|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2000//825 $5.00
The Journal of Cell Biology, Volume 151, Number 4,
, 2000 825-836
Original Article |
Exogenous Expression of the Amino-Terminal Half of the Tight Junction Protein Zo-3 Perturbs Junctional Complex Assembly
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and β-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100–soluble, signaling-active β-catenin was increased in NZO-3–expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or β-catenin.
Key Words: zonula occludens-3 protein • tight junction • cadherins • actin cytoskeleton • β-catenin
© 2000 The Rockefeller University Press
 |
Introduction |
|---|
 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
|---|
Since the identification and characterization of the membrane-associated guanylate kinase (MAGUK) protein ZO-1 (Stevenson et al. 1986), other TJ-associated proteins have been discovered, now totaling at least 15 peripheral and 3 integral membrane proteins including the multiple members of the claudin family (Morita et al. 1999). However, our understanding of the functions of these proteins, particularly those peripherally associated with the TJ membrane, is minimal. As a first step towards this understanding, determining how junctional proteins interact will help decipher TJ physiology at the molecular level. Two additional MAGUK family proteins, ZO-2 (Jesaitis and Goodenough 1994; Beatch et al. 1996) and ZO-3 (Haskins et al. 1998) have been shown to interact directly with ZO-1 (Haskins et al. 1998; Wittchen et al. 1999) but not with each other (Haskins et al. 1998). ZO-1/ZO-2 and ZO-1/ZO-3 likely exist as two distinct heterodimers in vivo and not as a trimeric complex as previously thought (Wittchen et al. 1999). All three ZO proteins interact directly with the carboxy-terminal 150 amino acids of occludin (Furuse et al. 1994; Haskins et al. 1998; Wittchen et al. 1999) and claudin 1–8 (Itoh et al. 1999a). ZO-1, ZO-2, and ZO-3 also bind actin filaments (Itoh et al. 1997; Fanning et al. 1998; Wittchen et al. 1999), providing multiple linkage sites to the actin cytoskeleton. Furthermore, ZO-1, ZO-2, and ZO-3 all interact with another TJ plaque protein, cingulin (Cordenonsi et al. 1999), and ZO-1 can associate with the Ras-effector molecule AF-6 (Yamamoto et al. 1997) and the integral membrane protein JAM (Bazzoni et al. 2000).
ZO-1, ZO-2, and ZO-3 contain a conserved series of well-known protein–protein interaction motifs. All three proteins have three amino-terminal PSD95/dlg/ZO-1 (PDZ) domains, followed by a Src homology 3 (SH3) domain and a guanylate kinase (GUK) domain. The carboxy-terminal portion of ZO-1 and ZO-2 consists of a proline-rich region; however, in ZO-3 this proline-rich region is located in the amino-terminal half of the protein between PDZ2 and PDZ3. The specific domains responsible for the various binding interactions of ZO-1, ZO-2, and ZO-3 have, in part been defined. ZO-1 and ZO-2 form a complex through reciprocal interactions between their PDZ2 domains (Fanning et al. 1998; Itoh et al. 1999b), and ZO-3 likely interacts with ZO-1 in the same manner (Itoh et al. 1999a). ZO-1 interaction with occludin likely occurs through the GUK domain, and actin binding via a region that encompasses the proline-rich domain of ZO-1 (Fanning et al. 1998). ZO-1, ZO-2, and ZO-3 binding to the carboxy-terminal YV sequence of claudin 1–8 requires the first PDZ domain of each protein (Itoh et al. 1999a). Finally, the amino-terminal region of ZO-1 and ZO-2 binds to 
-catenin, and ZO-1 and ZO-2 are components of the adherens junction (AJ) in certain nonepithelial cells (Itoh et al. 1997, Itoh et al. 1999b). No further information on individual domain binding characteristics or overall protein function(s) is available for ZO-3.
One current model for TJ assembly involves the coordinate assembly of the AJ and TJ. This model proposes that the first step in de novo junction formation involves E-cadherin–mediated cell adhesion (Gumbiner et al. 1988), followed by ZO-1 recruitment to the lateral surface of the cell via a transient interaction with the -/β-catenin complex (Rajasekaran et al. 1996). Indeed, it was subsequently found that ZO-1 binds -catenin in vitro (Itoh et al. 1997), providing the means by which ZO-1 is associated with the catenin complex during this early stage in junction formation. As epithelial polarization proceeds, ZO-1 is sorted out of the AJ and forms part of a separate TJ complex (Ando-Akatsuka et al. 1999). In vitro binding experiments have shown that ZO-2 can also bind -catenin (Itoh et al. 1999b), indicating the possibility that ZO-2 may follow a similar path during initial stages of junctional complex assembly, either directly via interaction with -catenin or indirectly via its association with ZO-1 (Wittchen et al. 1999). Recent data suggest that this hierarchy of junctional complex assembly is not absolute. Troxell et al. 2000 have shown that TJ assembly was extensive in MDCK cells expressing a mutant E-cadherin protein lacking the extracellular domain required for cell–cell adhesion, suggesting the possibility that TJs can assemble in the absence of an initial E-cadherin–mediated cell adhesion event.
Here we pursue the role of ZO-3 in epithelial cell physiology by expressing mutant ZO-3 constructs in MDCK cells. Through studies on TJ assembly and the domains responsible for protein–protein interactions, we have demonstrated that exogenous expression of the PDZ domain-containing, amino-terminal half of ZO-3 perturbs both TJ and AJ assembly. Moreover, such expression also affects actin dynamics and the biochemical properties of at least one adherens junction protein. Through in vitro binding assays we show that this amino-terminal half of ZO-3 preferentially binds ZO-1 and actin filaments. These studies provide the first functional information on ZO-3 and help to define how the molecular constituents of the junctional complex may interact and assemble in epithelial cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
15 h) to allow disassembly of the junctional complex. Cells were then switched back to 1.8 mM calcium-containing media at t = 0, and junctional reassembly was followed for various times. Control cells used for determination of steady-state levels of NZO-3 were trypsinized, replated in normal calcium media, and incubated for 65 h. Cytochalasin D (cD) (4 µg/ml) in Hepes-buffered saline plus glucose (HBSG; 10 mM Hepes, pH 7.4, 5.4 mM KCl, 137 mM NaCl, 1.3 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose), was added to filter-grown monolayers for 60 min and then was washed out by rinsing filters 3x with HBSG. Cells were then incubated in HBSG without cD for another 2 h.
Transepithelial Resistance Measurements
TER was measured with a Millicell-ERS apparatus (Millipore) in HBSG as previously described (Stevenson and Begg 1994). In the results depicted in Fig. 3 (A and B), duplicate filters from each cell line were measured for each time point, and the experiment was performed at least twice. Data from at least two experiments (n > 4 filters) are presented as mean ± SEM. Data from single clones of parental, MDCK/NZO-3, MDCK/CZO-3, and MDCK/FLZO-3 cells are shown. Two additional, independently selected MDCK/NZO-3 cell lines were tested in both the calcium switch and cD experiments and showed results essentially identical to the first MDCK/NZO-3 cell line (data not shown). For calcium switch experiments (see Fig. 3 A), TER is plotted in ohm x cm2. For cD experiments (see Fig. 3 B), TER is expressed as a percentage normalized to t = 0. The steady-state TER of all cell lines examined was 50–60 ohm x cm2.
|
Immunohistochemistry
For immunofluorescence staining with anti-VSVG, cells grown on collagen-coated coverslips were fixed with 5% acetic acid in ethanol for 5 min at –20°C and permeabilized with 0.2% Triton X-100 (TX-100) in TBS plus 1.8 mM calcium (TBS+) for 4 min at room temperature. For all other staining, cells grown on filters were fixed with 2.5% paraformaldehyde in TBS and permeabilized with 0.2% TX-100. Rhodamine-conjugated phalloidin (Sigma-Aldrich) was used to stain F-actin. Coverslips and filters were viewed on a Zeiss Axioskop (Carl Zeiss, Inc.).
Protein Expression Analysis and TX-100 Extraction
Parental or MDCK/NZO-3 cells grown on 35-mm tissue culture dishes were subjected to a calcium switch, and total lysates were made from cells 6, 12, 24, and 48 h after the re-addition of calcium. The steady-state level of NZO-3 was assessed on control cells not subjected to calcium switch that were maintained in normal calcium media for 65 h. Monolayers were rinsed 2x with ice-cold TBS+ and scraped up into 1 ml ice-cold TBS+ plus protease inhibitor cocktail (1 µg/ml aprotinin, 1 µg/ml chymostatin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM Pefabloc SC; Boehringer). Scraped cells were then harvested by centrifugation at 6,500 rpm for 5 min at 4°C. Cell pellets were resuspended in hot SDS lysis buffer (10 mM Tris, pH 7.8, 1% SDS) plus protease inhibitors and boiled for 5 min, shearing with a narrow bore pipette tip. Protein concentration was determined by BCA protein assay (Pierce Chemical Co.) and 20 µg total protein was loaded per lane on SDS-PAGE. For immunoblots, gels were electrophoretically transferred to nitrocellulose, immunoblotted with corresponding antibodies, and proteins detected via ECL (Amersham Pharmacia Biotech).
For separation of TX-100–soluble and –insoluble β-catenin, parental and MDCK/NZO-3 cells subjected to a calcium switch were extracted with CSK buffer (10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, 0.5% TX-100 plus 0.1 mg/ml DNAse, 0.1 mg RNAse, and protease inhibitor cocktail; Troxell et al. 2000). In brief, cell monolayers grown on 35-mm dishes were rinsed 2x with ice-cold TBS+ and then extracted for 10 min on ice with 1.5 ml CSK buffer. Insoluble material was scraped from the dish, and the extracts were centrifuged 10 min at 13,000 rpm at 4°C. Supernatants contain the detergent-soluble pool of β-catenin. Pellets containing detergent-insoluble material were resuspended in 1.5 ml hot SDS lysis buffer and boiled for 10 min. Protein concentration in each fraction was determined by BCA protein assay, and samples were loaded for SDS-PAGE and immunoblot analysis as above.
Recombinant Protein Expression and In Vitro Binding Studies
Recombinant 6-histidine–tagged NZO-3 and CZO-3 were expressed in Sf9 insect cells using a Baculovirus eukaryotic expression system (GIBCO BRL) and purified according to previously published methods (Haskins et al. 1998; Wittchen et al. 1999). Empty expression vector encoding the 6-histidine tag plus 36 nonspecific amino acids served as the negative control. Equal amounts of purified NZO-3 and CZO-3 were used in the binding experiments. [35S]Methionine-labeled in vitro–transcribed/translated human ZO-1 was prepared as described (Haskins et al. 1998; Wittchen et al. 1999). Expression of GST-occludin encoding the carboxy-terminal 150 amino acids of the cytoplasmic tail of chicken occludin (Furuse et al. 1993) was described previously (Haskins et al. 1998; Wittchen et al. 1999). GST–N-cingulin cDNA encoding the amino-terminal head region (amino acids 1–378) of Xenopus cingulin in pGEX-4T1 (a gift from Dr. Sandra Citi, University of Geneva, Geneva, Switzerland) was expressed according to published methods (Cordenonsi et al. 1999).
The binding of NZO-3 and CZO-3 to actin, ZO-1, and occludin was performed as previously described (Wittchen et al. 1999). Cingulin binding experiments used a modification of the methods of Cordenonsi et al. 1999. In brief, GST–N-cingulin or GST alone was immobilized on glutathione-Sepharose 4B beads, and equal amounts of purified NZO-3 or CZO-3 were added. Binding buffer comprised of PBS plus 1% TX-100 and protease inhibitors. Binding was allowed to proceed overnight at 4°C, followed by washing with the same buffer, and eluting bound protein by boiling the beads in gel sample buffer. NZO-3 and CZO-3 binding was detected by immunoblot, and in the case of occludin and cingulin binding experiments, the amount of bound NZO-3 and CZO-3 was compared by Coomassie blue staining of the bound fraction.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
50 ohm x cm2 by 96 h. Cell lines expressing the CZO-3 and FLZO-3 constructs followed a similar trend, plateauing at the same steady-state level as parental cells. In contrast, NZO-3–expressing cells exhibited delayed TER development, with no initial overshoot typical of the other cell lines. However, regardless of the delay, NZO-3–expressing cells reached a steady-state TER similar to that of the other cell lines by 48–96 h. The TER of all cells at 96 h is similar to that of untreated cells (data not shown). We corroborated the results obtained in the calcium switch experiments by determining the effect of ZO-3 construct expression on TJ reformation in MDCK cells treated with the actin-disrupting drug cD. In cells treated with cD, the apical actin cytoskeletal ring associated with the junctional complex is disrupted into large punctate aggregates that colocalize with similarly disrupted TJ proteins ZO-1, ZO-2, ZO-3, and occludin (Stevenson and Begg 1994; Wittchen et al. 1999). The cD-treated cells also show a sharp drop in TER, indicating a loss of functional TJs. It was shown that this effect is reversible, and upon washout of cD, TJs reassemble synchronously (Madara et al. 1986; Stevenson and Begg 1994). We treated our MDCK cell lines with cD for 60 min, and the drug was then washed out (Fig. 3 B). Upon cD treatment all cell lines exhibited an immediate and substantial decrease in TER. After cD washout, parental, FLZO-3–, and CZO-3–expressing cells recovered TER values equal to or greater than t = 0 values within 1 h. However, NZO-3–expressing cells showed a delayed TER recovery from cD treatment and failed to reach t = 0 levels for the duration of the experiment. This result is in agreement with the calcium switch experiment (Fig. 3 A) and demonstrates that MDCK cells expressing the NZO-3 construct display an impaired ability to assemble functional TJs.
Junctional Protein Localization
The physiological data suggesting impaired TJ-forming ability in NZO-3–expressing cells prompted us to assess the localization of proteins associated with both TJs and AJs in the calcium switch experimental system. Parental and MDCK/NZO-3 cells grown on filters were subjected to a calcium switch and protein localization was determined 6, 12, 24, and 48 h after re-addition of calcium. As shown in Fig. 4 A, ZO-1 is localized at cell–cell contacts within 6 h after re-addition of calcium. In the 12–48-h time period, the parental cells develop the cobblestone uniformity of shape and size that is typical of an MDCK epithelial cell monolayer at steady-state. The appearance of ZO-1 at cellular contacts 6 h after re-addition of calcium correlates well with the rapid increase in TER observed in the parental cells (Fig. 3 A). In contrast, cells expressing NZO-3 showed a significant delay in ZO-1 recruitment to cell borders after re-addition of calcium (Fig. 4 A). At 6 h, only small, isolated clusters of cells showed ZO-1 staining at cell borders. Over time the areas of cell monolayer that show ZO-1 localization at cell–cell contacts increase in size. At 6, 12, and 24 h when ZO-1 is not yet fully localized to cell borders, ZO-1 also localizes to large intracellular aggregates and linear planes of contact between neighboring cells. Complete localization of ZO-1 at the junctional membrane in NZO-3–expressing cells did not occur until 48 h after re-addition of calcium. ZO-1 localization in both cell types at 48 h is indistinguishable from untreated, steady-state cells (data not shown). Both parental and MDCK/NZO-3 cells displayed no detectable differences in expression levels of ZO-1 (or any other junctional protein examined; see Fig. 9 immunoblot) at 6, 12, 24, and 48 h. The delayed targeting of the TJ protein ZO-1 in MDCK/NZO-3 cells correlates with the delay in TER recovery after calcium switch (Fig. 3 A). The localization of the TJ proteins ZO-2, endogenous ZO-3, and the integral membrane protein occludin was identical to that of ZO-1 in the MDCK/NZO-3 cells (data not shown).
|
|
The disrupted localization of TJ proteins during junctional assembly after calcium switch in cells expressing NZO-3 indicates that this construct interferes with the formation of the complex of proteins found at the TJ. We therefore examined the localization of the construct itself during this process. The anti-VSVG antibody only weakly recognizes its epitope on NZO-3 in immunofluorescence and was not visible in filter-grown cells. Hence, MDCK/NZO-3 cells grown on coverslips and subjected to a calcium switch were costained with the anti–ZO-1 and anti-VSVG (NZO-3) antibodies. Parental MDCK cells stained with anti-VSVG serve as a negative control and show that this antibody exhibits nonspecific nuclear staining in untransfected cells (Fig. 5 A). At the earliest time points (6, 12, and 24 h), the NZO-3 construct appears at only those cell borders that also show ZO-1 localization. MDCK/NZO-3 cells stained with anti-VSVG antibody also exhibit a faint, granular cytoplasmic staining above background. NZO-3 staining is only faintly visible at 48 h, likely due to the lower levels of construct expression at this time point (see Fig. 9 immunoblot). In areas where NZO-3 is visible, it was colocalized with ZO-1 at all cell–cell contacts. Interestingly, NZO-3 was absent from the intracellular aggregates and linear planes of ZO-1 staining observed at early time points, and was only colocalized with ZO-1 at points of cell–cell contact formed by mature TJs (Fig. 5 B).
The actin cytoskeleton is an important structural and a functional element of the TJ (Hirokawa and Tilney 1982; Madara 1987). Actin filaments have been shown to interact directly with multiple TJ proteins (Itoh et al. 1997; Fanning et al. 1998; Wittchen et al. 1999). Disruption of the actin cytoskeleton is associated with impaired barrier function of the TJ (Madara et al. 1986; Madara and Pappenheimer 1987; Stevenson and Begg 1994), whereas the fence function appears to be unaffected (Takakuwa et al. 2000). Actin localization during TJ assembly in the calcium switch system was studied by rhodamine-phalloidin staining of actin filaments (Fig. 6). In parental MDCK cells, actin recruitment to a ring-like structure at the apical aspect of the lateral cell surface occurs rapidly after re-addition of calcium; as early as 6 h all parental cells show a circumferential actin ring. This ring thickens and condenses as the cells become more tightly packed and uniform in size in the 12–48-h time period. However, exogenous expression of NZO-3 delays recruitment of actin to the apical ring in a manner similar to that observed with ZO-1. Development of an apical actin ring at points of cell–cell contact in MDCK/NZO-3 cells occurred only in isolated patches of cells at 6 h, with the final localization not complete until 48 h. In addition, at the earlier time points (6, 12, and 24 h), actin also localized to large aggregates in cells which show underdeveloped actin rings. The delayed association of actin filaments with the junctional complex in MDCK/NZO-3 cells is likely a factor in the delayed TJ barrier formation in these cells.
|
|
|
In epithelial cells, β-catenin is found in two distinct pools: a TX-100–insoluble pool, which comprises the β-catenin molecules which are bound to E-cadherin and linked to the actin cytoskeleton via -catenin, and a TX-100–soluble pool that represents the non–AJ-associated, cytoplasmic pool of free β-catenin (Fagotto et al. 1996; Funayama et al. 1995). Because these TX-100–soluble and –insoluble pools of β-catenin are functionally distinct, we examined the expression levels of β-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 β-catenin in MDCK/NZO-3 cells relative to parental cells. This difference 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 β-catenin levels. No differences were observed in the amount of -catenin 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 interactions of ZO-3 could be mapped to the amino- and carboxy-terminal halves of the molecule. It has been previously 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 experiments. 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 visible 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 binding 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 showing that ZO-1 and ZO-2 bind each other via their second PDZ domains (Fanning et al. 1998; Itoh et al. 1999b). To define the occludin binding site on ZO-3, in vitro binding 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 occludin appear approximately equivalent, as assayed by Coomassie blue staining of the bound fractions. This indicates 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 carboxy-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 approximately equal affinity. In summary, the binding studies 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 (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 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 (Fig. 3 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 (Fig. 7) and β-catenin (Fig. 8) 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 (Fig. 4, Fig. 7, Fig. 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 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 (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 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 (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). 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 (Fig. 6). 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.
|
Acknowledgments |
|---|
We thank Alan Fanning, Warren Gallin, Barry Gumbiner, Lijie Gu, Tom Hobman, Brigitte Keon, Carolyn Machamer, Manijeh Pasdar, Sandra Citi, and Alpha Yap for providing reagents and/or advice. We also thank Naveen Basappa for his expert assistance in this project.
This work was supported by grants from the Medical Research Council of Canada, the Kidney Foundation of Canada, and the Canadian Association of Gastroenterology/Janssen-Ortho Inc. E.S. Wittchen holds a Medical Research Council studentship.
Submitted: 18 July 2000
Revised: 14 September 2000
Accepted: 4 October 2000
Abbreviations used in this paper: AJ, adherens junction; cD, cytochalasin D; CZO-3, carboxy-terminal half of ZO-3; FLZO-3, full-length ZO-3; GUK, guanylate kinase; MAGUK, membrane-associated guanylate kinase; NZO-3, amino-terminal half of ZO-3; PDZ, PSD95/dlg/ZO-1; SH3, Src homology 3; TER, transepithelial resistance; TJ, tight junction; TX-100, Triton X-100; VSVG, vesicular stomatitis virus glycoprotein.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Aberle H., Bauer A., Stappert J., Kispert A. & Kemler R.. Beta-catenin is a target for the ubiquitin-proteasome pathway, EMBO (Eur. Mol. Biol. Organ.) J., 16, 1997, 3797–3804.[Medline]
Anderson J.M., Stevenson B.R., Jesaitis L.A., Goodenough D.A. & Mooseker M.S.. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells, J. Cell Biol., 106, 1988, 1141–1149.
Ando-Akatsuka Y., Yonemura S., Itoh M., Furuse M. & Tsukita S.. Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity, J. Cell Physiol., 179, 1999, 115–125.[Medline]
Bazzoni G., Martinez-Estrada O.M., Orsenigo F., Cordenonsi M., Citi S. & Dejana E.. Interaction of Junctional Adhesion Molecule with the tight junction components ZO-1, cingulin, and occludin, J. Biol. Chem., 275, 2000, 20520–20526.
Beatch M., Jesaitis L.A., Gallin W.J., Goodenough D.A. & Stevenson B.R.. The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs-large/ZO-1) domains and an alternatively spliced region, J. Biol. Chem., 271, 1996, 25723–25726.
Behrens J., von Kries J.P., Kuhl M., Bruhn L., Wedlich D., Grosschedl R. & Birchmeier W.. Functional interaction of beta-catenin with the transcription factor LEF-1, Nature., 382, 1996, 638–642.[Medline]
Cereijido M., Robbins E.S., Dolan W.J., Rotunno C.A. & Sabatini D.D.. Polarized monolayers formed by epithelial cells on permeable and translucent support, J. Cell Biol., 77, 1978, 853–880.
Cordenonsi M., D'Atri F., Hammar E., Parry D.A., Kendrick-Jones J., Shore D. & Citi S.. Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin, J. Cell Biol., 147, 1999, 1569–1582.
Fagotto F., Funayama N., Gluck U. & Gumbiner B.M.. Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus, J. Cell Biol., 132, 1996, 1105–1114.
Fanning A.S., Jameson B.J., Jesaitis L.A. & Anderson J.M.. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton, J. Biol. Chem., 273, 1998, 29745–29753.
Farquhar M.G. & Palade G.E.. Junctional complexes in various epithelia, J. Cell Biol., 17, 1963, 375–412.
Funayama N., Fagotto F., McCrea P. & Gumbiner B.M.. Embryonic axis induction by the armadillo repeat domain of beta-cateninevidence for intracellular signaling, J. Cell Biol., 128, 1995, 959–968.
Furuse M., Hirase T., Itoh M., Nagafuchi A., Yonemura S., Tsukita Sa. & Tsukita Sh.. Occludina novel integral membrane protein localizing at tight junctions, J. Cell Biol., 123, 1993, 1777–1788.
Furuse M., Itoh M., Hirase T., Nagafuchi A., Yonemura S., Tsukita Sa. & Tsukita Sh.. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions, J. Cell Biol., 127, 1994, 1617–1626.
Gumbiner B. & Simons K.. A functional assay for proteins involved in establishing an epithelial occluding barrieridentification of an uvomorulin-like polypeptide, J. Cell Biol., 102, 1986, 457–468.
Gumbiner B., Stevenson B.R. & Grimaldi A.. The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex, J. Cell Biol., 107, 1988, 1575–1587.
Haskins J., Gu L., Wittchen E.S., Hibbard J. & Stevenson B.R.. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin, J. Cell Biol., 141, 1998, 199–208.
Hirokawa N. & Tilney L.G.. Interactions between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear, J. Cell Biol., 95, 1982, 249–261.
Howarth A.G., Hughes M.R. & Stevenson B.R.. Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types, Am. J. Physiol., 262, 1992, C461–C469.[Medline]
Huber O., Korn R., McLaughlin J., Ohsugi M., Herrmann B.G. & Kemler R.. Nuclear localization of beta-catenin by interaction with transcription factor LEF-1, Mech. Dev., 59, 1996, 3–10.[Medline]
Itoh M., Nagafuchi A., Moroi S. & Tsukita S.. Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments, J. Cell Biol., 138, 1997, 181–192.
Itoh M., Furuse M., Morita K., Kubota K., Saitou M. & Tsukita S.. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins, J. Cell Biol., 147, 1999, 1351–1363a.
Itoh M., Morita K. & Tsukita S.. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin, J. Biol. Chem., 274, 1999, 5981–5986b.
Jesaitis L.A. & Goodenough D.A.. Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the drosophila discs-large tumor suppressor protein, J. Cell Biol., 124, 1994, 949–961.
Madara J.L.. Intestinal absorptive cell tight junctions are linked to cytoskeleton, Am. J. Physiol., 253, 1987, C171–C175.[Medline]
Madara J.L., Barenberg D. & Carlson S.. Effects of cytochalasin D on occluding junctions of intestinal absorptive cellsfurther evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity, J. Cell Biol., 102, 1986, 2125–2136.
Madara J.L. & Pappenheimer J.R.. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia, J. Membr. Biol., 100, 1987, 149–164.[Medline]
Morita K., Furuse M., Fujimoto K. & Tsukita S.. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands, Proc. Natl. Acad. Sci. USA., 96, 1999, 511–516.
Rajasekaran A.K., Hojo M., Huima T. & Rodriguez-Boulan E.. Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions, J. Cell Biol., 132, 1996, 451–463.
Reichert M., Muller T. & Hunziker W.. The PDZ domains of zonula occludens-1 induce an epithelial to mesenchymal transition of Madin-Darby canine kidney I cells. Evidence for a role of beta-catenin/Tcf/Lef signaling, J. Biol. Chem., 275, 2000, 9492–9500.
Rimm D.L., Koslov E.R., Kebriaei P., Cianci C.D. & Morrow J.S.. Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex, Proc. Natl. Acad. Sci. USA., 92, 1995, 8813–8817.
Sakakibara A., Furuse M., Saitou M., Ando-Akatsuka Y. & Tsukita Sh.. Possible involvement of phosphorylation of occludin in tight junction formation, J. Cell Biol., 137, 1997, 1393–1401.
Stevenson B.R. & Begg D.A.. Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells, J. Cell Sci., 107, 1994, 367–375.[Abstract]
Stevenson B.R., Siliciano J.D., Mooseker M.S. & Goodenough D.A.. Identification of ZO-1a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia, J. Cell Biol., 103, 1986, 755–766.
Stewart D.B., Barth A.I. & Nelson W.J.. Differential regulation of endogenous cadherin expression in Madin-Darby Canine Kidney cells by cell–cell adhesion and activation of beta-catenin signaling, J. Biol. Chem., 275, 2000, 20707–20716.
Takakuwa R., Kokai Y., Kojima T., Akatsuka T., Tobioka H., Sawada N. & Mori M.. Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B, Exp. Cell Res., 257, 2000, 238–244.[Medline]
Tetsu O. & McCormick F.. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature., 398, 1999, 422–426.[Medline]
Troxell M.L., Gopalakrishnan S., McCormack J., Poteat B.A., Pennington J., Garringer S.M., Schneeberger E.E., Nelson W.J. & Marrs J.A.. Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly, J. Cell Sci., 113, 2000, 985–996.[Abstract]
Wittchen E.S., Haskins J. & Stevenson B.R.. Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3, J. Biol. Chem., 274, 1999, 35179–35185.
Wong V. & Gumbiner B.M.. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier, J. Cell Biol., 136, 1997, 399–409.
Yamamoto T., Harada N., Kano K., Taya S.-I., Canaani E., Matsuura Y., Mizoguchi A., Ide C. & Kaibuchi K.. The ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells, J. Cell Biol., 139, 1997, 785–795.
Yonemura S., Itoh M., Nagafuchi A. & Tsukita Sh.. Cell-to-cell adherens junction formation and actin filament organizationsimilarities and differences between non-polarized fibroblasts and polarized epithelial cells, J. Cell Sci., 108, 1995, 127–142.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
