Figure 7. NZO-3 expression de- lays recruitment of E-cadherin to cell–cell contacts. Parental (P) and MDCK/NZO-3 (NZO- 3) cells were subjected to a cal- cium switch, and E-cadherin and ZO-1 localization was mon- itored by costaining at 6, 12, 24, and 48 h after the re-addition of calcium. In parental cells, E-cadherin is localized to cell borders by 6 h. By 48 h, cell monolayers have an organized morphology. NZO-3–express- ing cells costained with anti– ZO-1 and anti–E-cadherin antibodies show a delayed E-cadherin recruitment to cell borders. The clusters of cells that show E-cadherin localization at cell peripheries at early time points coincide with cells showing a similar distribution of ZO-1. Some cells (arrow) show E-cadherin at cell borders preceding ZO-1. E-cadherin staining at cell borders is complete by 24 h after the readdition of calcium, whereas ZO-1 staining at cell peripheries is not complete until 48 h. Bar, 30 mm. showing that ZO-1 and ZO-2 bind each other via their sec- ond PDZ domains (Fanning et al., 1998; Itoh et al., 1999b). To define the occludin binding site on ZO-3, in vitro bind- ing assays were performed using NZO-3 and CZO-3 added to affinity columns containing immobilized GST- occludin or GST alone, and bound proteins were detected by immunoblot (Fig. 10 D). Both NZO-3 and CZO-3 were specifically retained on the occludin column. Although the NZO-3 immunoblot reaction is stronger than that for CZO-3, the amounts of NZO-3 and CZO-3 bound to oc- cludin appear approximately equivalent, as assayed by Coomassie blue staining of the bound fractions. This indi- cates that occludin binding domains of approximately equal affinity are found in both the amino- and carboxy- terminal halves of ZO-3. It has recently been reported that the amino-terminal head and to a lesser degree, the car- boxy-terminal tail of cingulin bind to full-length ZO-3 in vitro (Cordenonsi et al., 1999). Here we determined which half of ZO-3 binds the amino-terminal portion of cingulin. Equivalent amounts of NZO-3 or CZO-3 were added to GST-cingulin or GST affinity columns. As shown in Fig. 10 E, both NZO-3 and CZO-3 are specifically retained by cingulin. Coomassie blue staining of the bound fractions revealed no significant difference between the amounts of NZO-3 or CZO-3 binding to cingulin, indicating that both halves of ZO-3 bind the head region of cingulin with ap- proximately equal affinity. In summary, the binding stud- ies shown in Fig. 10 reveal that while both halves of ZO-3 bind to occludin and cingulin equally well, F-actin and ZO-1 bind preferentially to NZO-3. Discussion In this study we have shown that exogenous expression of the amino-terminal half of ZO-3 delays assembly of both TJs and AJs. Analysis of protein expression showed that whereas the overall levels of the junctional proteins ZO-1, ZO-2, ZO-3, occludin, and E-cadherin remain constant during 48 h of junction assembly, NZO-3 expression in- creases in response to the calcium switch and then returns to stable, steady-state levels over the same time period. This return of NZO-3 expression to steady-state levels is concomitant with recovery of TER and localization of junctional proteins and actin to the membrane. The amount of b-catenin in the TX-100–soluble pool is also el- evated in NZO-3 expressing cells. Finally, in vitro binding Figure 8. NZO-3 expression delays recruitment of b-cate- nin to cell–cell contacts. b-Catenin and ZO-1 local- ization in MDCK/NZO-3 cells was monitored at 6, 12, 24, and 48 h after the re- addition of calcium in a cal- cium switch experiment. NZO-3–expressing cells show a delayed b-catenin recruit- ment to intercellular junc- tions, which parallels the de- lay of E-cadherin recruitment (Fig. 7). The cells that show b-catenin localization at cell peripheries at early time points coincide with cells showing similar distribution of ZO-1, although many cells show b-catenin at cell borders preceding ZO-1. b-Catenin staining at cell borders is not complete until 24 h after the re-addition of calcium in MDCK/NZO-3 cells. Bar, 30 mm. The Journal of Cell Biology, Volume 151, 2000 832