tagged protein used for in vitro binding experiments. A construct composed of the entire carboxy-termi- nal half of ZO-3 (CZO-3) containing the SH3 and GUK domains, fol- lowed by the acidic region (amino acids 455–899) was subcloned into pBK-CMV with a VSVG-epitope tag at the carboxy terminus and stably transfected into MDCK cells. CZO-3 was also inserted into the pFastBac HT vector series to generate a 6-histidine–tagged recombinant protein for binding analyses. For each stable cell transfection, multiple clones were selected and analyzed for expression of constructs by immunofluores- cence and immunoblotting. Calcium Switch and Cytochalasin D Treatment For transepithelial resistance (TER) measurements and immunohis- tochemistry, untransfected or transfected MDCK cells grown as previ- ously described (Howarth et al., 1992) were plated on Transwell polycar- bonate membrane filter inserts (0.4 mm pore size; Costar Corp.) coated with rat tail collagen at a cell density of 3 3 105 cells/cm2. Filters were fed every day. Calcium switch experiments were performed with minor modi- fications of previously published methods (Cereijido et al., 1978; Gum- biner and Simons, 1986). In brief, cells were trypsinized and replated on filters in DME containing 1.8 mM calcium for 2–3 h to allow attachment. Filters were then rinsed 23 with PBS without added calcium (PBS2) be- fore switching to low calcium (5 mM) DME overnight (z15 h) to allow disassembly of the junctional complex. Cells were then switched back to 1.8 mM calcium-containing media at t 5 0, and junctional reassembly was followed for various times. Control cells used for determination of steady- state levels of NZO-3 were trypsinized, replated in normal calcium media, and incubated for z65 h. Cytochalasin D (cD) (4 mg/ml) in Hepes-buffered saline plus glucose (HBSG; 10 mM Hepes, pH 7.4, 5.4 mM KCl, 137 mM NaCl, 1.3 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose), was added to filter-grown mono- layers for 60 min and then was washed out by rinsing filters 33 with HBSG. Cells were then incubated in HBSG without cD for another 2 h. Transepithelial Resistance Measurements TER was measured with a Millicell-ERS apparatus (Millipore) in HBSG as previously described (Stevenson and Begg, 1994). In the results de- picted in Fig. 3 (A and B), duplicate filters from each cell line were mea- sured for each time point, and the experiment was performed at least twice. Data from at least two experiments (n . 4 filters) are presented as mean 6 SEM. Data from single clones of parental, MDCK/NZO-3, MDCK/CZO-3, and MDCK/FLZO-3 cells are shown. Two additional, in- dependently selected MDCK/NZO-3 cell lines were tested in both the cal- cium switch and cD experiments and showed results essentially identical to the first MDCK/NZO-3 cell line (data not shown). For calcium switch experiments (see Fig. 3 A), TER is plotted in ohm 3 cm2. For cD experi- ments (see Fig. 3 B), TER is expressed as a percentage normalized to t 5 0. The steady-state TER of all cell lines examined was 50–60 ohm 3 cm2. Antibodies The following antibodies were used: rat anti–ZO-1 mAb R40.76 (Ander- son et al., 1988), rabbit anti–ZO-2 polyclonal Ab R9989 (Jesaitis and Goodenough, 1994), rabbit anti-VSVG (a gift from Dr. Carolyn Macha- mer, Johns Hopkins University, Baltimore, MD), rabbit anti-occludin The Journal of Cell Biology, Volume 151, 2000 826