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Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan

	Abstract

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The formation of tight junctions (TJs) is dependent on the formation of adherens junctions (AJs) in MDCK cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin, occludin and junctional adhesion molecule (JAM) are major CAMs at TJs. When MDCK cells precultured at 2 µM Ca²⁺ are cultured at 2 mM Ca²⁺, nectin first forms cell-cell adhesion and recruits E-cadherin to the nectin-based cell-cell adhesion sites to form AJs. Thereafter, nectin recruits first JAM-A and then claudin-1 and occludin to the apical side of AJs to form TJs. In contrast, when MDCK cells precultured at 2 µM Ca²⁺ are cultured at 2 µM Ca²⁺ in the presence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a TJ-like structure is formed without the formation of the E-cadherin-based AJs. We showed here that GFP-E-cadherin, which did not trans-interact due to 2 µM Ca²⁺ but associated with - and ß-catenins and p120^ctn, was recruited to the nectin-based cell-cell adhesion sites by the action of TPA. The nectin inhibitors, which inhibited the trans-interaction of nectin, inhibited the recruitment of GFP-E-cadherin and their associating catenins by the action of TPA. Microbeads coated with the extracellular fragment of nectin recruited not only cellular nectin but also GFP-E-cadherin and their associating catenins by the action of TPA. These results indicate that when the TJ-like structure is formed by the action of TPA, non-trans-interacting E-cadherin and its associating catenins are recruited to the nectin-based cell-cell adhesion sites and that the trans-interaction of E-cadherin is not essential for the formation of TJs.

	Introduction

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In polarized epithelial cells, cell-cell adhesion is mediated through a junctional complex comprised of tight junctions (TJs), cell-cell adherens junctions (AJs) and desmosomes (Farquhar & Palade 1963). These junctional structures are typically aligned from the apical to the basal side, although desmosomes are independently distributed in other areas (Farquhar & Palade 1963). TJs are likely to serve as a barrier that prevents solutes and water from passing through the paracellular pathway and as a fence between the apical and basolateral plasma membranes in epithelial cells (Tsukita et al. 1999, 2001). AJs are closely apposed plasma membrane domains reinforced by a dense cytoplasmic plaque where actin filament (F-actin) bundles are undercoated (Farquhar & Palade 1963). Desmosomes connect intermediate filaments from cell to cell. The formation and maintenance of TJs and desmosomes are generally dependent on the formation and maintenance of AJs (Yap et al. 1997).

At TJs, claudin is a key Ca²⁺-independent cell-cell adhesion molecule (CAM), which constitutes a family of over 27 members (Tsukita et al. 1999, 2001). Occludin is another CAM at TJs, but its function has not been established. Claudin and occludin are associated with the actin cytoskeleton through peripheral membrane proteins, such as ZO-1, -2 and -3. Junctional adhesion molecule (JAM) that belongs to the Ca²⁺-independent immunoglobulin (Ig)-like CAM also localizes at TJs and interacts with ZO proteins. JAM comprises a family consisting of four members (Ebnet et al. 2004). At AJs, E-cadherin, a member of the cadherin superfamily consisting of over 80 members, is a key Ca²⁺-dependent CAM (Takeichi 1995; Gumbiner 1996; Perez-Moreno et al. 2003). E-Cadherin is associated with the actin cytoskeleton through peripheral membrane proteins, including - and ß-catenins, vinculin and -actinin (Gumbiner 2000; Nagafuchi 2001; Perez-Moreno et al. 2003). This association strengthens the cell-cell adhesion of AJs.

Nectin, which constitutes a family of four members, has recently emerged as a Ca²⁺-independent Ig-like CAM at AJs (Takai & Nakanishi 2003; Takai et al. 2003). All nectin is associated with the actin cytoskeleton through afadin, an F-actin- and nectin-binding protein. Each nectin first forms homo-cis-dimers and then homo- or hetero-trans-dimers through the extracellular region in a Ca²⁺-independent manner, causing cell-cell adhesion. Nectin then recruits non-trans-interacting cadherin to the nectin-based cell-cell adhesion sites, where non-trans-interacting cadherin trans-interacts to form AJs. In addition, each nectin induces activation of Cdc42 and Rac small G proteins. Nectin first forms cell-cell adhesion and recruits and activates c-Src at the nectin-based cell-cell adhesion sites. c-Src then tyrosine-phosphorylates Cdc42-GEF FRG (Fukuhara et al. 2004). In addition, c-Src induces activation of Rap1 through the Crk-C3G complex (Fukuyama et al. 2005). Rap1 then induces the activation of tyrosine-phosphorylated FRG locally at the nectin-based cell-cell adhesion sites, eventually causing the activation of Cdc42. Moreover, c-Src tyrosine-phosphorylates a Rac-GEF, Vav2 (Kawakatsu et al. 2005). Cdc42 then activates tyrosine-phosphorylated Vav2 locally at the nectin-based cell-cell adhesion sites. Cdc42 then increases the number of filopodia and cell-cell contact sites. Rac induces the formation of lamellipodia, which efficiently expands the cell-cell adhesion between filopodia, acting like a ‘zipper.’ In these ways, these small G proteins enhance the formation of AJs.

In addition, after or during the formation of AJs, nectin recruits first JAM-A and then claudin and occludin to the apical side of AJs in cooperation with E-cadherin, resulting in the formation of TJs (Takai & Nakanishi 2003; Takai et al. 2003). JAM-A binds the cell-polarity protein complex, consisting of Par-3, atypical PKC and Par-6, by directly binding Par-3 and recruits them to TJs (Ohno 2001). This complex is essential for the formation of TJs. It remains, however, unknown how nectin recruits the TJ components to the apical side of AJs, but it may be noted that nectin-1 and -3, but not nectin-2, directly bind Par-3. Cdc42 activated by the action of trans-interacting nectin is likely to bind to Par-6 and to activate the polarity protein complex (Takai et al. 2003).

It has been shown that when MDCK cells precultured at 2 µM Ca²⁺ are cultured at 2 µM Ca²⁺ in the presence of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a TJ-like structure is formed, and that nectin, afadin, ZO-1, but not E-cadherin, accumulate there (Balda et al. 1993; Asakura et al. 1999; Fukuhara et al. 2002b). These results implicate that TPA induces the TJ formation in the absence of the E-cadherin-based AJs. Here we re-examined whether a TJ-like structure was formed in the absence of the E-cadherin assembly at the nectin-based cell-cell adhesion sites. We found that E-cadherin, which did not trans-interact due to 2 µM Ca²⁺ but associated with - and ß-catenins and p120^ctn, was recruited to the nectin-based cell-cell adhesion sites when a TJ-like structure was formed at 2 µM Ca²⁺ by the action of TPA.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Concentration of GFP-E-cadherin associated with - and ß-catenins and p120^ctn at the nectin-based cell-cell contact sites by the action of TPA

When MDCK cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at 2 mM Ca²⁺, the immunofluorescence signals for nectin-1, afadin, E-cadherin, - and ß-catenins and p120^ctn are all concentrated at the cell-cell contact sites (Takahashi et al. 1999; Fukuhara et al. 2002a, 2002b; Honda et al. 2003a, 2003b). The signals for JAM-A, claudin-1, occludin and ZO-1 are also concentrated at the cell-cell contact sites. The sites of the signals for nectin-1 and E-cadherin correspond to AJs, while the sites of the signals for JAM-A, claudin-1 and occludin correspond to TJs. When nectin-1-MDCK cells are cultured at 2 µM Ca²⁺, the signals for E-cadherin, - and ß-catenins, p120^ctn, JAM-A, claudin-1 and occludin disappear from the cell-cell contact sites, although those for nectin-1, afadin and ZO-1 remain there as described (Fukuhara et al. 2002a, 2002b; Honda et al. 2003a, 2003b). When these cells are re-cultured at 2 mM Ca²⁺, the signals for E-cadherin, - and ß-catenins, p120^ctn, JAM-A, claudin-1 and occludin are re-concentrated at the cell–cell contact sites where nectin-1 is concentrated, resulting in the formation of AJs and TJs. We first confirmed these earlier observations by staining of E-cadherin, - and ß-catenins, p120^ctn, JAM-A, claudin-1 and occludin (Fig. 1, Normal Ca²⁺). When nectin-1-MDCK cells precultured at 2 µM Ca²⁺ were cultured at 2 µM Ca²⁺ in the presence of TPA, the signals for JAM-A, claudin-1 and occludin, but not that for E-cadherin, were apparently re-concentrated at the nectin-1-based cell-cell contact sites as described (Asakura et al. 1999; Fukuhara et al. 2002b) (Fig. 1, Low Ca²⁺+TPA). However, the signals for - and ß-catenins and p120^ctn were re-concentrated there. The essentially same results were obtained for wild-type MDCK cells (Fig. 2). In this experiment, endogenous nectin-3 and afadin were stained instead of nectin-1 because endogenous nectin-1 was only faintly stained in wild-type MDCK cells as previously described (Fukuhara et al. 2002a, 2002b; Honda et al. 2003a, 2003b). These results suggest that E-cadherin associated with - and ß-catenins and p120^ctn is re-concentrated at the nectin-based cell-cell adhesion sites at 2 µM Ca²⁺ in the presence of TPA, but the signal for E-cadherin is not detected in an immunofluorescence microscopy. Alternatively, - and ß-catenins and p120^ctn, which are not associated with E-cadherin, are re-concentrated there.

View larger version (85K): [in this window] [in a new window]   Figure 1  Concentration of - and ß-catenins, p120ctn and the TJ components, but not E-cadherin, at the nectin-based cell-cell adhesion sites by the action of TPA in nectin-1-MDCK cells. Nectin-1-MDCK cells were precultured at 2 µM Ca2+ for 3 h and then cultured at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA for 1 h. The cells were triple stained with various combinations of the anti-nectin-1, anti-FLAG, anti-E-cadherin (ECCD-2), anti-claudin-1, anti-occludin, anti-JAM-A, anti-ZO-1, anti-afadin, anti--catenin, anti-ß-catenin or anti-p120ctn Abs. Normal Ca2+, the cells precultured at 2 µM Ca2+ were cultured at 2 mM Ca2+; Low Ca2++TPA, the cells precultured at 2 µM Ca2+ were cultured at 2 µM Ca2+ in the presence of TPA; and Bars, 10 µM. The results are representative of three independent experiments.

View larger version (107K): [in this window] [in a new window]   Figure 2  Concentration of - and ß-catenins, p120ctn and the TJ components, but not E-cadherin, at the nectin-based cell-cell adhesion sites by the action of TPA in wild-type MDCK cells. Wild-type MDCK cells were precultured at 2 µM Ca2+ for 3 h and then incubated at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA for 1 h. The cells were triple stained with various combinations of the anti-nectin-3, anti-E-cadherin (ECCD-2), anti-claudin-1, anti-occludin, anti-JAM-A, anti-ZO-1, anti-afadin, anti--catenin, anti-ß-catenin or anti-p120ctn Abs. Normal Ca2+, the cells precultured at 2 µM Ca2+ were cultured at 2 mM Ca2+; Low Ca2++TPA, the cells precultured at 2 µM Ca2+ were cultured at 2 µM Ca2+ in the presence of TPA; and Bars, 10 µM. The results are representative of three independent experiments.

View larger version (58K): [in this window] [in a new window]   Figure 3  Detection of E-cadherin by other anti-cadherin Abs in the cells cultured at 2 µM Ca2+ in the presence of TPA. Wild-type MDCK cells were precultured at 2 µM Ca2+ for 3 h and then incubated at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA for 1 h. The cells were triple stained with various combinations of the anti-afadin mAb, the anti-afadin pAb, the anti-E-cadherin mAb (ECCD-2), the anti-E-cadherin mAb (610182), the anti-pan-cadherin pAb and the anti-uvomorulin mAb (DECMA-1). Normal Ca2+, the cells precultured at 2 µM Ca2+ were cultured at 2 mM Ca2+; Low Ca2++TPA, the cells precultured at 2 µM Ca2+ were cultured at 2 µM Ca2+ in the presence of TPA; and Bars, 10 µM. The results are representative of three independent experiments.

View larger version (65K): [in this window] [in a new window]   Figure 4  Concentration of GFP-E-cadherin associated with - and ß-catenins and p120ctn at the nectin-based cell-cell adhesion sites by the action of TPA. GFP-E-cadherin-MDCK cells were precultured at 2 µM Ca2+ for 3 h and then incubated at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA for 1 h. The cells were triple stained with various combinations of the anti-afadin, anti--catenin, anti-ß-catenin or anti-p120ctn Abs. Normal Ca2+, the cells precultured at 2 µM Ca2+ were cultured at 2 mM Ca2+; Low Ca2++TPA, the cells precultured at 2 µM Ca2+ were cultured at 2 µM Ca2+ in the presence of TPA; and Bars, 10 µM. The results are representative of three independent experiments.

Recruitment of E-cadherin and their associating catenins to the Nef-3-coated bead-cell contact sites at 2 µM Ca²⁺ by the action of TPA

We have previously shown that when microbeads coated with the extracellular region of nectin-3 fused to the Fc portion of IgG (Nef-3) are put on the surface of nectin-1-MDCK cells, the immunofluorescence signals for cellular nectin-1, afadin, E-cadherin and - and ß-catenins are concentrated at the contact sites between the Nef-3 beads and the cells at 2 mM Ca²⁺ (Honda et al. 2003b). Similarly, the signals for cellular nectin-1, afadin, GFP-E-cadherin, - and ß-catenins and p120^ctn were concentrated at the contact sites between the Nef-3 beads and the nectin-1-MDCK cells expressing GFP-E-cadherin at 2 µM Ca²⁺ by the action of TPA (Fig. 5, Nef-3). The signal for cellular nectin-1, afadin, GFP-E-cadherin, - and ß-catenins or p120^ctn was not concentrated at the contact sites between the concanavalin A (ConA)-coated beads and the cells at 2 µM Ca²⁺ by the action of TPA (Fig. 5, ConA). These results have provided another line of evidence that non-trans-interacting GFP-E-cadherin is recruited to the nectin-based cell-cell adhesion sites at 2 µM Ca²⁺ by the action of TPA.

View larger version (47K): [in this window] [in a new window]   Figure 5  Recruitment of E-cadherin and their associating catenins to the Nef-3-coated bead-cell contact sites at 2 µM Ca2+ by the action of TPA. The Nef-3- or ConA-coated beads were added to nectin-1-MDCK cells expressing GFP-E-cadherin, followed by incubation at 2 µM Ca2+ for 3 h. The cells were further incubated at 2 µM Ca2+ in the presence of 100 nM TPA for 1 h. The cells were triple stained with various combinations of the anti-nectin-1, anti-FLAG, anti-afadin, anti--catenin, anti-ß-catenin, or anti-p120ctn Abs. DIC, a differential interference contrast image; and Bars, 10 µM. The results are representative of three independent experiments.

Association of E-cadherin with - and ß-catenins and p120^ctn in the cells cultured at 2 µM Ca²⁺ in the presence of TPA

We next examined the amount of GFP-E-cadherin and endogenous E-cadherin on the plasma membrane in the GFP-E-cadherin-MDCK cells cultured at 2 µM Ca²⁺ in the presence of TPA. The extracellular regions of GFP-E-cadherin and endogenous E-cadherin of GFP-E-cadherin-MDCK cells were labeled with sulfo-NHS-SS-biotin. After free sulfo-NHS-SS-biotin was removed by extensive washing, the detergent-soluble, surface-biotinylated proteins on the plasma membrane were recovered on streptavidin beads and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting with the anti-E-cadherin mAb (610182). GFP-E-cadherin and endogenous E-cadherin remained on the plasma membrane in the GFP-E-cadherin-MDCK cells cultured at 2 µM Ca²⁺ in the presence of TPA, although the amounts were somewhat reduced than those in the cells cultured at 2 mM Ca²⁺ (Fig. 6A).

View larger version (46K): [in this window] [in a new window]   Figure 6  Association of E-cadherin with - and ß-catenins and p120ctn in the cells cultured at 2 µM Ca2+ in the presence of TPA. (A) The amount of GFP-E-cadherin and endogenous E-cadherin on the plasma membrane in GFP-E-cadherin-MDCK cells cultured at 2 µM Ca2+ in the presence of TPA. GFP-E-cadherin-MDCK cells were surface-biotinylated at 4 °C. Detergent-soluble, surface-biotinylated proteins on the plasma membrane were recovered on streptavidin beads and analyzed by SDS-PAGE, followed by Western blotting with the anti-GFP pAb and the anti-E-cadherin mAb (610182). Surface, surface-biotinylated protein; Total, total cell extracts; N, 2 mM Ca2+; and LT, 2 µM Ca2+ in the presence of TPA. (B) Association of E-cadherin with - and ß-catenins and p120ctn in the cells cultured at 2 µM Ca2+ in the presence of TPA. The extracts of GFP-E-cadherin MDCK cells cultured at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA were immunoprecipitated with the anti-GFP pAb and analyzed by Western blotting with the anti-GFP, anti--catenin, anti-ß-catenin and anti-p120ctn Abs. Total, total cell extracts; IP, immunoprecipitates with the anti-GFP pAb; N, 2 mM Ca2+; and LT, 2 µM Ca2+ in the presence of TPA. (C) Cis-dimerization of E-cadherin in the GFP-E-cadherin-MDCK cells cultured at 2 µM Ca2+ in the presence of TPA. GFP-E-cadherin-MDCK cells cultured at 2 mM Ca2+ or 2 µM Ca2+ in the presence of 100 nM TPA were treated with BS3. Total cell extracts were subjected to SDS-PAGE, followed by Western blotting with the anti-E-cadherin mAb (610182). N, 2 mM Ca2+; LT, 2 µM Ca2+ in the presence of TPA; +, in the presence of 5 mM BS3; –, in the absence of 5 mM BS3; open arrowheads, dimers; and closed arrowheads, monomers. The results are representative of three independent experiments.

E-Cadherin forms first cis-dimers and then trans-dimers, causing cell-cell adhesion (Koch et al. 1999). The formation of cis-dimers and trans-dimers is dependent on 2 mM Ca²⁺, suggesting that non-trans-interacting E-cadherin recruited to the nectin-based cell-cell contact sites by the action of TPA does not form cis-dimers. To confirm this issue, GFP-E-cadherin-MDCK cells cultured at 2 mM Ca²⁺ or at 2 µM Ca²⁺ in the presence of TPA were cross-linked by BS³, followed by SDS-PAGE and Western blotting with the anti-E-cadherin mAb (610182). E-Cadherin formed cis-dimers in both the cells cultured at 2 mM Ca²⁺ and the cells cultured at 2 µM Ca²⁺ in the presence of TPA, whereas the amounts of the cis-dimers were somewhat reduced in the cells cultured at 2 µM Ca²⁺ in the presence of TPA (Fig. 6C). This is inconsistent with the previous observation that E-cadherin does not form cis-dimers at the 2 µM Ca²⁺ in EL cells (Takeda 2004). E-Cadherin formed cis-dimers in the cells cultured at 2 µM Ca²⁺ in the absence of TPA (data not shown), excluding the possibility that TPA induce the cis-dimer formation of E-cadherin. Thus, it may be caused by a difference of cell lines. These results indicate that E-cadherin, which forms cis-dimers, but not trans-dimers, is recruited to the nectin-based cell-cell adhesion sites by the action of TPA.

Necessity of the nectin-based cell-cell adhesion for the TPA-induced concentration of non-trans-interacting E-cadherin at the cell-cell contact sites

We have previously shown that the disruption of the nectin-based cell-cell adhesion by the nectin inhibitors, gD and Nef-3, inhibits the recruitment of E-cadherin and - and ß-catenins to the nectin-based cell-cell adhesion sites and of claudin-1, occludin, JAM-A and ZO-1 to the apical side of the nectin-based cell-cell adhesion sites at 2 mM Ca²⁺ (Fukuhara et al. 2002a, 2002b; Honda et al. 2003b). gD is a fragment of glycoprotein D fused to the Fc portion of IgG. Kinetically, they reduce the velocities of the recruitment of these components (Honda et al. 2003a). Similarly, these inhibitors disrupted the nectin-based cell-cell adhesion and inhibited the TPA-induced concentration of non-trans-interacting GFP-E-cadherin, - and ß-catenins, p120^ctn, claudin-1, occludin, JAM-A and ZO-1 at the cell-cell contact sites (Fig. 7). These results indicate that the nectin-based cell-cell adhesion is necessary for the TPA-induced recruitment of non-trans-interacting E-cadherin associated with - and ß-catenins and p120^ctn to the cell-cell adhesion sites.

View larger version (101K): [in this window] [in a new window]   Figure 7  Inhibition of the TPA-induced concentration of GFP-E-cadherin associated with - and ß-catenins and p120ctn at the nectin-based cell-cell contact sites by the nectin inhibitors. Nectin-1-MDCK cells expressing GFP-E-cadherin were precultured at 2 µM Ca2+ for 3 h. After the culture, 100 nM TPA was added to the medium and the cells were further cultured in the presence or absence of 60 µg/mL gD and 60 µg/mL Nef-3 for 1 h. The cells were triple stained with various combinations of the anti-nectin-1, anti-FLAG, anti-claudin-1, anti-occludin, anti-JAM-A, anti-ZO-1, anti-afadin, anti--catenin, anti-ß-catenin or anti-p120ctn Abs. Control, in the absence of gD and Nef-3; gD +Nef-3, in the presence of gD and Nef-3; and Bars, 10 µM. The results are representative of three independent experiments.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

The disruption of the nectin-based cell-cell adhesion by the nectin inhibitors inhibits the accumulation of E-cadherin, - and ß-catenins and p120^ctn by the action of TPA. In addition, GFP-E-cadherin, - and ß-catenins and p120^ctn also accumulate at the Nef-3-bead-cell contact sites by the action of TPA. Thus, the trans-interaction of nectin is necessary for the accumulation of non-trans-interacting E-cadherin which associates with - and ß-catenins and p120^ctn by the action of TPA. Nectin is necessary for the recruitment of the TJ components such as claudin-1, occludin, JAM-A and ZO-1, to the nectin-based cell-cell contact sites by the action of TPA (Fukuhara et al. 2002b). It remains unknown whether nectin is independently involved in the accumulation of the AJ and TJ components by the action of TPA. It also remains unknown how TPA exerts the assembly of the AJ and TJ components. Since TPA is a potent activator of conventional protein kinase C (cPKC) and novel PKC (nPKC), these PKCs are likely to mediate its action of TPA in the assembly of AJ and TJ components. It has been reported that PKCs are involved in both assembly and disassembly of junctional complexes (Matter & Balda 2003). It should be clarified how PKCs exert their action. It has been reported that the activity of PKC increases during the formation of cell-cell junctions in Ca²⁺ switch experiment of MDCK cells (Stuart & Nigam 1995). It should also be clarified how PKC is activated during the formation of cell-cell junctions under normal conditions.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Antibodies, inhibitors and chemicals

A rabbit anti-nectin-1 pAb was prepared as described (Takahashi et al. 1999). A mouse anti-afadin mAb and a rabbit anti-afadin pAb were prepared as described (Sakisaka et al. 1999). A rat anti-E-cadherin mAb (ECCD-2) was supplied from Dr M. Takeichi (Center for Developmental Biology, RIKEN, Kobe, Japan). A rabbit anti-JAM-A pAb was supplied by Dr T. Kita (Kyoto University, Kyoto, Japan). A rabbit anti-nectin-3 pAb was supplied from Dr K. E. Mostov (University of California, San Francisco, CA, USA). A mouse anti-FLAG M1 mAb, a rat anti-uvomorulin mAb (DECMA-1), a rabbit anti-pan-cadherin pAb, a rabbit anti--catenin pAb, a goat anti-human IgG (Fc specific) Ab and TPA were purchased from Sigma. Mouse anti-p120^ctn and anti-E-cadherin (610182) mAbs were purchased from BD Transduction Laboratories. A mouse anti-ß-catenin mAb was purchased from Santa Cruz. Rabbit anti-claudin-1 and anti-JAM-A pAbs and a mouse anti-occludin mAb were purchased from Zymed. A rat anti-occludin mAb and a mouse anti-ZO-1 mAb were purchased from SANKO JUNYAKU. A rabbit anti-GFP pAb was purchased from MBL. Secondary Abs for immunofluorescence microscopy were purchased from Chemicon. gD (1–285 amino acids; a fragment of glycoprotein D fused to the Fc portion of IgG) and Nef-3 (56–400 amino acids; an extracellular region of nectin-3 fused to the Fc portion of IgG) were prepared as described (Tachibana et al. 2000; Satoh-Horikawa et al. 2000). Cell membrane-impermeable chemical cross-linker bis-(sulfosuccinimidyl) suberate (BS³) (a spacer arm length of 1.14 nM) was purchased from Pierce Chemical Co.

Cell culture and DNA transfection

MDCK cells were kindly supplied from Dr W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany). Nectin-1-MDCK cells (MDCK cells stably expressing FLAG-tagged nectin-1) were prepared as described (Takahashi et al. 1999). GFP-E-cadherin-MDCK cells (MDCK cells stably expressing GFP-E-cadherin) were prepared as described (Hoshino et al. 2004). Nectin-1-MDCK cells were transfected with pMXII-GFP-E-cadherin using LipofectAMINE 2000 Reagent (Invitrogen).

Ca²⁺ switch assay

Ca²⁺ switch experiments using nectin-1-MDCK, wild-type MDCK or GFP-E-cadherin-MDCK cells were done as described (Kartenbeck et al. 1991). Briefly, the cells (1 x 10⁵) were seeded on 18-mm glass coverslips in 12-well culture dishes. Forty-eight h later, the cells were washed with phosphate buffered saline (PBS) and cultured at 2 mM Ca²⁺ in Dulbecco's modified Eagle's medium (DMEM) without serum for 1 h. The cells were then cultured at 2 µM Ca²⁺ (DMEM with 5 mM EGTA) for 3 h. After the culture, the cells were cultured in DMEM at 2 mM Ca²⁺ for 1 h. When the cells were treated with TPA, the cells were washed with PBS and cultured at 2 mM Ca²⁺ in DMEM without serum for 1 h. The cells were then cultured at 2 µM Ca²⁺ (DMEM with 5 mM EGTA) for 3 h. After the culture, 100 nM TPA was added to the medium and the cells were further cultured in the presence or absence of 60 µg/mL gD and 60 µg/mL Nef-3 for 1 h.

Immunofluorescence microscopy

Immunofluorescence microscopy was done as described (Mandai et al. 1997; Takahashi et al. 1999). Briefly, the cells were fixed in the mixture of 50% acetone and 50% methanol at –20 °C for 1 min or in PBS containing 1% formaldehyde for 15 min and PBS containing 0.2% Triton X-100 for 15 min at room temperature. After being blocked in Tris buffered saline (TBS) containing 1% BSA and 1 mM CaCl₂ for 1 h, the cells were incubated in the same buffer with various combinations of Abs for 1 h. The samples were washed three times with TBS containing 1 mM CaCl₂ for 5 min and incubated for 30 min in TBS containing 1% BSA and 1 mM CaCl₂ with the secondary pAbs. The samples were then washed three times with TBS containing 1 mM CaCl₂ for 5 min and mounted in GEL/MOUNT (Biomeda). The samples were analyzed by a Radiance 2100 confocal laser scanning microscope (Bio-Rad Laboratories) and LSM 510 META confocal microscope (Carl Zeiss).

Assay for bead-cell contact

Nectin-1-MDCK cells (5 x 10⁴) were seeded on 14-mm glass coverslips in 24-well culture dishes. After 16 h, the cells were transfected with pMXII-GFP-E-cadherin using the Lipofect AMINE 2000 reagent (Invitrogen) according to the manufacturer's protocol. After 24 h of transfection, the cells were washed with PBS and cultured at 2 mM Ca²⁺ in DMEM without serum for 1 h. The cells were then cultured at 2 µM Ca²⁺ (DMEM with 5 mM EGTA) with latex-sulfate microbeads coated with Nef-3 or ConA for 3 h and followed to stimulate with 100 nM TPA for 1 h. The cells were fixed and immunostained as described (Honda et al. 2003b).

Immunoprecipitation

Co-immunoprecipitation experiments using GFP-E-cadherin-MDCK cells were done as follows: GFP-E-cadherin-MDCK cells (2 x 10⁶) were seeded on 10-cm dishes. Forty-eight h later, the cells were subjected to the Ca²⁺ switch. After the Ca²⁺ switch, the cells were washed twice with TBS-C (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM CaCl₂) or TBS and then suspended in 1 mL of buffer A (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 µg/mL leupeptin, 100 µg/mL PMSF, 10 µg/mL aprotinin, 50 µM ALLN), followed by ultracentrifugation at 100 000 g for 15 min. The cell extract was incubated with 5 µL of the anti-GFP pAb-coated protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences) at 4 °C for 18 h. After the beads were washed with buffer A, the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mM Tris-HCl at pH 6.7, 3% SDS, 2% 2-mercaptoethanol, 5% glycerol) for 10 min. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-GFP, anti--catenin, anti-ß-catenin and anti-p120^ctn Abs.

Cell surface biotinylation

GFP-E-cadherin-MDCK cells grown on 24-mm diameter Transwell^TM filters were subjected to the Ca²⁺ switch. After the Ca²⁺ switch, the cells were incubated with 0.5 mg/mL sulfosuccinimidyl 2-(biotinamido) ethyl-dithioproprionate (sulfo-NHS-SS-biotin) (Pierce Chemical Co.) which was applied to both apical and basal sides of the filter, followed by washing with PBS containing 50 mM NH₄Cl to quench free sulfo-NHS-SS-biotin, followed by several further washes in PBS. The cells were then scraped off the filters and suspended in 150 µL of buffer A. The cell lysates were centrifuged and the supernatant was incubated with streptavidin beads (Sigma) to collect bound, biotinylated protein. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-E-cadherin mAb (610182).

Chemical cross-linking

Chemical cross-linking was done as described (Takeda 2004). GFP-E-cadherin-MDCK cells grown on 24-mm diameter Transwell^TM filters were subjected to the Ca²⁺ switch. After the Ca²⁺ switch, the cells were washed with PBS containing 0.5 mM CaCl₂ (PBS-C) or PBS. 5 mM BS³-PBS-C or 5 mM BS³-PBS were applied to both apical and basal-lateral surfaces and incubated for 1 h at room temperature. The reaction was stopped by washing the cells with 10 mM Tris-HCl (pH 7.4) for 15 min. Cross-linked and control cells were solubilized directly in the SDS sample buffer. The samples were then subjected to SDS-PAGE followed by Western blotting with the anti-E-cadherin mAb (610182).

Cell dissociation assay

The cell dissociation assay was done as described (Nagafuchi et al. 1994). In brief, GFP-E-cadherin-MDCK cells (2 x 10⁵) grown in a 35-mm dish were subjected to the Ca²⁺ switch. After the Ca²⁺ switch, the cells were washed with HEPES-buffered saline (HBS, pH 7.4) and treated with 0.01% trypsin supplemented with 1 mM CaCl₂ in HBS (TC treatment) or 0.01% trypsin supplemented with 1 mM EDTA in HBS (TE treatment) at 37 °C for 3 h, followed by dissociation by 10-time pipetting. The extent of dissociation of cells was represented by the index N_TC/N_TE, where N_TC and N_TE were the total particle number after the TC and TE treatments, respectively.

Other procedures

Protein concentrations were determined with BSA as a reference protein (Bradford 1976). SDS-PAGE was done as described (Laemmli 1970).

	Acknowledgements

 
We thank Dr M. Takeichi (Center for Developmental Biology, RIKEN, Kobe, Japan) for providing us with the anti-E-cadherin mAb, Dr T. Kita (Kyoto University, Kyoto, Japan) for providing us with the anti-JAM-A pAb, Dr K. E. Mostov (University of California, San Francisco, CA) for providing us with the anti-nectin-3 pAb and Dr W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science & Technology, Japan (2003, 2004).

	Footnotes

 
Communicated by: Eisuke Nishida

^* Correspondence: E-mail: ytakai{at}molbio.med.osaka-u.ac.jp

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Received: 24 December 2004
Accepted: 24 January 2005

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