(Wong and Gumbiner, 1997; a gift from Drs. Alpha Yap and Barry Gum- biner, Memorial Sloan-Kettering Cancer Center, New York, NY), mouse anti–E-cadherin mAb 3G8, (a gift from Dr. Warren Gallin, University of Alberta, Edmonton, Canada), and rabbit anti–b-catenin (Sigma-Aldrich; No. C2206). For detection of ZO-3 constructs, the following antibodies were used: guinea pig anti–ZO-3 (Haskins et al., 1998) and mouse anti–ZO-3 mAB3260 (Chemicon International, Inc.), which both recognize an epitope in the amino terminus of ZO-3 and were used to detect full-length ZO-3 and NZO-3; rabbit anti–ZO-3 polyclonal AB3220 (Chemicon Interna- tional, Inc.) recognizes an epitope in the extreme carboxy terminus of ZO-3 and was used to detect CZO-3. HRP-conjugated goat anti–rabbit and HRP-conjugated goat anti–mouse (Bio-Rad Laboratories), HRP-conju- gated goat anti–guinea pig and HRP-conjugated anti–rat (Jackson Immu- noResearch Laboratories) were used as secondary antibodies for immuno- blots. Rhodamine-conjugated donkey anti–rabbit, rhodamine-conjugated donkey anti–guinea pig, rhodamine-conjugated goat anti–rat, rhodamine- conjugated goat anti–mouse, FITC-conjugated goat anti–rat, and Texas red–conjugated goat anti–mouse (Jackson ImmunoResearch Laboratories) were used as secondary antibodies for indirect immunofluorescence. Immunohistochemistry For immunofluorescence staining with anti-VSVG, cells grown on col- lagen-coated coverslips were fixed with 5% acetic acid in ethanol for 5 min at 2208C and permeabilized with 0.2% Triton X-100 (TX-100) in TBS plus 1.8 mM calcium (TBS1) for 4 min at room temperature. For all other staining, cells grown on filters were fixed with 2.5% paraformalde- hyde in TBS and permeabilized with 0.2% TX-100. Rhodamine-conju- gated phalloidin (Sigma-Aldrich) was used to stain F-actin. Coverslips and filters were viewed on a Zeiss Axioskop (Carl Zeiss, Inc.). Protein Expression Analysis and TX-100 Extraction Parental or MDCK/NZO-3 cells grown on 35-mm tissue culture dishes were subjected to a calcium switch, and total lysates were made from cells 6, 12, 24, and 48 h after the re-addition of calcium. The steady-state level of NZO-3 was assessed on control cells not subjected to calcium switch that were maintained in normal calcium media for z65 h. Monolayers were rinsed 23 with ice-cold TBS1 and scraped up into 1 ml ice-cold TBS1 plus protease inhibitor cocktail (1 mg/ml aprotinin, 1 mg/ml chymo- statin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mM Pefabloc SC; Boeh- ringer). Scraped cells were then harvested by centrifugation at 6,500 rpm for 5 min at 48C. Cell pellets were resuspended in hot SDS lysis buffer (10 mM Tris, pH 7.8, 1% SDS) plus protease inhibitors and boiled for 5 min, shearing with a narrow bore pipette tip. Protein concentration was deter- mined by BCA protein assay (Pierce Chemical Co.) and 20 mg total pro- tein was loaded per lane on SDS-PAGE. For immunoblots, gels were electrophoretically transferred to nitrocellulose, immunoblotted with cor- responding antibodies, and proteins detected via ECL (Amersham Phar- macia Biotech). For separation of TX-100–soluble and –insoluble b-catenin, parental and MDCK/NZO-3 cells subjected to a calcium switch were extracted with CSK buffer (10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, 0.5% TX-100 plus 0.1 mg/ml DNAse, 0.1 mg RNAse, and protease inhibi- tor cocktail; Troxell et al., 2000). In brief, cell monolayers grown on 35- mm dishes were rinsed 23 with ice-cold TBS1 and then extracted for 10 min on ice with 1.5 ml CSK buffer. Insoluble material was scraped from the dish, and the extracts were centrifuged 10 min at 13,000 rpm at 48C. Supernatants contain the detergent-soluble pool of b-catenin. Pellets con- taining detergent-insoluble material were resuspended in 1.5 ml hot SDS lysis buffer and boiled for 10 min. Protein concentration in each fraction was determined by BCA protein assay, and samples were loaded for SDS- PAGE and immunoblot analysis as above. Recombinant Protein Expression and In Vitro Binding Studies Recombinant 6-histidine–tagged NZO-3 and CZO-3 were expressed in Sf9 insect cells using a Baculovirus eukaryotic expression system (GIBCO BRL) and purified according to previously published methods (Haskins et al., 1998; Wittchen et al., 1999). Empty expression vector encoding the 6-histidine tag plus 36 nonspecific amino acids served as the negative con- trol. Equal amounts of purified NZO-3 and CZO-3 were used in the bind- ing experiments. [35S]Methionine-labeled in vitro–transcribed/translated human ZO-1 was prepared as described (Haskins et al., 1998; Wittchen et al., 1999). Expression of GST-occludin encoding the carboxy-terminal 150 amino acids of the cytoplasmic tail of chicken occludin (Furuse et al., 1993) was described previously (Haskins et al., 1998; Wittchen et al., 1999). GST–N-cingulin cDNA encoding the amino-terminal head region (amino acids 1–378) of Xenopus cingulin in pGEX-4T1 (a gift from Dr. Sandra Citi, University of Geneva, Geneva, Switzerland) was expressed according to published methods (Cordenonsi et al., 1999). The binding of NZO-3 and CZO-3 to actin, ZO-1, and occludin was performed as previously described (Wittchen et al., 1999). Cingulin bind- ing experiments used a modification of the methods of Cordenonsi et al. (1999). In brief, GST–N-cingulin or GST alone was immobilized on glu- tathione-Sepharose 4B beads, and equal amounts of purified NZO-3 or CZO-3 were added. Binding buffer comprised of PBS plus 1% TX-100 and protease inhibitors. Binding was allowed to proceed overnight at 48C, followed by washing with the same buffer, and eluting bound protein by boiling the beads in gel sample buffer. NZO-3 and CZO-3 binding was de- tected by immunoblot, and in the case of occludin and cingulin binding ex- periments, the amount of bound NZO-3 and CZO-3 was compared by Coomassie blue staining of the bound fraction. Results The Effect of Truncated ZO-3 Constructs on Tight Junction Physiology We first determined which region of ZO-3 encodes the in- formation for targeting the protein to the TJ. MDCK cells were stably transfected with the VSVG-tagged FLZO-3, NZO-3, and CZO-3 constructs (Fig. 1). Subcellular local- ization of the constructs was detected by indirect immuno- fluorescence using anti-VSVG antibodies and costaining for ZO-1 as a marker for the TJ. FLZO-3 correctly targets to cell borders, colocalizing with the TJ marker ZO-1 (data not shown). The NZO-3 construct also colocalizes with en- dogenous ZO-1 at cell borders (Fig. 2); furthermore, its lo- calization was also indistinguishable from endogenous ZO-1 as assayed by 0.2 mm Z-section confocal microscopy (data not shown). The nuclear staining visible with the anti- VSVG staining is nonspecific as the same staining pattern is seen in untransfected parental MDCK cells (see Fig. 5 A). Fig. 2 also shows that when expressed in MDCK cells, CZO-3 is absent from cell borders and is instead diffusely distributed in the cytoplasm. Expression of the CZO-3 con- struct in these cells was confirmed by immunoblot (data not shown). Combined, these data indicate that the infor- mation necessary for proper targeting of ZO-3 to the TJ is contained within the amino-terminal half of the protein. We next examined the effect of exogenous expression of these constructs on TJ formation using two experimental paradigms. The first approach is based on the observation Figure 1. Schematic diagram of the ZO-3 constructs used in this study. Full-length ZO-3 (FLZO-3) contains three PDZ domains, an arginine-rich basic region (1), a proline-rich region (pro), an SH3 domain, a GUK domain, and an acidic region (2). NZO-3 comprises the amino-terminal half of ZO-3 and contains the three PDZ domains, basic region and proline-rich region. The CZO-3 construct corresponds to the carboxy-terminal half of the molecule and contains the SH3, GUK, and acidic domains. The constructs used for generating stably transfected MDCK cells have a VSVG epitope tag at their carboxy terminus. Wittchen et al. Delayed Junctional Complex Assembly 827