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Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S - J. Cell Biol. (1999)

Bottom Line: We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4.At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased.These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

View Article: PubMed Central - PubMed

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

ABSTRACT
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

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Effects of C-CPE on the subcellular distribution of claudin-3 and the morphology of reconstituted TJ strands in L transfectants expressing claudin-3 (C3L cells). (a and b) Immunofluorescence microscopy. When C3L cells were stained with anti–claudin-3 pAb (a), claudin-3 was shown to be concentrated at cell–cell borders as planes (arrows). At 4 h after incubation with 2.5 μg/ml C-CPE, these claudin-3-positive planes were fragmented into punctate structures and gradually disappeared (b). (c and d) Freeze-fracture replica electron microscopy. In the absence of C-CPE, a well-developed network of TJ strands was reconstituted between adjacent C3L cells (c). When cells were incubated with C-CPE for 4 h, TJ strands were disintegrated into fragmented belt-like aggregates of intramembranous particles (d). Bars: (a and b) 20 μm; (c and d) 200 nm.
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Figure 3: Effects of C-CPE on the subcellular distribution of claudin-3 and the morphology of reconstituted TJ strands in L transfectants expressing claudin-3 (C3L cells). (a and b) Immunofluorescence microscopy. When C3L cells were stained with anti–claudin-3 pAb (a), claudin-3 was shown to be concentrated at cell–cell borders as planes (arrows). At 4 h after incubation with 2.5 μg/ml C-CPE, these claudin-3-positive planes were fragmented into punctate structures and gradually disappeared (b). (c and d) Freeze-fracture replica electron microscopy. In the absence of C-CPE, a well-developed network of TJ strands was reconstituted between adjacent C3L cells (c). When cells were incubated with C-CPE for 4 h, TJ strands were disintegrated into fragmented belt-like aggregates of intramembranous particles (d). Bars: (a and b) 20 μm; (c and d) 200 nm.

Mentions: In good agreement with previous observations (Horiguchi et al. 1986, Horiguchi et al. 1987; Hanna et al. 1991, Hanna et al. 1992), the COOH-terminal fragment (184-319 amino acids) of CPE (C-CPE) did not show any cytotoxicity to C3L or C4L cells (Fig. 2 A). The non-cytotoxicity of C-CPE allowed us to follow the subsequent behavior of claudin-3 and -4 after their specific binding to C-CPE within cells. Thus, in C1L, C2L, and C3L cells, we examined the effects of C-CPE on the subcellular distribution of claudin-1 to -3, respectively, by immunofluorescence microscopy, since for as yet unknown reasons in C4L cells exogenous claudin-4 was not concentrated efficiently into cell–cell borders. Similarly to claudin-1 and -2 (Furuse et al. 1998b), claudin-3 was also concentrated at cell–cell contact planes to reconstitute TJ strand networks when introduced into L fibroblasts. When these L transfectants were incubated with 2.5 μg/ml C-CPE for 4 h, claudin-3, but not claudin-1 and -2, which were concentrated at cell–cell contact planes, showed punctate distribution and gradually disappeared (Fig. 3, a and b). We then observed the cell–cell contact planes of C3L cells before and after 4-h incubation with C-CPE by conventional freeze-fracture replica electron microscopy. As shown in Fig. 3 d, in the presence of C-CPE, the well-developed network of claudin-3–based TJ strands disintegrated into thick belt-like aggregates of intramembranous particles of various lengths, and then disappeared.


Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S - J. Cell Biol. (1999)

Effects of C-CPE on the subcellular distribution of claudin-3 and the morphology of reconstituted TJ strands in L transfectants expressing claudin-3 (C3L cells). (a and b) Immunofluorescence microscopy. When C3L cells were stained with anti–claudin-3 pAb (a), claudin-3 was shown to be concentrated at cell–cell borders as planes (arrows). At 4 h after incubation with 2.5 μg/ml C-CPE, these claudin-3-positive planes were fragmented into punctate structures and gradually disappeared (b). (c and d) Freeze-fracture replica electron microscopy. In the absence of C-CPE, a well-developed network of TJ strands was reconstituted between adjacent C3L cells (c). When cells were incubated with C-CPE for 4 h, TJ strands were disintegrated into fragmented belt-like aggregates of intramembranous particles (d). Bars: (a and b) 20 μm; (c and d) 200 nm.
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Related In: Results  -  Collection

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Figure 3: Effects of C-CPE on the subcellular distribution of claudin-3 and the morphology of reconstituted TJ strands in L transfectants expressing claudin-3 (C3L cells). (a and b) Immunofluorescence microscopy. When C3L cells were stained with anti–claudin-3 pAb (a), claudin-3 was shown to be concentrated at cell–cell borders as planes (arrows). At 4 h after incubation with 2.5 μg/ml C-CPE, these claudin-3-positive planes were fragmented into punctate structures and gradually disappeared (b). (c and d) Freeze-fracture replica electron microscopy. In the absence of C-CPE, a well-developed network of TJ strands was reconstituted between adjacent C3L cells (c). When cells were incubated with C-CPE for 4 h, TJ strands were disintegrated into fragmented belt-like aggregates of intramembranous particles (d). Bars: (a and b) 20 μm; (c and d) 200 nm.
Mentions: In good agreement with previous observations (Horiguchi et al. 1986, Horiguchi et al. 1987; Hanna et al. 1991, Hanna et al. 1992), the COOH-terminal fragment (184-319 amino acids) of CPE (C-CPE) did not show any cytotoxicity to C3L or C4L cells (Fig. 2 A). The non-cytotoxicity of C-CPE allowed us to follow the subsequent behavior of claudin-3 and -4 after their specific binding to C-CPE within cells. Thus, in C1L, C2L, and C3L cells, we examined the effects of C-CPE on the subcellular distribution of claudin-1 to -3, respectively, by immunofluorescence microscopy, since for as yet unknown reasons in C4L cells exogenous claudin-4 was not concentrated efficiently into cell–cell borders. Similarly to claudin-1 and -2 (Furuse et al. 1998b), claudin-3 was also concentrated at cell–cell contact planes to reconstitute TJ strand networks when introduced into L fibroblasts. When these L transfectants were incubated with 2.5 μg/ml C-CPE for 4 h, claudin-3, but not claudin-1 and -2, which were concentrated at cell–cell contact planes, showed punctate distribution and gradually disappeared (Fig. 3, a and b). We then observed the cell–cell contact planes of C3L cells before and after 4-h incubation with C-CPE by conventional freeze-fracture replica electron microscopy. As shown in Fig. 3 d, in the presence of C-CPE, the well-developed network of claudin-3–based TJ strands disintegrated into thick belt-like aggregates of intramembranous particles of various lengths, and then disappeared.

Bottom Line: We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4.At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased.These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

View Article: PubMed Central - PubMed

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

ABSTRACT
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Show MeSH
Related in: MedlinePlus