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Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
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-catenin, ß-catenin, vinculin, -actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin-1, occludin and JAM-1, or the PMPs at TJs, ZO-1 and MAGI-1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin-afadin unit with other CAMs and PMPs at AJs and TJs. |
Introduction |
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-catenin, ß-catenin, vinculin, and -actinin (Gumbiner 2000; Nagafuchi 2001; Perez-Moreno et al. 2003). Cadherins directly binds ß-catenin, which in turn binds -catenin. -Catenin then binds to vinculin and -actinin. Of these PMPs, -catenin, vinculin, and -actinin are actin filament (F-actin)-binding proteins (Nagafuchi 2001). At TJs, claudins are key Ca2+-independent CAMs (Tsukita & Furuse 1999; Tsukita et al. 1999, 2001). Occludin is another CAM at TJs, but its function has not been established (Tsukita & Furuse 1999; Tsukita et al. 1999). Junctional adhesion molecules (JAMs), Ca2+-independent immunoglobulin-like CAMs, also localize at TJs (Tsukita et al. 1999, 2001). Claudins, occludin, and JAMs interact with ZO-1/-2/-3 which are F-actin-binding proteins (Tsukita et al. 1999, 2001). In addition, JAMs directly interact with one of the cell polarity proteins, Par-3, which forms a ternary complex with Par-6 and atypical protein kinase C (aPKC), suggesting that JAMs are involved in the formation of cell polarity through these proteins (Ohno 2001). Nectins, which constitute a family of four members, have recently emerged as Ca2+-independent Ig-like CAMs at AJs (Takai & Nakanishi 2003; Takai et al. 2003). Nectins form homo-cis-dimers and then homo- and hetero-trans-dimers through the extracellular region, causing cell-cell adhesion. The cytoplasmic tail of nectins interacts with afadin which is an F-actin-binding protein. Nectins first form cell-cell adhesion where cadherins are recruited, eventually forming AJs in epithelial cells and fibroblasts. In addition, nectins recruit first JAMs and then claudins and occludin to the apical side of AJs, eventually forming TJs in epithelial cells. Furthermore, nectin-1 and -3, but not nectin-2, bind Par-3, suggesting that nectins are involved in the formation of cell polarity.
The association of nectins and cadherins are mediated through their binding PMPs. The detailed molecular mechanisms for this association have not fully been understood, but afadin and -catenin are essential for this association and several proteins which connect afadin and -catenin have been identified (Takai & Nakanishi 2003; Takai et al. 2003). The first putative connector unit for nectins and E-cadherin is a ponsin-vinculin unit (Mandai et al. 1999). Ponsin is an afadin- and vinculin-binding protein and vinculin is an F-actin- and -catenin-binding protein. The second one is an afadin DIL domain-interacting protein (ADIP)--actinin unit (Asada et al. 2003). ADIP is an afadin- and -actinin-binding protein and -actinin is an -catenin-binding protein. The third one is a LIM domain only 7 (LMO7)--actinin unit (Ooshio et al. 2004). LMO7 is also an afadin- and -actinin-binding protein. The molecular mechanisms of association of nectins with the CAMs at TJs remain unknown, either, but both afadin and ZO-1/-2/-3 are likely to be involved in it (Fukuhara et al. 2002a).
On the other hand, nectins induce activation of Cdc42 and Rac small G proteins (Shimizu & Takai 2003; Takai et al. 2003). Nectins first recruit c-Src to the nectin-based cell-cell adhesion sites and activate it, and c-Src phosphorylates FRG, a Cdc42-guanine nucleotide exchange factor (GEF) (Fukuhara et al. 2004). In addition, c-Src induces activation of another small G protein Rap1 through the Crk-C3G complex, and Rap1 then induces activation of FRG, eventually causing activation of Cdc42 (T. Fukuyama, H. Ogita, T. Kawakatsu, T. Fukuhara, T. Yamada, K. Shimizu, T. Nakamura, M. Matsuda & Y. Takai). The activation of c-Src and Cdc42 moreover induces activation of Rac through an unidentified Rac GEF. Rac is also activated by E-cadherin in phosphatidylinositol-3 (PI3) kinase-dependent and -independent manners in epithelial cells (Yap & Kovacs 2003). Cdc42 then increases the number of the actin cytoskeleton-based filopodia and cell-cell contact sites. Rac induces formation of the actin cytoskeleton-based lamellipodia and efficiently zips the cell-cell adhesion between the filopodia like ‘Zipper.’ Thus, nectins increase the velocity of the formation of AJs through the activation of these small G proteins. In addition, Cdc42 activated by nectins may bind to Par-6 and regulate the formation of TJs (Ohno 2001; Takai et al. 2003).
Cortical F-actin underlying AJs in epithelial cells form a bundle around the apical end of the cell (Farquhar & Palade 1963; Hirano et al. 1987). It has previously been believed that the cadherin-catenin unit may organize this actin cytoskeleton structure directly via -catenin or indirectly via vinculin and -actinin. However, there are several reports showing that the depletion or significant reduction of the cadherin-catenin unit does not hinder the formation of the apparently normal AJ structure (Tepass et al. 2000). Therefore, at least in some cases, an additional adhesive mechanism(s) is required to determine the precise size and localization of the AJ structure. The nectin-afadin unit is an excellent candidate because of the following reasons: (1) the nectin-afadin unit is concentrated even more strictly at epithelial AJs than the cadherin-catenin unit; (2) in afadin (-/-) mice, the organization of AJs is impaired in epithelial cells of embryos; and (3), afadin interacts directly with both nectins and F-actin and indirectly with the cadherin-catenin unit (Takai & Nakanishi 2003; Takai et al. 2003). The nectin-based adhesive membrane microdomains show one-to-one linkage with each F-actin bundle at the Sertoli cell-spermatid junctions in the testis (Ozaki-Kuroda et al. 2002). The structure of F-actin bundles at the Sertoli cell-spermatid junctions differs from that at typical AJs; F-actin is bundled tightly in parallel at the Sertoli cell-spermatid junctions and does not seem contractile (Vogl et al. 1991), while they are bundled loosely in anti-parallel at typical AJs and exhibit the contractile property (Hirokawa & Tilney 1982). Nevertheless, their side-to-side association to the plasma membrane and their function to scaffold the contact sites are comparable. The nectin-afadin unit may couple adhesion sites and F-actin bundles at AJs in cooperation with the E-cadherin-catenin unit in epithelial cells.
It has been shown by use of F-actin-disrupting agents, such as cytochalasin D and latrunculin A, that the actin cytoskeleton is necessary for the organization and maintenance of AJs and TJs (Stevenson & Begg 1994; Adams et al. 1998; Quinlan & Hyatt 1999; Wittchen et al. 1999; Ma et al. 2000; Vasioukhin et al. 2000). The connection of E-cadherin to the actin cytoskeleton through PMPs has also been shown to strengthen the cell-cell adhesion of AJs (Nagafuchi 2001). In contrast, we have recently found that the nectin- and E-cadherin-based cell-cell adhesion is formed and maintained in fibroblasts even when the actin cytoskeleton is mostly disrupted by cytochalasin D or latrunculin A (Honda et al. 2003b). We therefore studied here whether the actin cytoskeleton is necessary for the association of the nectin-afadin unit with other CAMs and their binding PMPs at AJs and TJs in wild-type MDCK cells and MDCK cells stably expressing exogenous nectin-1 (nectin-1-MDCK cells).
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We have previously shown that the immunofluorescence signals for nectin-1, E-cadherin, and F-actin are concentrated at the cell-cell contact sites of nectin-1-MDCK cells cultured at 2 mM Ca2+ (Fukuhara et al. 2002a,b; Honda et al. 2003a,c). The sites of the signals for nectin-1 and E-cadherin would correspond to AJs. We first confirmed these earlier observations as controls (Fig. 1A, Control). When the cells were cultured at 2 mM Ca2+ in the presence of latrunculin A, the signal for F-actin was markedly impaired, but that for nectin-1 remained at the cell-cell contact sites (Fig. 1A, Lat A). The signal for E-cadherin mostly disappeared from the contact sites. The residual punctuated signal for E-cadherin at the plasma membranes was mostly overlapped with that for F-actin. When the PMPs, afadin, -catenin, ß-catenin, vinculin, -actinin, ADIP and LMO7, which connect nectins to E-cadherin (Nagafuchi 2001; Takai & Nakanishi 2003; Takai et al. 2003; Ooshio et al. 2004), were stained, the signals for all of them were concentrated at the cell-cell contact sites in the absence of latrunculin A (Fig. 2A–G, Control). In the presence of latrunculin A, the signal for afadin remained at the cell-cell contact sites, whereas the signals for other PMPs, -catenin, ß-catenin, vinculin, -actinin, ADIP, and LMO7, disappeared from them (Fig. 2A–G, Lat A). The punctuated residual signals for -catenin and ß-catenin at the plasma membranes were mostly overlapped with that for F-actin. Ponsin was not stained by our antibody (Ab) used irrespective of the presence and absence of latrunculin A in nectin-1-MDCK cells (data not shown). The essentially same results were obtained with cytochalasin D (data not shown). These results indicate that at least the latrunculin A- and cytochalasin D-sensitive actin cytoskeleton is necessary for the concentration of E-cadherin and its associated PMPs, but not those of nectin and afadin, at the cell-cell contact sites of nectin-1-MDCK cells.
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We have previously shown that when nectin-1-MDCK cells are cultured at 2 µM Ca2+ for 2 h, the immunofluorescence signal for nectin-1 remains at cell-cell contact sites, whereas the signal for E-cadherin disappears from the contact sites (Fukuhara et al. 2002a; Honda et al. 2003a,c). When these cells are re-cultured at 2 mM Ca2+, the signal for E-cadherin is concentrated at the sites where the signal for nectin-1 is concentrated, resulting in the formation of AJs (Fukuhara et al. 2002a; Honda et al. 2003a,c). We confirmed these earlier observations as controls (Fig. 3A, Control). In the presence of latrunculin A, however, the signal for nectin-1 remained at the cell-cell contact sites, but that for E-cadherin was not recruited to the cell-cell contact sites and AJs was not formed (Fig. 3A, Lat A). Under these conditions, no punctuated signal for E-cadherin was observed at the plasma membrane, whereas that for F-actin was observed there. We next examined the concentration of PMPs, afadin, -catenin, ß-catenin, vinculin, -actinin, ADIP, and LMO7, when nectin-1-MDCK cells precultured at 2 µM Ca2+ were re-cultured at 2 mM Ca2+. In the absence of latrunculin A, the signals for all of them were concentrated at the cell-cell contact sites (Fig. 4A–G, Control). In the presence of this agent, the signal for afadin remained at the cell-cell contact sites, whereas the signals for other PMPs, -catenin, ß-catenin, vinculin, -actinin, ADIP, and LMO7, were not recruited there (Fig. 4A–G, Lat A). The essentially same results were obtained with cytochalasin D (data not shown). These results indicate that the latrunculin A- and cytochalasin D-sensitive actin cytoskeleton is necessary for the concentration of E-cadherin and its associated PMPs at the cell-cell contact sites of nectin-1-MDCK cells during the formation of AJs.
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We have previously shown that the immunofluorescence signals for claudin-1, occludin, JAM-1, ZO-1, and MAGI-1 are concentrated at the cell-cell contact sites of nectin-1- MDCK cells cultured at 2 mM Ca2+ (Ide et al. 1999; Fukuhara et al. 2002a,b). The contact sites where the signals for claudin-1, occludin, JAM-1, ZO-1, and MAGI-1 are concentrated would correspond to TJs. We first confirmed these earlier observations as controls (Figs 1B,C and 2H,I, Control and data not shown). When the cells were cultured at 2 mM Ca2+ in the presence of latrunculin A, the signal for F-actin was markedly impaired, but the signals for nectin-1 and afadin remained at the cell-cell contact sites (Figs 1A and 2A, Lat A). The signals for claudin-1, occludin, JAM-1, ZO-1 and MAGI-1 as well as those for E-cadherin and -catenin mostly disappeared from the contact sites (Figs 1B,C and 2H,I, Lat A and data not shown). The residual punctuated signals for claudin-1, occludin, JAM-1, ZO-1 and MAGI-1 at the plasma membranes were mostly overlapped with that for F-actin. The essentially same results were obtained with cytochalasin D (data not shown). These results indicate that at least the latrunculin A- and cytochalasin D-sensitive actin cytoskeleton is necessary for the concentration of the TJ components at the cell-cell contact sites of nectin-1-MDCK cells.
Inhibition of the formation of the claudin-1-, occludin- and JAM-1-based TJs by the F-actin-disrupting agents in nectin-1-MDCK cells
We have previously shown that when nectin-1-MDCK cells are cultured at 2 µM Ca2+ for 2 h, the immunofluorescence signals for ZO-1 and MAGI-1 as well as those for nectin-1 and afadin remain at cell-cell contact sites, whereas the signals for claudin-1, occludin, and JAM-1 as well as those for E-cadherin and -catenin disappear from them (Fukuhara et al. 2002a,b). When these cells are re-cultured at 2 mM Ca2+, the signal for E-cadherin is concentrated at the sites where nectin-1, afadin, ZO-1 and MAGI-1 are concentrated, whereas the signals for claudin-1, occludin, and JAM-1 are concentrated to the apical side of those for nectin-1 and afadin (Fukuhara et al. 2002a,b). The signal for ZO-1 translocates from AJs to TJs (Fukuhara et al. 2002a). We confirmed these earlier observations as controls (Figs 3B,C and 4H,I, Control). In the presence of latrunculin A, the signals for nectin-1 and afadin remained at the cell-cell contact sites, but the signal for claudin-1, occludin, JAM-1, ZO-1 or MAGI-1 as well as that for E-cadherin or -catenin was not recruited to the cell-cell contact sites and TJs were not formed (Figs 3A–C and 4A,B,H,I, Lat A and data not shown). Under these conditions, no punctuated signals for claudin-1 and JAM-1 were observed at the plasma membrane, whereas that for F-actin was observed. The essentially same results were obtained with cytochalasin D (data not shown). These results indicate that the latrunculin A- and cytochalasin D-sensitive actin cytoskeleton is necessary for the concentration of the TJ components at the cell-cell contact of nectin-1-MDCK cells during the formation of TJs.
Disruption of the nectin- and E-cadherin-based AJs by F-actin-disrupting agents in wild-type MDCK cells
In the last set of experiments, we examined the effect of the F-actin-disrupting agents on the localization of CAMs and PMPs in wild-type MDCK cells, which did not express exogenous nectin-1. Wild-type MDCK cells expressed nectin-1, -2 and -3 as estimated by Western blotting, but nectin-1 or -2 was not stained by any currently available Ab (data not shown). The immunofluorescence signal for nectin-3 as well as those for afadin, E-cadherin, and F-actin was concentrated at the cell-cell contact sites of wild-type MDCK cells cultured at 2 mM Ca2+ (Honda et al. 2003a,c) (Fig. 5A,B, Control). The signals for claudin-1, occludin, and JAM-1 were also concentrated at the cell-cell contact sites of wild-type MDCK cells cultured at 2 mM Ca2+ (Honda et al. 2003c) (Fig. 5C,D, Control and data not shown). These staining patterns were similar to those of nectin-1-MDCK cells. When wild-type MDCK cells were cultured at 2 mM Ca2+ in the presence of latrunculin A, the signal for F-actin was markedly impaired (Fig. 5A–D, Lat A). The signals for nectin-3 and afadin mostly disappeared at the cell-cell contact sites of wild-type MDCK cells (Fig. 5A–D, Lat A), although the signals for nectin-1 and afadin remained at the cell-cell contact sites of nectin-1-MDCK cells (see Figs 1A and 2A, Lat A). The signals for E-cadherin, claudin-1, occludin, and JAM-1 also mostly disappeared from the contact sites of wild-type MDCK cells as well as those of nectin-1-MDCK cells (Fig. 5B–D, Lat A and data not shown). When wild-type MDCK cells precultured at 2 µM Ca2+ were re-cultured at 2 mM Ca2+ in the presence of latrunculin A, the signal for nectin-3 or afadin did not remain at the cell-cell contact sites (Fig. 6A–D, Lat A), although the signals for nectin-1 and afadin remained at the cell-cell contact sites of nectin-1-MDCK cells (see Figs 3A and 4A, Lat A). The signal for E-cadherin, claudin-1, occludin, or JAM-1 was not recruited to the cell-cell contact sites of wild-type MDCK cells as well as in nectin-1-MDCK cells (Fig. 6A–D, Lat A). Thus, the remaining and concentration of nectin-1 and afadin at the cell-cell contact sites in the presence of the F-actin-disrupting agents requires the exogenous expression of nectin-1 in MDCK cells.
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The result that the nectin-1-based cell-cell adhesion is not disrupted by the F-actin-disrupting agents in nectin-1-MDCK cells is consistent with our previous observation that the nectin-1-based cell-cell adhesion is not disrupted by the F-actin-disrupting agents in L cells expressing nectin-1 and E-cadherin (nectin-1-EL cells) (Honda et al. 2003b). We have then shown here that E-cadherin, which is concentrated at the nectin-1-based cell-cell adhesion sites, disappears from there in the presence of the F-actin-disrupting agents in nectin-1-MDCK cells. We have also shown here that E-cadherin does not concentrate at the nectin-1-based cell-cell adhesion sites during the formation of AJs in nectin-1-MDCK cells, suggesting that at least the cytochalasin D- and latrunculin A-sensitive actin cytoskeleton is necessary for the formation and maintenance of AJs. This result is consistent with the earlier observations that the formation and maintenance of AJs are disrupted by the F-actin-disrupting agents in primary epithelial cells (Quinlan & Hyatt 1999). However, we have previously shown that the concentration of E-cadherin at the nectin-1-based cell-cell adhesion sites is not inhibited by the F-actin-disrupting agent in nectin-1-EL cells (Honda et al. 2003b). Thus, the results are different between fibroblasts and epithelial cells. Exact molecular mechanisms for this difference between nectin-1-MDCK and nectin-1-EL cells are unknown, but in epithelial cells, AJs are undercoated with F-actin bundles, forming belt-like structures, whereas in fibroblasts, AJs are less developed than those in epithelial cells and belt-like structures are not observed at AJs (Vasioukhin & Fuchs 2001). Therefore, it is likely that the actin cytoskeleton is necessary for the formation and maintenance of AJs which are undercoated with F-actin bundles in epithelial cells, whereas it is not necessary for the concentration of nectin and E-cadherin at the cell-cell adhesion sites where AJs are not undercoated with F-actin bundles in fibroblasts. The result that the maintenance or the formation of AJs is not disrupted by the F-actin-disrupting agents in nectin-1-EL cells, suggest that the association between nectin-1 and E-cadherin through many PMPs does not require the actin cytoskeleton (Honda et al. 2003b). On the other hand, the actin cytoskeleton may be necessary for the association between nectin-1 and E-cadherin in nectin-1-MDCK cells. It remains unknown how the actin cytoskeleton is involved in the association between nectin-1 and E-cadherin in epithelial cells. It should be noted that only afadin remains at the nectin-1-based cell-cell adhesion sites even in the presence of the F-actin-disrupting agents, whereas other PMPs at AJs, including -catenin, ß-catenin, vinculin, -actinin, ADIP, and LMO7, disappear from them. The association of the nectin-1-afadin unit with these PMPs requires the actin cytoskeleton.
Moreover, we have shown here that the TJ components, which are concentrated at the apical area of the nectin-1-based cell-cell adhesion sites, disappear from them in the presence of the F-actin-disrupting agents in nectin-1-MDCK cells, and that they do not concentrate there during the formation of TJs, suggesting that at least the cytochalasin D- and latrunculin A-sensitive actin cytoskeleton is necessary for the formation and maintenance of TJs. These results are consistent with the previous observation that transepithelial resistance is reduced by cytochalasin D in MDCK cells and Caco-2 cells and that the concentration of ZO-1 at TJs is inhibited by this agent (Stevenson & Begg 1994; Wittchen et al. 1999; Ma et al. 2000). It has been shown that the maintenance and formation of TJs are generally dependent on the formation and maintenance of AJs in epithelial cells, such as MDCK cells (Gumbiner et al. 1988; Stevenson & Begg 1994). Therefore, the inhibitory effects of the F-actin-disrupting agents on the maintenance and formation of TJs may be due to their inhibitory effects on the maintenance and formation of AJs or the actin cytoskeleton may be necessary for the association of the nectin-afadin and E-cadherin-catnin units with the TJ components. It may be noted that nectins recruit ZO-1 to the nectin-based cell-cell adhesion sites in L cells expressing nectin-2 and that the localization of ZO-1 at the nectin-2-based cell-cell adhesion sites does not require the actin cytoskeleton (Yokoyama et al. 2001). The association of ZO-1 with other AJ components such as -catenin and afadin may sustain ZO-1 at the cell-cell adhesion sites in an F-actin-independent manner in fibroblasts.
It may be noted that there are two types of F-actin-binding proteins: one is involved in the formation of AJs and TJs through the formation of filopodia and lamellipodia; and the other is involved in the maintenance of AJs and TJs. Moreover, even after AJs and TJs are formed, they may dynamically repeat disruption and formation. Filopodia and lamellipodia are formed by the action of Cdc42 and Rac activated by nectins and E-cadherin (Shimizu & Takai 2003; Takai et al. 2003; Yap & Kovacs 2003). Many downstream effectors have been identified for these small G proteins and some of these effectors are F-actin-binding proteins; IQGAP1 and IRSp53/WAVE for Rac and IQGAP1, NWASP and WASP for Cdc42 (Takenawa & Miki 2001; Takai et al. 2001). Therefore, the nectin-afadin and E-cadherin-catenin units regulate organization of the peripheral actin cytoskeleton in two different ways: one is organized by F-actin-binding PMPs associated with nectins and cadherins, such as afadin, -catenin, -actinin, and vinculin; the other is organized by Cdc42 and Rac through their downstream effector F-actin-binding proteins. It is unknown which F-actin-binding proteins are involved in the formation or maintenance of AJs and TJs. It is necessary for our understanding of the roles and mechanisms of the actin cytoskeleton based on these actin-binding proteins in the formation and maintenance of AJs and TJs in epithelial cells.
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Experimental procedures |
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Rabbit anti-MAGI-1, anti-nectin-1, anti-ADIP, and anti-LMO7 polyclonal Abs (pAbs) were prepared as described (Ide et al. 1999; Takahashi et al. 1999; Asada et al. 2003; Ooshio et al. 2004). A rabbit anti-afadin pAb and a mouse anti-afadin monoclonal Ab (mAb) were prepared as described (Mandai et al. 1997; Sakisaka et al. 1999). A rat anti-E-cadherin mAb (ECCD-2) was supplied by Dr M. Takeichi (Centre for Developmental Biology, RIKEN, Kobe, Japan). A rabbit anti-JAM pAb was supplied from 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). Mouse anti-human vinculin, anti--actinin (sarcomeric), anti-FLAG M1 mAbs, and a rabbit anti--catenin pAb were obtained from Sigma. A rabbit anti-ß-catenin mAb was obtained from Santa Cruz Biotechnology, Inc. A mouse anti-occludin mAb and a rabbit anti-claudin-1 pAb were obtained from Zymed. A mouse anti-ZO-1 mAb was obtained from SANKO JUNYAKU. Secondary Abs for immunofluorescence microscopy were obtained from Chemicon International, Inc.
Cell culture and DNA transfection
MDCK cells were kindly supplied from Dr W. Birchmeier (Max-Delbruck-Centre for Molecular Medicine, Berlin, Germany). An MDCK cell line stably expressing FLAG-nectin-1 (nectin-1-MDCK cells) was prepared as described (Takahashi et al. 1999). Briefly, MDCK cells were transfected with pCAGI-puro-FLAG-nectin-1 using the LipofectAMINE reagent (Invitrogen). The cells were then cultured for 24 h, replated, and selected by culturing in the presence of 5 µg/mL puromycin (Sigma).
Disassembly of the actin cytoskeleton
Nectin-1-MDCK cells (1 x 105) were seeded on an 18-mm glass cover slip in 12-well culture dishes. Forty-eight h later, the cells were washed with phosphate buffered saline, pH 7.4 (PBS) and cultured at 2 mM Ca2+ in DMEM without serum for 1 h. Nectin-1-MDCK cells were then cultured at 2 mM Ca2+ in DMEM with 200 nM latrunculin A (Wako) for 1 h. On the other hand, after being 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 2 h. After the culture, nectin-1-MDCK cells were cultured at 2 µM Ca2+ in DMEM with 5 mM EGTA and 200 nM latrunculin A for 1 h. Nectin-1-MDCK cells were then cultured at 2 mM Ca2+ in DMEM with 200 nM latrunculin A for 2 h.
Immunofluorescence microscopy
Immunofluorescence microscopy was performed as described (Mandai et al. 1997). Briefly, the cells were fixed with 1% formaldehyde in PBS for 15 min. The fixed cells were then treated with 0.2% Triton X-100 in PBS for 15 min. After being blocked in PBS containing 1% bovine serum albumin (BSA) for 1 h, the cells were incubated in the same buffer with various combinations of the anti-afadin, anti-E-cadherin, anti-vinculin, anti--actinin, anti-FLAG, anti-ß-catenin, anti-occludin, and anti-ZO-1 mAbs, and the anti-nectin-1, anti-afadin, anti-ADIP, anti-LMO7, anti-MAGI-1, anti-JAM, anti--catenin, and anti-claudin-1 pAbs for 1 h. The samples were washed with PBS for 5 min three times and incubated for 30 min in PBS containing 1% BSA with various combinations of the secondary pAbs, and rhodamine-phalloidin. The samples were then washed with PBS for 5 min three times and mounted in GEL/MOUNT (Biomeda). The samples were analysed by Radiance 2100 confocal laser scanning microscope (Bio-Rad Laboratories).
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* Correspondence: E-mail: ytakai{at}molbio.med.osaka-u.ac.jp
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References |
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Received: 27 May 2004
Accepted: 22 June 2004
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