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Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S - J. Cell Biol. (1999)

Bottom Line: Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

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Recruitment of endogenous ZO-1 to claudin-based networks in L transfectants expressing claudin-1 or -2. Parental L cells (L cell; a and b) and L transfectants expressing claudin-1 (C1L cell; c and d) were double stained with rat anti–claudin-1 mAb (claudin; a and c) and mouse anti–ZO-1 mAb (ZO-1; b and d). L transfectants expressing claudin-2 (C2L cell; e and f) were double stained with anti–claudin-2 mAb (e) and anti–ZO-1 mAb (f). L transfectants expressing claudin-1 mutant lacking the COOH-terminal YV sequence (C1ΔYVL cell; g and h) were double stained with anti–claudin-1 pAb (g) and anti–ZO-1 mAb (h). ZO-1 showed no characteristic concentration in L cells (b), whereas in C1L and C2L cells ZO-1 was coconcentrated with claudin-1 (c and d) and claudin-2 (e and f) as planes at cell–cell borders. Close inspection revealed that in these cells both claudins and ZO-1 were concentrated in elaborate network patterns, and that their network patterns were mostly overlapped (insets in c–f). In C1ΔYVL cells, mutant claudin-1 was concentrated at cell–cell borders (g), whereas ZO-1 showed no concentration (h). Bar: (a–h) 10 μm; (insets) 15 μm.
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Figure 7: Recruitment of endogenous ZO-1 to claudin-based networks in L transfectants expressing claudin-1 or -2. Parental L cells (L cell; a and b) and L transfectants expressing claudin-1 (C1L cell; c and d) were double stained with rat anti–claudin-1 mAb (claudin; a and c) and mouse anti–ZO-1 mAb (ZO-1; b and d). L transfectants expressing claudin-2 (C2L cell; e and f) were double stained with anti–claudin-2 mAb (e) and anti–ZO-1 mAb (f). L transfectants expressing claudin-1 mutant lacking the COOH-terminal YV sequence (C1ΔYVL cell; g and h) were double stained with anti–claudin-1 pAb (g) and anti–ZO-1 mAb (h). ZO-1 showed no characteristic concentration in L cells (b), whereas in C1L and C2L cells ZO-1 was coconcentrated with claudin-1 (c and d) and claudin-2 (e and f) as planes at cell–cell borders. Close inspection revealed that in these cells both claudins and ZO-1 were concentrated in elaborate network patterns, and that their network patterns were mostly overlapped (insets in c–f). In C1ΔYVL cells, mutant claudin-1 was concentrated at cell–cell borders (g), whereas ZO-1 showed no concentration (h). Bar: (a–h) 10 μm; (insets) 15 μm.

Mentions: As shown in Fig. 6, ZO-1, but not ZO-2 or ZO-3, was expressed endogenously in parental L cells (and also in C1L and C2L cells). This endogenous ZO-1 did not show characteristic concentration in L cells (Fig. 7, a and b). In contrast, in both C1L and C2L cells in which claudin-1 and claudin-2 were concentrated at cell–cell borders in an elaborate network, respectively, endogenous ZO-1 was colocalized with claudins (Fig. 7, c–f). Close inspection revealed that ZO-1 was also concentrated as elaborate networks, which almost overlapped with those based on claudin (Fig. 7, c–f, insets). When claudin-1 mutant lacking its COOH-terminal YV was transfected into L cells (C1ΔYVL cells), introduced claudin-1 mutant was again concentrated at cell–cell borders as elaborate networks (Fig. 7 g). Interestingly, however, ZO-1 was not recruited to these claudin-based networks in C1ΔYVL cells (Fig. 7 h). These findings, together with the in vitro binding data suggested that ZO-1 also directly binds to the COOH-terminal YV of claudins through its PDZ1 domain inside cells.


Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S - J. Cell Biol. (1999)

Recruitment of endogenous ZO-1 to claudin-based networks in L transfectants expressing claudin-1 or -2. Parental L cells (L cell; a and b) and L transfectants expressing claudin-1 (C1L cell; c and d) were double stained with rat anti–claudin-1 mAb (claudin; a and c) and mouse anti–ZO-1 mAb (ZO-1; b and d). L transfectants expressing claudin-2 (C2L cell; e and f) were double stained with anti–claudin-2 mAb (e) and anti–ZO-1 mAb (f). L transfectants expressing claudin-1 mutant lacking the COOH-terminal YV sequence (C1ΔYVL cell; g and h) were double stained with anti–claudin-1 pAb (g) and anti–ZO-1 mAb (h). ZO-1 showed no characteristic concentration in L cells (b), whereas in C1L and C2L cells ZO-1 was coconcentrated with claudin-1 (c and d) and claudin-2 (e and f) as planes at cell–cell borders. Close inspection revealed that in these cells both claudins and ZO-1 were concentrated in elaborate network patterns, and that their network patterns were mostly overlapped (insets in c–f). In C1ΔYVL cells, mutant claudin-1 was concentrated at cell–cell borders (g), whereas ZO-1 showed no concentration (h). Bar: (a–h) 10 μm; (insets) 15 μm.
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Figure 7: Recruitment of endogenous ZO-1 to claudin-based networks in L transfectants expressing claudin-1 or -2. Parental L cells (L cell; a and b) and L transfectants expressing claudin-1 (C1L cell; c and d) were double stained with rat anti–claudin-1 mAb (claudin; a and c) and mouse anti–ZO-1 mAb (ZO-1; b and d). L transfectants expressing claudin-2 (C2L cell; e and f) were double stained with anti–claudin-2 mAb (e) and anti–ZO-1 mAb (f). L transfectants expressing claudin-1 mutant lacking the COOH-terminal YV sequence (C1ΔYVL cell; g and h) were double stained with anti–claudin-1 pAb (g) and anti–ZO-1 mAb (h). ZO-1 showed no characteristic concentration in L cells (b), whereas in C1L and C2L cells ZO-1 was coconcentrated with claudin-1 (c and d) and claudin-2 (e and f) as planes at cell–cell borders. Close inspection revealed that in these cells both claudins and ZO-1 were concentrated in elaborate network patterns, and that their network patterns were mostly overlapped (insets in c–f). In C1ΔYVL cells, mutant claudin-1 was concentrated at cell–cell borders (g), whereas ZO-1 showed no concentration (h). Bar: (a–h) 10 μm; (insets) 15 μm.
Mentions: As shown in Fig. 6, ZO-1, but not ZO-2 or ZO-3, was expressed endogenously in parental L cells (and also in C1L and C2L cells). This endogenous ZO-1 did not show characteristic concentration in L cells (Fig. 7, a and b). In contrast, in both C1L and C2L cells in which claudin-1 and claudin-2 were concentrated at cell–cell borders in an elaborate network, respectively, endogenous ZO-1 was colocalized with claudins (Fig. 7, c–f). Close inspection revealed that ZO-1 was also concentrated as elaborate networks, which almost overlapped with those based on claudin (Fig. 7, c–f, insets). When claudin-1 mutant lacking its COOH-terminal YV was transfected into L cells (C1ΔYVL cells), introduced claudin-1 mutant was again concentrated at cell–cell borders as elaborate networks (Fig. 7 g). Interestingly, however, ZO-1 was not recruited to these claudin-based networks in C1ΔYVL cells (Fig. 7 h). These findings, together with the in vitro binding data suggested that ZO-1 also directly binds to the COOH-terminal YV of claudins through its PDZ1 domain inside cells.

Bottom Line: Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

Show MeSH
Related in: MedlinePlus