Figure 4. NZO-3 expression alters the distribution of ZO-1 during the early stages of TJ assembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO- 3) cells were subjected to a calcium switch, and the localization of ZO-1 at 6, 12, 24, and 48 h after the re-addition of calcium was determined by immunofluo- rescence with anti–ZO-1 antibody. In parental cells, ZO-1 is completely local- ized to cell borders after 6 h in calcium- containing media. Over 48 h the cells be- come more tightly packed and uniform in size. In NZO-3 expressing cells, only scattered islands of cells showed ZO-1 localization at cell borders by 6 h. The num- ber of cells with ZO-1 localized to cell–cell contact sites increases over time and is complete by 48 h after re-addition of calcium. (B) No MDCK/NZO-3 cells show ZO-1 localization around their peripheries at t 5 0 in the calcium switch. Phase-con- trast image shows that cells form a confluent monolayer at t 5 0 of calcium switch. Anti–ZO-1 immunofluorescence shows ZO-1 staining in large intracellular aggre- gates and in thick bars at sites where two cells make contact. Bars: (A and B) 30 mm. to a calcium switch and protein localization was deter- mined 6, 12, 24, and 48 h after re-addition of calcium. As shown in Fig. 4 A, ZO-1 is localized at cell–cell contacts within 6 h after re-addition of calcium. In the 12–48-h time period, the parental cells develop the cobblestone unifor- mity of shape and size that is typical of an MDCK epithe- lial cell monolayer at steady-state. The appearance of ZO-1 at cellular contacts 6 h after re-addition of calcium corre- lates well with the rapid increase in TER observed in the parental cells (Fig. 3 A). In contrast, cells expressing NZO-3 showed a significant delay in ZO-1 recruitment to cell borders after re-addition of calcium (Fig. 4 A). At 6 h, only small, isolated clusters of cells showed ZO-1 staining at cell borders. Over time the areas of cell monolayer that show ZO-1 localization at cell–cell contacts increase in size. At 6, 12, and 24 h when ZO-1 is not yet fully localized to cell borders, ZO-1 also localizes to large intracellular aggregates and linear planes of contact between neighbor- ing cells. Complete localization of ZO-1 at the junctional membrane in NZO-3–expressing cells did not occur until 48 h after re-addition of calcium. ZO-1 localization in both cell types at 48 h is indistinguishable from untreated, steady-state cells (data not shown). Both parental and MDCK/NZO-3 cells displayed no detectable differences in expression levels of ZO-1 (or any other junctional pro- tein examined; see Fig. 9 immunoblot) at 6, 12, 24, and 48 h. The delayed targeting of the TJ protein ZO-1 in MDCK/NZO-3 cells correlates with the delay in TER re- covery after calcium switch (Fig. 3 A). The localization of the TJ proteins ZO-2, endogenous ZO-3, and the integral membrane protein occludin was identical to that of ZO-1 in the MDCK/NZO-3 cells (data not shown). To rule out the possibility that the isolated islands of MDCK/NZO-3 cells showing ZO-1 staining encircling cell peripheries at 6 h arose from cell clusters that did not dis- assemble their TJs during incubation in low calcium, and to demonstrate that the recruitment of ZO-1 to cell peripher- ies occurred de novo, we looked at ZO-1 localization in MDCK/NZO-3 cells before the re-addition of calcium (t 5 0). As viewed by phase-contrast microscopy, cells plated at an identical density on glass coverslips show a confluent distribution at t 5 0, although the cells are somewhat re- tracted from each other due to the dissociation of adherens and TJs in low calcium (Fig. 4 B). ZO-1 staining is com- pletely disrupted, and is present only in large intracellular aggregates and thick, linear planes of contact between neighboring cells. No cells show ZO-1 staining encircling their peripheries at t 5 0. Thus, ZO-1 (and ZO-2, endoge- nous ZO-3, and occludin; data not shown) is recruited from an intracellular location to cell–cell contacts upon re- addition of calcium, and this recruitment process is delayed in NZO-3–expressing MDCK cells. The disrupted localization of TJ proteins during junc- tional assembly after calcium switch in cells expressing NZO-3 indicates that this construct interferes with the for- mation of the complex of proteins found at the TJ. We therefore examined the localization of the construct itself during this process. The anti-VSVG antibody only weakly recognizes its epitope on NZO-3 in immunofluorescence and was not visible in filter-grown cells. Hence, MDCK/ NZO-3 cells grown on coverslips and subjected to a cal- cium switch were costained with the anti–ZO-1 and anti- VSVG (NZO-3) antibodies. Parental MDCK cells stained with anti-VSVG serve as a negative control and show that this antibody exhibits nonspecific nuclear staining in un- transfected cells (Fig. 5 A). At the earliest time points (6, 12, and 24 h), the NZO-3 construct appears at only those cell borders that also show ZO-1 localization. MDCK/ NZO-3 cells stained with anti-VSVG antibody also exhibit a faint, granular cytoplasmic staining above background. NZO-3 staining is only faintly visible at 48 h, likely due to the lower levels of construct expression at this time point (see Fig. 9 immunoblot). In areas where NZO-3 is visible, it was colocalized with ZO-1 at all cell–cell contacts. Inter- estingly, NZO-3 was absent from the intracellular aggre- gates and linear planes of ZO-1 staining observed at early time points, and was only colocalized with ZO-1 at points of cell–cell contact formed by mature TJs (Fig. 5 B). The actin cytoskeleton is an important structural and a functional element of the TJ (Hirokawa and Tilney, 1982; Madara, 1987). Actin filaments have been shown to inter- act directly with multiple TJ proteins (Itoh et al., 1997; Wittchen et al. Delayed Junctional Complex Assembly 829