In the current study we demonstrate that ZO-1 depletion in MDCK cells results in a size-selective increase in permeability for solutes that are larger than the claudin-based pores. In addition, ZO-1 depletion results in uncoupling of the normal barrier response to inhibition of myosin activity. The barrier defect does not involve a disruption of the small claudin-based pores as measured by the Papp of PEGs <4 Å or by TER. In fact, TER, a measure of instantaneous ionic permeability, is slightly but significantly increased in knockdown cells relative to controls. The increased permeability for larger solutes is rescued by expression of both full-length ZO-1 and a truncated construct containing amino-terminal domains that, among other things, binds to all of the know tight junction transmembrane proteins (). ZO-1 depletion also results in enhanced sensitivity to Ca2+-depletion and actin disruption with cytochalasin D. Taken together this is the first direct evidence that ZO-1 normally serves to stabilize the barrier and functionally link it to the actin–myosin cytoskeleton.
Figure 8. Diagram of reported ZO-1–binding proteins and their binding sites. Original references are cited in Fanning and Anderson (2009) . Many ZO-1–binding proteins also bind actin including AF-6, cingulin, ZO-2 and -3, α-catenin, Shroom (more ...)
Although ZO-1 is a universal component of tight junctions and interacts with multiple other tight junction proteins, two recent studies (Umeda et al., 2004
; McNeil et al., 2006
) reported limited physiological consequences after ZO-1 knockout or knockdown in epithelial cells. In contrast, in the current study we demonstrate a requirement for ZO-1 in normal barrier function in MDCK cells and show that ZO-1–depleted MDCK cells have an altered morphology characterized by a loss of tortuosity at the apical junction contacts and changes in apical actin localization. The reasons for the differences between previously published studies and our findings are likely a function of both cell type and methodology. Umeda et al. (2004)
used homologous recombination to knockout ZO-1 expression in Eph4 cells and reported no change in flux for 40-kDa FD; we report a consistent increase in flux for a smaller (3 kDa) FD, but like Umeda et al.
have also found that flux of larger 10-kDa FD was not significantly altered. Modest permeability changes (in our case up to twofold) are more difficult to detect as the tracer size increases, except cases where the barrier is catastrophically opened, for example, by Ca2+
depletion. The lack of effect on cell morphology may be related to cell type, because Eph4 cells normally lack the convoluted tight junction staining pattern that characterizes MDCK II cells. Macara and coworkers (McNeil et al., 2006
), on the other hand, did not measure flux in their ZO-1 knockdown cell lines; that they did not note a change in morphology is likely to be a function of the fact that they used transient transfection to deplete ZO-1 rather than generating stable cell lines as we have done. A third group (Aijaz et al., 2007
) has also generated stable MDCK ZO-1 knockdown cell lines. In contrast to our finding, they did not find an effect on paracellular flux. However, this group reported a decrease in ZO-1 levels only to 25% of control levels, compared with 2–5% remaining in our cell lines. We suspect the residual level of ZO-1 in their cells is the reason for their failure to see change in flux, because this level of ZO-1 expression in our ”rescued“ cell lines is sufficient to return flux to normal levels.
In spite of many studies which have reported changes in ZO-1 localization in association with alterations in paracellular permeability (Nusrat et al., 2000
; Wang et al., 2005
), the finding that ZO-1 depletion results in increased flux in the size-independent pathway is the first demonstration of a clear physiological role for this much studied protein. The observed increase in flux is modest, but it occurs in a background of some residual ZO-1 and minor or no changes in the distribution and levels of most other tight junction proteins. The increased flux is not associated with a decrease in TER or with a change in dilution potential; these outcomes are consistent with the immunolocalization and immunoblot findings that the tight junction transmembrane proteins such as claudins, occludin, and tricellulin are unchanged. Along with the change in flux, the observed alteration in actin localization in the knockdown cells provides clear evidence that ZO-1 is a crucial scaffolding protein, linking a physiologically relevant actin pool to the tight junction barrier. Evidence for a similar role for ZO-2 is lacking, because in our hands, ZO-2 knockdown had no observable physiological effect. This result is different from that reported elsewhere (Hernandez et al., 2007
), but could be due to the fact that Hernandez and coworkers tested transiently transfected cells, whereas we analyzed flux in stable knockdowns.
Increased flux for solutes which are larger than the claudin pores in the absence of decreased TER implies that the long-term dynamic characteristics of the barrier are altered by ZO-1 depletion without a compromise in the instantaneous electrical barrier. The tight junction is arranged as a series of continuous claudin-based cell–cell contacts. Multiple barriers presumably provide a fail-safe in case individual contacts transiently break. We speculate that the selective effect of ZO-1 depletion could result from more frequent transient breaks in the contacts but without the simultaneous loss of all of the contacts in series. This would increase permeability for solutes measured over long times but not affect a fast assessment of the barrier with TER. Future studies will test whether ZO-1–depleted junctions are more dynamic.
Previous studies have demonstrated that a ring of filamentous actin normally encircles the apical junctional complex and have indicated that contraction of the ring is associated with changes in cell shape and tight junction permeability (Madara, 1987
). Additionally, ultrastructural studies have detected direct contact between actin filaments and the tight junction, although the functional significance of these contacts is unknown (Hirokawa et al., 1983
; Madara, 1987
). Our studies indicate that F-actin localization is significantly altered in the ZO-1 knockdown cells, with an increase in actin staining at apical junctional complex and into scattered apical dots. These observations suggest that ZO-1 has very specific effects on actin dynamics at the apical junctional complex, perhaps by localizing the activity of cytoskeletal proteins [such as α actinin; Chen et al., 2006
), α catenin (Itoh et al., 1997
), or shroom2 (Etournay et al., 2007
)] or signaling pathways [such as the RhoGEF TUBA (Otani et al., 2006
) and Gα12
(Meyer et al., 2002
)] that regulate actin dynamics.
How these changes in actin dynamics or cortical F-actin architecture affect permeability is a matter of speculation. One possibility is that there is a specific cortical actin network that is associated with the barrier and that the assembly or plasticity of this network is organized by ZO-1 (via contacts with cytoskeletal proteins outlined in ). In the absence of ZO-1, the network does not maintain a normal barrier. It seems unlikely that ZO-1 acts as a direct transducer between the physical force generated by actomyosin activity and components of the barrier. The C-terminus of ZO-1 binds directly to F-actin (outlined in ); however, because the increased flux is rescued by the Nterm construct, which lacks the direct actin-binding site, direct interactions between ZO-1 and F-actin do not appear essential for barrier stabilization. Instead, our data suggest that indirect contacts via other actin-binding and/or -signaling proteins that bind to the Nterm construct are sufficient.
Whatever the mechanism, our observations suggest that one critical factor is the ability of ZO-1 to link the activity of myosin 2 to the state of the barrier. Perhaps surprisingly, the localization of myosin within the apical junctional complex is only slightly altered in ZO-1 knockdown cells. There is a tendency for it to become less continuous and more punctuate in staining pattern. This pattern, most evident in C, is again consistent with loss of normal interactions. Many studies (Nusrat et al., 1995
; Jou et al., 1998
; Walsh et al., 2001
; Benais-Pont et al., 2003
; Shen et al., 2006
) have implicated myosin activation in changes in paracellular flux downstream of RhoA and MLCK. Despite the subtle changes in myosin 2B localization, there is a clear difference between control and ZO-1 knockdown cells in their responses to pharmacologic inhibition of ROCK or myosin 2 ATPase. This suggests that ZO-1 does not localize myosin 2B, but may still act as a scaffolding protein indirectly regulating myosin activity. For example, ZO-1 knockdown decreases cingulin localization (Umeda et al., 2004
; Supplementary Figure S3). Cingulin (and the related protein, paracingulin, Guillemot et al., 2008
) have been demonstrated to bind the guanidine nucleotide exchange factor, GEF-H1, which inhibits Rho signaling (Benais-Pont et al., 2003
; Aijaz et al., 2005
). Decreased tight junction cingulin might increase Rho activation and thus myosin phosphorylation; however, as previously reported, neither cingulin knockout (Guillemot et al., 2004
) nor knockdown (Guillemot and Citi, 2006
) altered permeability. The role of paracingulin in flux regulation has not yet been tested. Another possibility is that ZO-1 might normally act as a sink for Gα12
, which can bind to the SH3 domain of ZO-1 (Meyer et al., 2002
; Sabath et al., 2008
; ) and that ZO-1 depletion would lead to increased activation of Rho A through an increase in accessible Gα12
In summary, this is the first direct experimental evidence of a role for ZO-1 in control of paracellular permeability through coupling it to perijunctional actin and myosin. However, providing the evidence that ZO-1 does in fact form a link between the barrier proteins and the cortical cytoskeleton is just a small piece of the puzzle. A cursory inspection of reveals just some of the other proteins that might be important links in this interaction. Unraveling the most relevant of these interactions in the physiological regulation of the tight junction will aid in understanding how barrier integrity is maintained and what components are likely to be involved when the barrier is pathologically compromised.