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Vol. 17, Issue 4, 1922-1932, April 2006
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Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908-0577
Submitted July 19, 2005;
Revised January 6, 2006;
Accepted January 12, 2006
Monitoring Editor: Asma Nusrat
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Gibson and Perrimon, 2003; Nelson, 2003). Typically, they create a barrier between the extracellular environment and the interior of the organism. Epithelia organize into layered sheets of cells with apical-basal polarity and strong cell-cell adhesions. The tight junction is the most apical adhesion between epithelial cells and is a complex structure that functions both as a barrier to the paracellular diffusion of ions and molecules and as a fence to separate the apical plasma membrane from the basolateral domain in polarized epithelia (D'Atri and Citi, 2002; Gonzalez-Mariscal et al., 2003; Matter and Balda, 2003). Tight junctions are fine-tuned to perform specific functions in different tissues and respond dynamically to changes in the environment and cell density (Tsukita and Furuse, 2002). The junctions between epithelial cells bear structural and functional relationships to endothelial cell junctions, neuromuscular junctions, and both immune and neural synapses. Similar proteins are present in all of these structures, and the same conserved groups of polarity proteins drive their formation. It is therefore important to understand the fundamental structural basis for tight junction formation and to learn the rules for their assembly.
Tight junctions contain at least 40 different proteins (Gonzalez-Mariscal et al., 2003). Some of these proteins, such as the claudins and occludin are transmembrane proteins that form intercellular homophilic and heterophilic adhesions, whereas others such as ZO-1, ZO-2, ZO-3, cingulin, MAGI-1, Pals1, and PATJ are intracellular plaque proteins that form a scaffold between the transmembrane proteins and the actin cytoskeleton. Certain of these proteins also engage in feedback regulation of gene expression. For example, ZO-1 can bind through its Src homology 3 (SH3) domain to a transcription factor called ZONAB, which controls epithelial cell proliferation (Balda and Matter, 2000; Balda et al., 2003). As epithelial cells differentiate and become more polarized, ZO-1 levels at the junctions increase, sequestering more ZONAB to the junctions and away from the nucleus, thereby repressing proliferation.
Polarity proteins such as Par-3, Par-6, aPKC, and Pals1 also localize to tight junctions, and in some cases, they have been shown to be essential for their normal assembly (Ohno, 2001; Chen and Macara, 2005; Shin et al., 2005). These same proteins are required for the polarization of diverse cell types, including neurons and astrocytes, the nematode zygote, fly neuroblasts and oocytes, endothelial cells, and T-lymphocytes (Nelson, 2003; Macara, 2004). Nonetheless, almost nothing is known about the basic steps in tight junction assembly. We do not know what most of the polarity proteins do, which proteins define the location of the junctions, or the order of assembly of the various junction components, or the structure of a junctional unit, or how these units link into a concatenated chain that encircles each epithelial cell. One problem is that many of the structural components of the tight junction are large, complex proteins with diverse domains of uncertain function. Junctions contain many MAGUK proteins, each of which possesses PSD-95, Discs large, ZO-1 (PDZ), SH3, and guanylate kinase-like (GUK) domains (Gonzalez-Mariscal et al., 2003; Funke et al., 2004). ZO-1, for example, contains three PDZ domains, which have been reported to bind to claudins, ZO-2, connexin36, and the junctional adhesion molecule-1 (Itoh et al., 1999; Ebnet et al., 2000; Li et al., 2004). The SH3 domain binds an unknown protein kinase, and to a transcription factor, ZONAB (Balda et al., 1996; Balda and Matter, 2000). An acidic sequence adjacent to the SH3 domain has been reported to bind to heterotrimeric G proteins; the GUK domain binds occludin (Furuse et al., 1994; Fanning et al., 1998); and the C terminus can bind actin, cortactin, and protein 4.1 (Fanning et al., 1998; Katsube et al., 1998; Meyer et al., 2002). ZO-1 can also associate with a coiled-coil protein called cingulin (Cordenonsi et al., 1999). However, it is unclear which, if any, of these domains and binding partners are important for the biological functions of ZO-1. Disruption of the occludin and cingulin genes in embryoid bodies derived from embryonic stem cells did not prevent tight junction formation (Saitou et al., 1998; Guillemot et al., 2004). Moreover, a comprehensive analysis of junctional complexes using density gradient fractionation found little association of ZO-1 with either occludin or claudins (Vogelmann and Nelson, 2005).
A knockout of ZO-1 in mouse epithelial cells recently demonstrated that ZO-1 is not essential for tight junction formation but that loss of ZO-1 causes a pronounced delay in junction assembly, as measured after withdrawal and replacement of calcium in the medium (Umeda et al., 2004). We now show that gene silencing of ZO-1 in canine MDCK epithelial cells by RNA interference (RNAi) causes the same phenotype and that the delay occurs at least in part during the initial spreading phase after the calcium switch, and during remodeling of the apical ring as cells form contacts with one another. We used rescue experiments with mouse ZO-1 to demonstrate that the SH3 is a key determinant for junction assembly, whereas the PDZ1 domain and C-terminal region seem to be nonessential. Several ZO-1 binding proteins, including occludin, ZO-2, and cingulin, lack detectable function in tight junction assembly, under the conditions of our experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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- isoform of murine ZO-1 using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). To create the PDZ domain mutation, R28 was mutated to a glutamate. The SH3 domain mutation was introduced into the ligand binding pocket by changing W557 and L558 to glycine and alanine, respectively. An EcoRV site is present at base 1999, close to the beginning of the GUK domain; so point mutations were introduced near the 3' end of the sequence that encodes the domain to create a second EcoRV restriction site that would retain the correct frame after digestion to remove the intervening sequence. The mutations were CGATT
ATATC (residues 2366-2370). Each of the mutant ZO-1 constructs was generated in pBluescript and then subcloned into the pK-YFP vector (Du and Macara, 2004). A C-terminal fragment of ZO-1, encoding residues 893-1666, was obtained by PCR and ligated into pKH3. The 1523 amino acid C-terminal truncation of ZO-1 was produced by digesting pK-YFP-ZO-1 with ClaI and inserting a linker that contained a stop codon. The presence of a unique Cla1 site 3' to the ZO-1 open reading frame (ORF) allowed removal of the C-terminal tail of ZO-1. Sequences encoding the isolated SH3 domains of ZO-1 and p120RasGAP were amplified by PCR and subcloned into pK-YFP. (A schematic of the mutants is shown in Figure 6A.)
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). Canine DNA data were obtained from GenBank, and short hairpin RNA (shRNA) targeting sequences were chosen using the Dharmacon Web site (http://dharmacon.com/siDESIGN/). Two shRNAs were designed for each gene (Table 1). Cells were transiently transfected using an electroporation technique (Amaxa Biosystems, Gaithersburg, MD). In total, 3-5 µg of DNA was electroporated into 2 x 106 cells.
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For calcium switch experiments, the cells (1 x 106/35-mm dish) were allowed to grow for 2 d posttransfection. Cells were then rinsed with warm phosphate-buffered saline (PBS), and the medium was changed to minimum essential medium lacking calcium (Invitrogen) supplemented with 2% heat-inactivated FCS that had been dialyzed against the same medium, plus penicillin and streptomycin. The following morning, the calcium-free medium was replaced by normal medium with serum.
Immunofluorescence/Immunoblotting
Transfected cells were plated on eight-well Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL). Approximately 4 x 105 cells were plated in each well. The remaining transfected cells were plated on 35-mm tissue culture dishes (Corning, Corning, NY) and used for immunoblotting to determine expression levels.
Cells were fixed using a 1:1 mixture of ice-cold methanol/acetone for 3 min, blocked for 1 h at room temperature with 3% bovine serum albumin/phosphate-buffered saline followed by a 1-h incubation at room temperature with anti-occludin monoclonal (1:500 dilution from 1 mg/ml; Invitrogen), and anti-green fluorescent protein (GFP) polyclonal antibodies (1:500, 2 mg/ml; Invitrogen). ZO-1 was stained with rabbit polyclonal antibodies (1:500, 0.25 mg/ml; Invitrogen). After a brief wash in PBS, cells were incubated with Alexa 594 anti-mouse and Alexa 488 anti-rabbit secondary antibodies (1:2000, 2 mg/ml; Invitrogen) for 1 h. The slides were mounted using 22 x 50-mm coverglasses (Fisher Scientific, Hampton, NH) and gel/mount aqueous mounting medium (Biomeda, Foster City, CA) and sealed with a coat of clear nail polish. Slides were viewed on a Nikon Eclipse inverted microscope equipped with a Hamamatsu Orca charge-coupled device camera (C4742-95-12NRB) and a 60x water immersion lens (numerical aperture [N.A.] 1.2). Images were collected at 12-bit depth with 1 x 1 binning, using Openlab software (Improvision, Boston, MA), and processed using Adobe Photoshop 7.0. Confocal images were collected using a Zeiss LSM510 META microscope, with a 100x oil-immersion lens (N.A. 1.3) and rendered with Volocity software (Improvision).
For immunoblotting, cells were scraped into 250 µl of lysis buffer (0.5% IGEPAL CA-630, 2 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride, and brought to a final volume in PBS). A fraction was removed for protein quantification by the Bradford assay, and then Laemmli buffer was added to the remainder, and they were boiled for 3 min. Proteins were separated by SDS PAGE on an 8% gel and transferred to nitrocellulose. Primary antibodies were rabbit polyclonals for ZO-1, ZO-2, claudin1, (1:1000, 0.25 mg/ml; Invitrogen), cingulin anti-serum (1:1000; Invitrogen), anti-E-cadherin monoclonal (1:2500, 0.25 mg/ml; BD Biosciences, San Jose, CA), anti-GFP polyclonal (1:500, 2 mg/ml; Invitrogen), and cortactin hybridoma supernatant (clone 4F11; a kind gift of Tom Parsons, University of Virginia, Charlottesville,VA). After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (1:20,000; Jackson ImmunoResearch Laboratories, West Grove, PA), bands were visualized using chemiluminescent reagents (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Image Processing
To improve the visualization of junction formation, images were processed in Adobe Photoshop by enhancing the contrast, and then inverting the pixel values, so that the junctions show up as black lines on a white background. To provide a more objective measure of junction assembly rescue, we developed a measure of junction staining integrity. Briefly, cell-cell boundaries were traced for a series of the images showing occludin staining, using ImageJ, which generates a value between 0 and 255 for each pixel along the traced line. We found it easiest to track cell boundaries in a continuous line from one edge of the image field to the other. We traced at least three such lines for each image, which collects data on the junctions for 10-15 cells. The mean pixel value was then calculated for all the traces in each image. In the absence of any detectable occludin at the boundaries, or at gaps in the junctions, the mean intensity value was close to zero (white pixels). For control cells with intact junctions, with no gaps, it was close to 140. Values from multiple images and experiments were pooled to obtain means and SEs and were compared using an unpaired, two-tailed t test.
Transepithelial Resistance Measurements
Cells (1 x 106) were plated and grown for 2 d on 12-mm Costar Transwell filters (0.4-µm pore size) to form confluent monolayers. They were depleted of calcium overnight. The resistance across the monolayers was measured, after readdition of calcium-containing medium, using an EVOM (WPI, Sarasota, FL). An empty filter was used to determine the background resistance. Three separate filters were used for each condition, and the mean resistance was calculated after background subtraction, in ohms · cm2 (Matter and Balda, 2003).
Time-Lapse Microscopy
Cells were transfected with pK-YFP-occludin or pK-YFP-ZO-1 and plated on Lab-Tek 4-well coverglasses. They were switched to calcium-free medium after 2 d, and then mounted on a Zeiss Axiophot inverted microscope and maintained at 37°C with a Nevek incubator system. The cells were switched to F-12 medium (with calcium) plus 20 mM HEPES, pH 7.2, plus Oxyrase (0.3 units/ml) to reduce phototoxicity. F-12 has lower background fluorescence than DMEM. Images (12-bit grayscale) were collected using a 40x objective lens (N.A. 0.95), with the mercury lamp at 50% power, and with a 34% neutral density filter in the excitation path. The frame-rate was 1/min. The YFP-ZO-1 cells were also imaged using a PerkinElmer spinning disk confocal microscope, at a frame rate of 0.5/min. Stacks of 10 images were collected at each time point, and each stack was flattened and processed using Volocity software. The movies were then converted to 8-bit and processed in Photoshop to enhance contrast.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Two distinct sequences were chosen, each of which differs from the corresponding DNA sequence of murine ZO-1 by at least two nucleotides. Both shRNAs efficiently knocked down expression of the endogenous ZO-1 in MDCK cells, without altering the expression of other proteins, including E-cadherin and atypical protein kinase C (PKC)
(Figure 1A). The ZO-1 level was reproducibly reduced by at least 90%. Gene silencing was most efficient with a mix of the two pSUPER constructs and so was used for most of the experiments described below. Loss of ZO-1 was apparent within 24 h, and persisted for almost a week (Figure 1B).
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), the silencing of ZO-1 expression did not cause any apparent defect in the tight junctions of polarized MDCK cells, as determined by staining for occludin (Figure 1D). Confocal z-stacks showed that the occludin remained apical and was indistinguishable in location between cells depleted of ZO-1 and control cells (Supplemental Figure S1).
Loss of ZO-1 Causes a Delay in Junction Assembly after Calcium Switch or Replating
To determine whether ZO-1 is required for normal junction assembly, cells were cotransfected with pSUPER or with the pSUPER-ZO-1 constructs plus the YFP vector, and then after 2 d were switched to a medium lacking calcium. Under these conditions, the E-cadherin interactions are lost, and the cells withdraw from one another (Matter and Balda, 2003). The next day, the cells were switched back to medium containing normal medium and then were fixed at intervals and stained for either occludin or claudin-1 (Figure 2A).
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In the control cells, occludin and claudin are initially concentrated in spots (presumably vesicles) within the cell bodies (Ivanov et al., 2004b), but they localize to the boundaries and form unbroken lines within 120 min after calcium replacement (Figure 2A). In cells lacking ZO-1, however, there is a pronounced delay in the accumulation of these proteins at boundaries, and they accumulate as broken lines that do not fuse into a continuous border around each cell. A similar phenotype was reported by Umeda et al. (2004) for Eph-4 cells from which the ZO-1 gene has been deleted. The quantitation of occludin at the cell boundaries showed, however, that once the junctions have started to form, the rate of assembly seems to be similar to that of control cells (Figure 2B).
To determine the functional consequences of the delay in junction assembly caused by loss of ZO-1, we measured the transepithelial resistance of MDCK cell monolayers grown on filters. Typically, our MDCK II cells exhibit a rapid increase in resistance, which peaks at 6-8 h. The resistance then gradually falls until it reaches a stable value of 100 ohms (Figure 2C). The molecular basis for this behavior is unknown. Interestingly, however, the silencing of ZO-1 expression abolishes the initial peak in transepithelial resistance, although by 6 h postcalcium switch the tight junctions seem to be morphologically almost normal as judged by staining for occludin (Figure 2C). By 24 h the resistance is the same irrespective of ZO-1 expression.
It seemed possible that the defect in cells lacking ZO-1 might be specific to the effects of calcium depletion, rather than to a fundamental change in the kinetics of junction assembly. Therefore, we trypsinized cells and replated them at high density onto collagen-coated slides. As shown in Figure 2D, within 6 h of replating, the normal cells had established junctions as assessed by occludin staining, whereas the cells lacking ZO-1 had not. Thus, the defect caused by silencing of ZO-1 expression is independent of the technique used to disrupt and permit junction assembly.
Junction Assembly Can Be Rescued by Expression of Murine ZO-1
To confirm that the delay in tight junction assembly is caused by loss of ZO-1 rather than by off-target effects of the shRNAs, we coexpressed murine ZO-1 as a YFP fusion protein together with the pSUPER-ZO-1 constructs. We used the - isoform of ZO-1 that lacks a 240-base pairs insert at position 2713 (see diagram, Figure 6A) (Willott et al., 1992; Balda and Anderson, 1993). This YFP-ZO-1 was targeted correctly to cell boundaries and efficiently rescued the formation of tight junctions as assessed by occludin staining (Figure 3A). Additionally, the presence of murine YFP-ZO-1 restored the normal time course for junction assembly (Figure 3B). When expressed in normal MDCK cells, the level of YFP-ZO-1 was comparable with that of the endogenous protein (Figure 3C). Note that the transfection efficiency was 75%, for visible expression of the fluorescent fusion protein. Importantly, the rescue of junction assembly was not caused by reexpression of the endogenous ZO-1 in cells transfected with the pK-YFP-ZO-1 vector (Figure 3C).
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). We think, therefore, that it is a suitable marker to track junction assembly. Cells were cotransfected with the control pSUPER or ZO-1 pSUPERs together with the YFP-occludin vector. After 2 d, calcium was withdrawn. Sixteen hours later, the cells were mounted on a microscope and imaged at a rate of 1 frame/min.
As shown in Figure 4 and Supplemental Video 1, the YFP-occludin in most cells is initially organized into a ring, within which are multiple spots. This ring coincides with actin and ZO-1, in fixed cells stained with phalloidin or anti-ZO-1 antibodies (our unpublished data). The structure is very similar to the apical ring observed in intestinal epithelial cells that express high levels of active LKB1 (Baas et al., 2004). Similar rings are also observed during the disassembly of junctions after calcium withdrawal from T84 epithelial cells (Ivanov et al., 2004a). It likely defines an apical surface that survives even when the cells do not contact one another. Interestingly, upon addition of calcium, the cells quickly begin to extend lamellipodia-like extensions. When these touch neighboring cells, the YFP-occludin ring disintegrates. The spots are no longer corralled and can spread throughout the cytoplasm. Also, shortly after the loss of the apical ring, broken lines of YFP-occludin occur between adjacent cells, which spread and consolidate into continuous junctional lines. (A diagram of these events is shown in Figure 8.) We did not observe the bright spots fusing with the junctions, but over the longer term, we assume that this material is incorporated into the junctions, because in cells with mature junctions such spots are no longer visible.
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To confirm that this behavior was not peculiar to YFP-occludin, we also made movies of YFP-ZO-1 in MDCK cells after a calcium switch. As shown in Supplemental Figure S2, the ZO-1 is organized into apical rings at early times, with several bright spots corralled within the rings. Within 8-20 min after calcium addition, the rings disintegrate and begin to reform at the cell periphery, where contact has been made with neighboring cells. The ZO-1 patches at the periphery spread and fuse to form a continuous band around each cell.
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Gene Silencing of Occludin, Cingulin, or ZO-2 Has No Effect on Tight Junction Assembly
We were concerned that the phenotype induced by ZO-1 knockdown might be a nonspecific or general effect induced by the loss of any junction protein. Therefore, we tested the response to silencing of occludin expression. Occludin has been shown to be dispensable for tight junction formation in mice, although it is essential for gastric epithelial differentiation (Saitou et al., 1998, 2000; Schulzke et al., 2005). Silencing by RNAi in MDCK cells has been shown previously to have little effect on tight junction integrity (Yu et al., 2005).
We designed pSUPER vectors targeted against two distinct sequences within the canine occludin ORF. Each vector knocked down the level of the endogenous protein by >90%, without effecting the levels of ZO-1, E-cadherin, or 
-catenin (our unpublished data). Immunofluorescence showed a clear reduction of occludin staining at the cell boundaries, but there was no apparent defect in ZO-1 localization, either in normal medium or 60 min after a calcium switch (our unpublished data). This result confirms previous data (Yu et al., 2005) that occludin expression is not required for the normal assembly of tight junctions in MDCK cells. Moreover, when the transepithelial resistance was measured after a calcium switch, the control and occludin-knockdown cells generated a similar profile, confirming that tight junctions lacking occludin are functionally intact, as has been concluded from studies in knockout mice (Saitou et al., 2000; Schulzke et al., 2005). Together, these data support the conclusion that the phenotype of cells lacking ZO-1 reflects a specific requirement for ZO-1 in junction assembly. Additionally, they suggest that the reported interaction of ZO-1 with occludin is not important for ZO-1 localization or for junction assembly.
We next tested the effects of silencing expression of two other known ZO-1 binding partners, ZO-2 and cingulin. Efficient silencing of ZO-2 expression had no discernible phenotype and did not potentiate the inhibition of junction assembly caused by knockdown of ZO-1 (Figure 5, A and C). Cingulin does not localize correctly in murine epithelial cells that lack ZO-1 (Umeda et al., 2004), so it seemed reasonable that this protein might play a role in junction assembly. However, disruption of the cingulin gene in murine embryoid bodies did not cause a loss of tight junctions (Guillemot et al., 2004), and, consistent with these data, knockdown of cingulin in MDCK cells did not alter ZO-1 localization nor did it inhibit junction reassembly after a calcium switch (Figure 5, B and C). Knockout of the cingulin gene did cause changes in the levels of gene expression for several junction proteins (Guillemot et al., 2004). The occludin protein in particular was dramatically increased in cingulin -/- cells. We therefore blotted for a number of junction proteins in cells depleted of cingulin, occludin, ZO-1, or ZO-2 (Figure 5D) but found no significant changes in level, compared with the control MDCK cells. Moreover, we observed no effects of knocking down any of the proteins on the paracellular diffusion of [3H]inulin (Figure 5E). We conclude that wild-type levels of cingulin, occludin, or ZO-2 are not required for the normal formation of tight junctions in MDCK cell monolayers.
Rescue of Tight Junction Assembly by Mutants and Fragments of ZO-1
ZO-1 is a multidomain protein, but the requirement for individual domains in junction assembly is not known. To address this issue, we used gene rescue with various mutants of the murine ZO-1. Initially, we tested several deletion mutants (Figure 6, A and B). We removed the C-terminal region of ZO-1 that contains the ZU-5 domain, and, as shown in Figure 7B, when YFP-ZO-1(1-1523) was cotransfected into cells with the pSUPER-ZO-1 constructs, it was able to rescue tight junction reassembly after calcium switch to an extent similar to that of the full-length protein. Surprisingly, however, it did not localize efficiently to the cell boundaries. This result suggests that localization might not be essential for the function of ZO-1 in forming tight junctions. In contrast, the isolated C-terminal region, HA3-ZO-1(893-1666), which contains both the actin binding domain and the ZU5 domain, did not rescue junction assembly (Figure 6, A and C). The fragments were expressed at similar levels to the full-length ZO-1 (Figure 6D).
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90% and full-length YFP-ZO-1 restores the intensity to the control value. Transfection of the C terminus raises the value only to 30% of control, whereas the N-terminal fragments can rescue junction formation by 80-100% of the control value.
To determine the role of distinct domains in junction assembly, we constructed several further mutants. A 
GUK mutant, in which most of the GUK domain has been deleted, was able to localize quite well to cell boundaries, but it did show a partial assembly defect (Figure 6, D and F). A point mutant in the N-terminal PDZ domain (PDZ1) was constructed that replaces the positively charged residue in the ligand binding site (Arg) with a negatively charged residue (Glu). The Arg normally forms a salt-bridge to the C-terminal carboxyl group on peptides that bind to PDZ domains (Doyle et al., 1996). A Glu is therefore expected to substantially reduce the affinity of the PDZ1 domain for its binding partners. This mutant was recruited to the cell boundaries even more efficiently than the wild-type YFP-ZO-1 and was able to rescue junction formation (Figure 6, D-F), indicating that the PDZ1 domain is not essential for ZO-1 function.
We next mutated the SH3 domain, converting the conserved Trp residue to Gly and the adjacent Leu to Ala. These residues are within the binding pocket of SH3 domains and are essential for interaction with PxxP ligands (Pawson and Gish, 1992). This mutant form of YFP-ZO-1 was only poorly localized to cell boundaries, and it did not efficiently rescue tight junction assembly, giving a mean occludin intensity value 55% compared with control cells (Figure 6, D-F; p = 0.01). Importantly, each of the mutants tested was of the predicted size and showed no evidence of proteolytic degradation (Figure 6C). However, the requirement for a functional SH3 domain was unexpected, because no known binding partners for this domain have been implicated in junction assembly. We were therefore concerned that we had introduced a spurious mutation elsewhere in the ZO-1 open reading frame. To test this possibility, we replaced the mutant SH3 domain with the wild-type version and retested the YFP-ZO-1. This revertant was capable of rescuing junction assembly with 90% efficiency (Figures 6E and 7A). Therefore, we are confident that the observed requirement for an intact SH3 domain is correct.
Finally, we wondered whether the isolated SH3 domain of ZO-1 might be sufficient to rescue junction assembly in cells depleted of endogenous ZO-1. To this end, we expressed a fusion of the SH3 domain and YFP. Surprisingly, junction assembly seemed to be relatively normal in knockdown cells that expressed this fusion protein (Figure 7B), even though this construct was diffuse within the cytoplasm (our unpublished data). The mutated SH3 domain, however, did not efficiently rescue junction assembly. In addition, as a control for specificity, we tested an SH3 domain from the p120 RasGAP. This domain did not rescue junction assembly (Figure 7B). Together, these data suggest that the SH3 domain of ZO-1 can regulate the formation of tight junctions even when it does not localize correctly to the junctions, perhaps by sequestering an assembly inhibitor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), and it is a target for the adenovirus type 9 E4 oncogenic protein (Glaunsinger et al., 2001); and ZO-1 expression is also lost in some breast tumors (Hoover et al., 1998). Nonetheless, the changes in expression level might be an effect of neoplastic transformation rather than a cause, and overexpressed proteins can mistarget and sequester binding partners away from their normal location in the cell, leading to defects unrelated to their normal function. Suppression of expression by RNAi can potentially avoid these concerns, and, when coupled with gene rescue and a quantitative assay, it can provide valuable information on structural requirements for function. Off-target effects and incomplete knockdown can complicate the interpretation of RNAi data (Jackson et al., 2003). In ZO-1, we have eliminated the concern about off-target effects by showing that expression of a human YFP-ZO-1 can reverse the phenotype caused by depletion of the endogenous protein. Moreover, ZO-1 RNAi phenocopies a homozygous disruption of the ZO-1 gene in Eph4 cells, suggesting that the knockdown is effectively eliminating ZO-1 function (Umeda et al., 2004).
The defect caused by loss of ZO-1 is kinetic—normal tight junctions do form in the absence of ZO-1, but the initial steps in assembly are unusually slow. Once initialization is complete, however, assembly proceeds at a similar rate to that seen in control cells, although the transepithelial resistance never peaks in ZO-1-depleted cells. Presumably, therefore, the event that triggers this overshoot in resistance occurs during the first 3 h. By time-lapse imaging, tight junction components were observed to be partially retained in an apical ring and also to be present in bright spots, presumably vesicles, which were corralled within this ring structure. The ring is reminiscent of a similar structure observed in isolated intestinal epithelial cells that ectopically express active LKB1. These cells possess an intrinsic apical/basal polarity, even though they are not in contact with neighboring cells (Baas et al., 2004). The same polarity seems to exist in the MDCK cells subjected to calcium withdrawal. This interpretation is supported by recent evidence that the apical marker gp135/podocalyxin is also confined to a "pre-apical" domain in MDCK cells plated onto coverslips (Meder et al., 2005). The apical ring rapidly disintegrates upon cell-cell contact and reassembles at these contacts, as some of the fragments form "primordial spot junctions," which spread into a tight junction belt that encircles each cell (Figure 8) (Ando-Akatsuka et al., 1999).
Interestingly, in the cells lacking ZO-1, the lamellipodia-like extensions induced by calcium addition were observed to be smaller than in control cells, and the apical ring remained intact and spread slowly to the cell periphery (Figure 8). We speculate that this behavior indicates a possible defect in actin dynamics. However, the mechanisms by which ZO-1 might regulate actin dynamics remain unclear. YFP-ZO-1 is also present in the apical ring and undergoes the same morphological transitions as were observed for YFP-occludin, so it is possible that the ZO-1 directly modulates actin in this ring or acts indirectly to do so through one of its binding partners. However, depletion of known partners for ZO-1, such as occludin, cingulin, and ZO-2, had no detectable effects on tight junction assembly.
The key regions of ZO-1 involved in junction assembly were, unexpectedly, the SH3 and (to a marginal extent) the GUK domain. These two domains are thought to interact and may regulate one another (Wu et al., 2000). What is their function? The GUK domain has been reported to bind occludin (Fanning et al., 1998; Gonzalez-Mariscal et al., 2003), but occludin is not required for junction assembly. The SH3 region of ZO-1 can bind G protein subunits, but these actually interact with an acidic region C-terminal to the domain, and the interaction is unlikely to be perturbed by the point mutations we introduced into the ligand binding site (Gonzalez-Mariscal et al., 2003). Finally, the SH3 domain can bind ZONAB, but this transcription factor seems to regulate cell proliferation rather than junction assembly (Balda et al., 2003). Overexpression of ZONAB did not alter the dynamics of junction assembly in our hands (our unpublished data). Therefore, we speculate that other binding partners exist, and the key to understanding ZO-1 function will be to determine their identity. The observation that the isolated SH3 domain of ZO-1 can rescue assembly even though it does not localize correctly suggests that the binding partner for the SH3 domain might not be a junction component but can interfere with assembly unless it is sequestered by ZO-1.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Abbreviations used: GUK, guanylate kinase-like; PDZ, PSD-95, Discs large, ZO-1; YFP, yellow fluorescent protein; ZO-1, zonula occludens-1.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Ian G. Macara (igm9c{at}virginia.edu).
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