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 increases 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 β-catenin in the TX-100–soluble pool is also elevated in NZO-3 expressing cells. Finally, in vitro binding 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 membrane 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 region. 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 diffusely 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 mesenchymal transition. Our results indicate that the PDZ domain-containing region of ZO-3 contains functional characteristics 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 apparent 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 compared 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 calcium ( 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 expression 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, although it can not be ruled out that other undetected phenotypic alterations may be occurring.
We used recovery from cD treatment as a second TJ assembly paradigm, and these experiments corroborated the calcium switch results. When parental cells or cell lines expressing 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 ( B). These results are consistent with the hypothesis that the NZO-3 construct exerts its effects on junction assembly through interactions with the actin cytoskeleton.
An unexpected result of NZO-3 expression during the calcium switch experiment was that AJ formation, as assessed by localization of E-cadherin () and β-catenin () to cell borders, was also delayed. The current model of junctional complex assembly in polarized epithelial cells is based on the discovery that E-cadherin–mediated 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 β-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 assembly 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 β-catenin were mislocalized during the early stages of junction assembly after calcium switch (, , , 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 β-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 ( 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 expression levels with sodium butyrate because such treatment was toxic to the cells after periods longer than overnight, 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 exhibited 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 (). 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 (). Because of the high nonspecific background staining, it is difficult to see whether another pool of NZO-3 exists in the cytoplasm. Based on the data presented here, and by confocal 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 construct is not found at the AJ in the calcium switch.
The involvement of the actin cytoskeleton in maintaining 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 means by which an actin filament network can be recruited to and organized in a functionally relevant manner at the TJ. The actin cytoskeleton is also a major structural and functional element of the AJ (Farquhar and Palade 1963
; Rimm et al. 1995
; Yonemura et al. 1995
), and is present in a bundled actin belt around the apical periphery cell at the level of the AJ. Interestingly, in MDCK/NZO-3 cells, there is a delay in actin recruitment and formation of this perijunctional apical actin ring (). Because the amino-terminal half of ZO-3 is responsible for binding F-actin, this may represent one mechanism whereby expression of this construct affects TJ and AJ assembly.
Not only did expression of the amino terminus of ZO-3 alter the distribution of β-catenin, but there was also an increase in the TX-100–soluble pool of signaling-active β-catenin. Presumably the presence of an increased level of the NZO-3 construct at early time points after calcium switch results in a downstream alteration of the E-cadherin/catenins complex at the adherens junction, releasing β-catenin from a cytoskeletal linkage into the TX-100–soluble pool. A corresponding change in the levels of β-catenin in the insoluble pool is not observed, although any possible change may be masked by the overall high levels of β-catenin present in these samples. Normally cytoplasmic β-catenin levels are strictly regulated via a ubiquitin-mediated proteolysis pathway requiring β-catenin interaction with the cytoplasmic tumor suppressor APC (Aberle et al. 1997
). Soluble β-catenin that escapes this targeted proteolysis is capable of translocating to the nucleus where it acts as a transcriptional transactivator in a complex with TCF/LEF family of transcription factors to direct transcription of a variety of genes that promote a proliferative phenotype (Behrens et al. 1996
; Huber et al. 1996
; Tetsu and McCormick 1999
). Recently, Stewart et al. 2000
, showed that expression of a mutant signaling-active (soluble) form of β-catenin in MDCK cells caused a delay in the establishment of tight confluent cell monolayers compared with control cells, and the cells appeared more motile and formed less compact colonies when plated at a low density. These results, taken together with our data showing that NZO-3 expression delays TER formation after a calcium switch and results in an increased level of signaling-active, soluble β-catenin in these cells, suggests that NZO-3 might act through β-catenin to exert its effects on epithelial junctional complex formation. At present we do not know if this action is direct or indirect.
In summary, through these studies we attempted to further elucidate the role of ZO-3 in epithelial cell physiology, including the role it plays in junctional complex assembly and its protein binding interactions. Exogenous expression of partial constructs is the experimental method we chose to approach the dissection of ZO-3 protein function and TJ physiology. This study demonstrates that exogenous expression of the PDZ domain-containing amino-terminal half of ZO-3 perturbs both TJ and AJ assembly. Moreover, expression of NZO-3 alters actin dynamics and increases the amount of soluble signaling-active β-catenin. We are currently testing the hypothesis that NZO-3 exerts its effect on junctional assembly via a mechanism that involves its F-actin and ZO-1 binding ability and/or the increase in soluble β-catenin.