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

Freeze-fracture replica images of TJs in MDCK I cells incubated with C-CPE. At 0, 4, or 8 h after incubation with 2.5 μg/ml C-CPE in the basolateral compartment, MDCK I cells plated at confluent density on filters were fixed with glutaraldehyde, and processed for freeze-fracture replica electron microscopy. In the absence of C-CPE (0 H), MDCK I cells were characterized by well-developed anastomosing networks of TJ strands. At 4 h after incubation with C-CPE (4 H), TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (arrows). Around 8 h after incubation (8 H), these belt-like particle aggregates had mostly disappeared, leaving a fairly simple TJ strand network. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164970&req=5

Figure 5: Freeze-fracture replica images of TJs in MDCK I cells incubated with C-CPE. At 0, 4, or 8 h after incubation with 2.5 μg/ml C-CPE in the basolateral compartment, MDCK I cells plated at confluent density on filters were fixed with glutaraldehyde, and processed for freeze-fracture replica electron microscopy. In the absence of C-CPE (0 H), MDCK I cells were characterized by well-developed anastomosing networks of TJ strands. At 4 h after incubation with C-CPE (4 H), TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (arrows). Around 8 h after incubation (8 H), these belt-like particle aggregates had mostly disappeared, leaving a fairly simple TJ strand network. Bar, 20 μm.

Mentions: The question naturally arose as to what types of morphological changes of TJ strands are associated with the C-CPE–induced disappearance of claudin-4 in MDCK I cells. MDCK I cells were incubated with C-CPE in their basolateral compartment for 4, 8, and 24 h, fixed with glutaraldehyde, and then examined by conventional freeze-fracture replica electron microscopy. TJs in nontreated MDCK I cells were characterized by well-developed anastomosing networks of TJ strands (Fig. 5 a). The mean strand number, which was determined by taking numerous counts along a line drawn perpendicular to the junctional axis (Stevenson et al. 1988), was 4.0 ± 1.3, and the complexity of TJs, defined as the number of branch points per unit length (1 μm) of TJ strands (Wolburg et al. 1994), was 11.4 (Table ). At 4 h after incubation with C-CPE, TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (Fig. 5 b). Around 8 h after incubation, these belt-like particle aggregates mostly disappeared, leaving a fairly simple TJ strand network (Fig. 5 c). The mean strand number and the complexity of TJs at this time point were 2.6 ± 0.9 and 5.9, respectively (Table ). The number of free ends per unit length (1 μm) of TJ strands also increased with incubation period (Table ). This type of simple network of TJs was maintained until 24 h after addition of C-CPE (Table ).


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)

Freeze-fracture replica images of TJs in MDCK I cells incubated with C-CPE. At 0, 4, or 8 h after incubation with 2.5 μg/ml C-CPE in the basolateral compartment, MDCK I cells plated at confluent density on filters were fixed with glutaraldehyde, and processed for freeze-fracture replica electron microscopy. In the absence of C-CPE (0 H), MDCK I cells were characterized by well-developed anastomosing networks of TJ strands. At 4 h after incubation with C-CPE (4 H), TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (arrows). Around 8 h after incubation (8 H), these belt-like particle aggregates had mostly disappeared, leaving a fairly simple TJ strand network. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Freeze-fracture replica images of TJs in MDCK I cells incubated with C-CPE. At 0, 4, or 8 h after incubation with 2.5 μg/ml C-CPE in the basolateral compartment, MDCK I cells plated at confluent density on filters were fixed with glutaraldehyde, and processed for freeze-fracture replica electron microscopy. In the absence of C-CPE (0 H), MDCK I cells were characterized by well-developed anastomosing networks of TJ strands. At 4 h after incubation with C-CPE (4 H), TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (arrows). Around 8 h after incubation (8 H), these belt-like particle aggregates had mostly disappeared, leaving a fairly simple TJ strand network. Bar, 20 μm.
Mentions: The question naturally arose as to what types of morphological changes of TJ strands are associated with the C-CPE–induced disappearance of claudin-4 in MDCK I cells. MDCK I cells were incubated with C-CPE in their basolateral compartment for 4, 8, and 24 h, fixed with glutaraldehyde, and then examined by conventional freeze-fracture replica electron microscopy. TJs in nontreated MDCK I cells were characterized by well-developed anastomosing networks of TJ strands (Fig. 5 a). The mean strand number, which was determined by taking numerous counts along a line drawn perpendicular to the junctional axis (Stevenson et al. 1988), was 4.0 ± 1.3, and the complexity of TJs, defined as the number of branch points per unit length (1 μm) of TJ strands (Wolburg et al. 1994), was 11.4 (Table ). At 4 h after incubation with C-CPE, TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles (Fig. 5 b). Around 8 h after incubation, these belt-like particle aggregates mostly disappeared, leaving a fairly simple TJ strand network (Fig. 5 c). The mean strand number and the complexity of TJs at this time point were 2.6 ± 0.9 and 5.9, respectively (Table ). The number of free ends per unit length (1 μm) of TJ strands also increased with incubation period (Table ). This type of simple network of TJs was maintained until 24 h after addition of C-CPE (Table ).

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