experiments revealed that the PDZ domain-containing amino-terminal half of ZO-3 binds to ZO-1 and F-actin, whereas both halves of ZO-3 appear to bind occludin and cingulin. These results suggest that a mechanism involving assembly of the actin cytoskeleton at the junctional mem- brane mediated through ZO-3 and/or ZO-1 underlies the inhibition of junctional assembly by NZO-3. The amino-terminal half of ZO-3 contains three PDZ domains, an arginine-rich domain and a proline-rich re- gion. This portion of the molecule correctly localizes to the TJ, whereas the carboxy-terminal half, which contains the SH3 domain, GUK domain, and acidic region, remains dif- fusely distributed in the cytoplasm. This is in contrast to the findings for ZO-1, where it was shown that the region encompassing the GUK and acidic region is required for correct targeting (Fanning et al., 1998). Furthermore, when just the three PDZ domains of ZO-1 were expressed in MDCK cells, this construct failed to localize to the TJ (Reichert et al., 2000) and induced an epithelial to mesen- chymal transition. Our results indicate that the PDZ do- main-containing region of ZO-3 contains functional char- acteristics distinct from those of ZO-1. Physiological data pertaining to the formation of the TJ barrier during junction assembly was obtained by using TER as an indicator of TJ integrity. Exogenous expression of NZO-3, CZO-3, or FLZO-3 did not alter steady-state TER levels (data not shown). However, when TER was measured during the time course of de novo TJ assembly in the calcium switch experimental system, it became ap- parent that expression of NZO-3 exerted a dominant- negative effect. Whereas CZO-3 or FLZO-3 expression did not affect TER recovery after calcium switch com- pared with untransfected parental MDCK cells, a calcium switch–induced increase in NZO-3 expression caused a significant lag in TER recovery after the re-addition of cal- cium (Fig. 3 A). It was not until 48 h that MDCK/NZO-3 cells approached the TER levels of the other cell lines, an effect that temporally correlated with the return of NZO-3 expression to steady-state levels. The fact that steady-state TER levels were not affected by steady-state NZO-3 ex- pression indicates that this construct exerts its effect only at the higher expression levels induced by the calcium switch and/or specifically during the assembly process, al- though it can not be ruled out that other undetected phe- notypic alterations may be occurring. We used recovery from cD treatment as a second TJ as- sembly paradigm, and these experiments corroborated the calcium switch results. When parental cells or cell lines ex- pressing FLZO-3, NZO-3, or CZO-3 were treated with the actin-disrupting drug, all showed the expected immediate drops in TER. However, when cD was washed out, the MDCK/NZO-3 cells displayed a lag in TER recovery (Fig. 3 B). These results are consistent with the hypothesis that the NZO-3 construct exerts its effects on junction assem- bly through interactions with the actin cytoskeleton. An unexpected result of NZO-3 expression during the calcium switch experiment was that AJ formation, as as- sessed by localization of E-cadherin (Fig. 7) and b-catenin (Fig. 8) to cell borders, was also delayed. The current model of junctional complex assembly in polarized epithe- lial cells is based on the discovery that E-cadherin–medi- ated cell adhesion provides the initial step that must occur before TJs can form (Gumbiner et al., 1988). Our data support the novel concept that exogenous expression of a truncated TJ protein component can exert effects on the AJ-associated proteins E-cadherin and b-catenin. The fact that both TJ and AJ proteins are similarly delayed in their assembly at the membrane lends credence to the notion that the two types of junctions share a coordinated assem- bly process, and that NZO-3 affects one or more steps in this process. Immunofluorescence localization experiments showed that the TJ proteins ZO-1, ZO-2, endogenous ZO-3, and occludin, and the AJ proteins E-cadherin and b-catenin were mislocalized during the early stages of junction as- sembly after calcium switch (Figs. 4, 7, 8, and data not shown). Moreover, the recovery of the typical localization of these proteins at cell borders temporally correlated with TER recovery after calcium switch. The fact that at later time points the cells were able to overcome the inhibitory effect exerted by NZO-3, as determined by the recovery of TER and correct targeting of ZO-1, ZO-2, ZO-3, occludin, E-cadherin and b-catenin to cell–cell contacts, is likely due to the observed return of NZO-3 protein expression to steady-state levels at later time points (Fig. 9 A). The mechanism(s) by which the calcium switch treatment causes NZO-3 expression to increase and then return to stable, steady-state levels is not known, although the effect was observed in repeated experiments with the same cell line (data not shown). Regardless, our results demonstrate that the NZO-3 construct has a dominant-negative effect on junctional complex assembly. Immunofluorescence staining of the NZO-3 construct was faint at the later time points, likely due to the lower levels of NZO-3 expression. We did not try to boost ex- pression levels with sodium butyrate because such treat- ment was toxic to the cells after periods longer than over- night, and overnight pulses of sodium butyrate treatment during calcium switch caused TER levels in parental cells to fluctuate significantly (data not shown). In addition, the anti-VSVG antibody only weakly reacted with our epitope-tagged construct in immunofluorescence and ex- hibited nonspecific nuclear staining, making it difficult to interpret the finer details of NZO-3 localization. However, we were able to determine that NZO-3 colocalizes with ZO-1 at cell borders at more mature TJs during calcium switch (Fig. 5). The NZO-3 construct did not colocalize with ZO-1 in the large intracellular aggregates that were observed in cells lacking cell border staining or with thick bars of ZO-1 at single cell–cell contacts that may represent two adjacent cells beginning TJ formation (Fig. 5). Be- cause of the high nonspecific background staining, it is dif- ficult to see whether another pool of NZO-3 exists in the cytoplasm. Based on the data presented here, and by con- focal Z-section analysis which shows precise colocalization of NZO-3 and ZO-1 at the TJ and no overlap of NZO-3 with E-cadherin more basally situated along the lateral membrane (data not shown), we believe the NZO-3 con- struct is not found at the AJ in the calcium switch. The involvement of the actin cytoskeleton in maintain- ing TJ integrity and regulating permeability has been well documented; this involvement is underscored by the fact that actin has multiple protein binding partners at the TJ (Itoh et al., 1997; Fanning et al., 1998; Wittchen et al., 1999), which themselves interact in various ways. It can be envisioned that this molecular architecture provides the The Journal of Cell Biology, Volume 151, 2000 834