Figure 2. The NZO-3 construct correctly targets to the TJ; the CZO-3 does not. MDCK cells were stably transfected with VSVG- tagged NZO-3 (MDCK/NZO-3) or CZO-3 (MDCK/CZO-3) con- structs. Transfected cells were costained with anti–ZO-1 and anti- VSVG antibodies to mark the TJ and localize the constructs by indirect immunofluorescence. NZO-3 precisely colocalizes with ZO-1 at cell borders, with additional staining visible in the cyto- plasm. CZO-3 is found mainly in the cytoplasm and is absent from cell borders. The nuclear staining in the MDCK/NZO-3 cells stained with anti-VSVG is nonspecific (see Fig. 5). Bar, 15 mm. that AJs and TJs are disrupted if epithelial cells are grown in medium containing subphysiological calcium concentration (5 mM). Subsequent restoration of physiological levels of calcium (1.8 mM) results in the synchronous de novo assem- bly of both junction types (Cereijido et al., 1978; Gumbiner and Simons, 1986). Filter-grown monolayers of parental, NZO-3, CZO-3, or FLZO-3–expressing MDCK cells were grown overnight in low calcium media. Physiological con- centration of calcium was added back at t 5 0, and TJ as- sembly was monitored by measuring TER over a 48-h time period. As shown in Fig. 3 A, parental MDCK cells undergo an initial rapid increase in TER to over 175 ohm 3 cm2 by 12 h. Resistance peaked at 12–24 h, before declining and lev- eling off to z50 ohm 3 cm2 by 96 h. Cell lines expressing the CZO-3 and FLZO-3 constructs followed a similar trend, plateauing at the same steady-state level as parental cells. In contrast, NZO-3–expressing cells exhibited delayed TER development, with no initial overshoot typical of the other cell lines. However, regardless of the delay, NZO-3–express- ing cells reached a steady-state TER similar to that of the other cell lines by 48–96 h. The TER of all cells at 96 h is similar to that of untreated cells (data not shown). We corroborated the results obtained in the calcium switch experiments by determining the effect of ZO-3 con- struct expression on TJ reformation in MDCK cells treated with the actin-disrupting drug cD. In cells treated with cD, the apical actin cytoskeletal ring associated with the junctional complex is disrupted into large punctate ag- gregates that colocalize with similarly disrupted TJ pro- teins ZO-1, ZO-2, ZO-3, and occludin (Stevenson and Begg, 1994; Wittchen et al., 1999). The cD-treated cells also show a sharp drop in TER, indicating a loss of func- tional TJs. It was shown that this effect is reversible, and upon washout of cD, TJs reassemble synchronously (Ma- dara et al., 1986; Stevenson and Begg, 1994). We treated our MDCK cell lines with cD for 60 min, and the drug was then washed out (Fig. 3 B). Upon cD treatment all cell Figure 3. Expression of NZO-3 delays TJ formation. (A) NZO-3 expression in MDCK cells subjected to a calcium switch delays re- establishment of TER. Filter-grown confluent monolayers of un- transfected parental cells or MDCK cell lines stably expressing NZO-3, CZO-3, or FLZO-3 were incubated in calcium-free media for z15 h to disrupt intercellular junctions and then switched to me- dia containing 1.8 mM calcium at t 5 0 to induce synchronous as- sembly of intercellular junctions. TER was measured over 96 h after calcium switch. CZO-3 and FLZO-3 expressing cell lines show TER recovery dynamics similar to parental cells. NZO-3–expressing cells display a delay in TER recovery, but reach the same TER as the other cell lines by 48–96 h. (B) Expression of NZO-3 delays TER recovery in MDCK cells after cD treatment. cD was added to filter- grown confluent monolayers at t 5 0 and then washed out after 60 min. TER measurements were taken every 30 min to monitor TJ breakdown and reformation. TER for each cell line is expressed as percentages of the values at t 5 0. After 60 min of cD treatment, all cell lines exhibit a significant drop in TER. After cD washout, MDCK/CZO-3, MDCK/FLZO-3 and parental cells recover TER to values greater than t 5 0, whereas MDCK/NZO-3 cells do not fully recover TER in the duration of the experiment. lines exhibited an immediate and substantial decrease in TER. After cD washout, parental, FLZO-3–, and CZO- 3–expressing cells recovered TER values equal to or greater than t 5 0 values within 1 h. However, NZO- 3–expressing cells showed a delayed TER recovery from cD treatment and failed to reach t 5 0 levels for the dura- tion of the experiment. This result is in agreement with the calcium switch experiment (Fig. 3 A) and demonstrates that MDCK cells expressing the NZO-3 construct display an impaired ability to assemble functional TJs. Junctional Protein Localization The physiological data suggesting impaired TJ-forming ability in NZO-3–expressing cells prompted us to assess the localization of proteins associated with both TJs and AJs in the calcium switch experimental system. Parental and MDCK/NZO-3 cells grown on filters were subjected The Journal of Cell Biology, Volume 151, 2000 828