Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of C-CPE on the TJ barrier function of MDCK I cells. (A–C) TER measurements. MDCK I cells were plated at confluent density on 12-mm filters. When 2.5 μg/ml C-CPE was added to the apical compartment at t = 0 (C-CPE), the TER was not affected but remained at the level of 8,000–10,000 Ωcm2 (A; n = 10 for each condition). Addition of C-CPE (2.5 μg/ml) in the basolateral compartment (at t = 0; C-CPE) resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment (at t = 24 h; wash) induced gradual recovery of TER to the level of nontreated cells within one day (B; n = 10 for each condition). In B, asterisks denote significant difference from nonincubated cells at the corresponding time point (P < 0.01). The C-CPE–induced reduction of TER was dose-dependent (C; n = 4 for each condition). (D) Paracellular tracer flux assay (n = 4 for each condition). C-CPE (2.5 μg/ml) in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K (*P < 0.01), but not 40K. All error bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164970&req=5

Figure 6: Effects of C-CPE on the TJ barrier function of MDCK I cells. (A–C) TER measurements. MDCK I cells were plated at confluent density on 12-mm filters. When 2.5 μg/ml C-CPE was added to the apical compartment at t = 0 (C-CPE), the TER was not affected but remained at the level of 8,000–10,000 Ωcm2 (A; n = 10 for each condition). Addition of C-CPE (2.5 μg/ml) in the basolateral compartment (at t = 0; C-CPE) resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment (at t = 24 h; wash) induced gradual recovery of TER to the level of nontreated cells within one day (B; n = 10 for each condition). In B, asterisks denote significant difference from nonincubated cells at the corresponding time point (P < 0.01). The C-CPE–induced reduction of TER was dose-dependent (C; n = 4 for each condition). (D) Paracellular tracer flux assay (n = 4 for each condition). C-CPE (2.5 μg/ml) in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K (*P < 0.01), but not 40K. All error bars represent standard deviations.

Mentions: Next, we examined the effects of C-CPE on TER of MDCK I cells plated at confluent density on filters. When 2.5 μg/ml C-CPE was added in the apical compartment, the TER was not affected but remained at a level of 8,000–10,000 Ωcm2 (Fig. 6 A). In contrast, addition of C-CPE to the basolateral compartment resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment induced gradual recovery of TER to the level of nontreated controls within one day (Fig. 6 B). Furthermore, this C-CPE–induced reduction of TER was dose dependent (Fig. 6 C). Finally, to exclude the possibility that this reduction of TER was caused by an increase in transcellular plasma membrane permeability to ions, we assessed the flux of membrane-impermeable paracellular tracers (FITC-dextran 4K, 10K, and 40K) across MDCK I cell monolayers. As shown in Fig. 6 D, C-CPE in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K, but not 40K. These findings indicated that C-CPE in the basolateral compartment downregulated the TJ barrier itself of MDCK I cells significantly with a similar time course to the disappearance of claudin-4 (see Fig. 4 A).


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 TJ barrier function of MDCK I cells. (A–C) TER measurements. MDCK I cells were plated at confluent density on 12-mm filters. When 2.5 μg/ml C-CPE was added to the apical compartment at t = 0 (C-CPE), the TER was not affected but remained at the level of 8,000–10,000 Ωcm2 (A; n = 10 for each condition). Addition of C-CPE (2.5 μg/ml) in the basolateral compartment (at t = 0; C-CPE) resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment (at t = 24 h; wash) induced gradual recovery of TER to the level of nontreated cells within one day (B; n = 10 for each condition). In B, asterisks denote significant difference from nonincubated cells at the corresponding time point (P < 0.01). The C-CPE–induced reduction of TER was dose-dependent (C; n = 4 for each condition). (D) Paracellular tracer flux assay (n = 4 for each condition). C-CPE (2.5 μg/ml) in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K (*P < 0.01), but not 40K. All error bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164970&req=5

Figure 6: Effects of C-CPE on the TJ barrier function of MDCK I cells. (A–C) TER measurements. MDCK I cells were plated at confluent density on 12-mm filters. When 2.5 μg/ml C-CPE was added to the apical compartment at t = 0 (C-CPE), the TER was not affected but remained at the level of 8,000–10,000 Ωcm2 (A; n = 10 for each condition). Addition of C-CPE (2.5 μg/ml) in the basolateral compartment (at t = 0; C-CPE) resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment (at t = 24 h; wash) induced gradual recovery of TER to the level of nontreated cells within one day (B; n = 10 for each condition). In B, asterisks denote significant difference from nonincubated cells at the corresponding time point (P < 0.01). The C-CPE–induced reduction of TER was dose-dependent (C; n = 4 for each condition). (D) Paracellular tracer flux assay (n = 4 for each condition). C-CPE (2.5 μg/ml) in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K (*P < 0.01), but not 40K. All error bars represent standard deviations.
Mentions: Next, we examined the effects of C-CPE on TER of MDCK I cells plated at confluent density on filters. When 2.5 μg/ml C-CPE was added in the apical compartment, the TER was not affected but remained at a level of 8,000–10,000 Ωcm2 (Fig. 6 A). In contrast, addition of C-CPE to the basolateral compartment resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm2 to ∼2,000 Ωcm2 within 4 h, and removal of C-CPE from the compartment induced gradual recovery of TER to the level of nontreated controls within one day (Fig. 6 B). Furthermore, this C-CPE–induced reduction of TER was dose dependent (Fig. 6 C). Finally, to exclude the possibility that this reduction of TER was caused by an increase in transcellular plasma membrane permeability to ions, we assessed the flux of membrane-impermeable paracellular tracers (FITC-dextran 4K, 10K, and 40K) across MDCK I cell monolayers. As shown in Fig. 6 D, C-CPE in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K, but not 40K. These findings indicated that C-CPE in the basolateral compartment downregulated the TJ barrier itself of MDCK I cells significantly with a similar time course to the disappearance of claudin-4 (see Fig. 4 A).

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