Figure 6. NZO-3 expression disrupts actin filament organization during early stages of junction as- sembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch. Actin filaments were visualized with rhodamine-phalloidin at 6, 12, 24, and 48 h after the re-addition of calcium. In parental cells, actin is concentrated in an apical circumferential ring at the junctional complex in all cells after 6 h in normal calcium. MDCK/NZO-3 cells show actin staining present in large aggregates at early time points (6, 12, and 24 h), with small clusters of cells containing a prominent apical actin ring, similar to the observations with ZO-1 and NZO-3 (Figs. 4 and 5). Actin is not localized to the apical circumferential ring in all cells until 48 h after calcium re-addition. Boxed areas of 12 h images are enlarged in B. (B) In MDCK/NZO-3 (NZO- 3) cells at 12 h, F-actin aggregates (arrow) are seen in cells which have not fully developed an api- cal actin ring. At the same time point, parental cells (P) have conspicuous apical actin rings and in- tracellular aggregates are absent. Bars: (A) 30 mm; (B) 15 mm. addition of calcium were immunoblotted for the NZO-3 construct, endogenous ZO-3, ZO-1, ZO-2, occludin, and E-cadherin (Fig. 9 A). As expected, no NZO-3 was de- tected in untransfected parental cells. NZO-3 expression in MDCK/NZO-3 cells increased in response to the cal- cium jump, with highest expression at 6 h. Construct ex- pression then gradually decreased over time and re- turned to stable, steady-state levels. This decrease over time correlates well with recovery of TER and the nor- mal localizations of junctional proteins over the same time period. The expression levels of all other proteins examined did not change over the course of the calcium switch in either parental or MDCK/NZO-3 cells. There was also no detectable difference in the expression levels between parental and MDCK/NZO-3 cells at any of the time points, indicating that the effects on junctional as- sembly caused by NZO-3 expression were not due to al- tered expression of other junctional proteins. We ob- served a shift in mobility of occludin in both parental and MDCK/NZO-3 cells to a higher molecular weight species over time, corresponding to an increase in phosphoryla- tion of this protein as the TJ assembles, as previously de- scribed (Sakakibara et al., 1997). In epithelial cells, b-catenin is found in two distinct pools: a TX-100–insoluble pool, which comprises the b-catenin molecules which are bound to E-cadherin and linked to the actin cytoskeleton via a-catenin, and a TX- 100–soluble pool that represents the non–AJ-associated, cytoplasmic pool of free b-catenin (Fagotto et al., 1996; Funayama et al., 1995). Because these TX-100–soluble and –insoluble pools of b-catenin are functionally distinct, we examined the expression levels of b-catenin in equivalent protein loadings of TX-100–extracted parental and NZO- 3–expressing cells. Fig. 9 B shows that at 12 and 24 h after re-addition of calcium there is more soluble b-catenin in MDCK/NZO-3 cells relative to parental cells. This differ- ence is present but less pronounced at 48 h. Equivalent samples of the TX-100–insoluble fractions of parental and MDCK/NZO-3 cells show no difference in b-catenin lev- els. No differences were observed in the amount of a-cate- nin in the soluble or insoluble pools of TX-100–extracted parental or MDCK/NZO-3 cells (data not shown). Protein Binding Analyses We determined which of the known protein–protein inter- actions of ZO-3 could be mapped to the amino- and car- boxy-terminal halves of the molecule. It has been previ- ously shown that ZO-3 binds directly to actin filaments in vitro (Wittchen et al., 1999). In addition, ZO-3 also binds ZO-1, occludin, and the amino terminus of cingulin, but not to ZO-2 (Haskins et al., 1998; Cordenonsi et al., 1999). Fig. 10 A shows Coomassie-stained samples of the purified NZO-3 and CZO-3 proteins, demonstrating the equal amounts of these proteins used in subsequent binding ex- periments. As shown by actin cosedimentation assays in Fig. 10 B, substantial NZO-3 is found in the pellet in the presence of F-actin whereas a much smaller amount is visi- ble in the pellet in the absence of actin, likely due to a low level of fusion protein aggregation. CZO-3 remained in the supernatant fraction in both the presence and absence of actin filaments. Therefore, the actin binding site(s) of ZO-3 reside in the amino terminus of the protein. To map the binding site(s) of ZO-1 on ZO-3, we performed a bind- ing assay using 35S-labeled in vitro–transcribed/translated human ZO-1 added to NZO-3 or CZO-3 affinity columns. As shown in Fig. 10 C, NZO-3 retains ZO-1, whereas CZO-3 and the negative control do not. This demonstrates that the amino-terminal half of ZO-3 is responsible for binding to ZO-1. This is consistent with previous studies Wittchen et al. Delayed Junctional Complex Assembly 831