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Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells.

Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S - J. Cell Biol. (2005)

Bottom Line: In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts.When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized.These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

View Article: PubMed Central - PubMed

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

ABSTRACT
For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

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Tricellular localization of tricellulin. (A) Immunostaining of various epithelial cell lines—mouse MTD1A, mouse CSG1, and dog MDCK II cells—with anti-tricellulin pAb (red) and anti-occludin mAb (green). Tricellulin was concentrated at tricellular contacts. (B) Immunostaining of Eph4 cells expressing HA-tagged tricellulin with anti-HA mAb (red) and anti-occludin pAb (green). HA–tricellulin was recruited to the tricellular contacts in large amounts and also to occludin-positive bTJs in small amounts. (A and B) Bars, 10 μm. (C) Immunofreeze-fracture replica electron microscopy. Freeze-fracture replicas obtained from tricellular contacts of Eph4 cells were immunolabeled with anti-tricellulin pAb. In the left panel, the central sealing elements (arrow), which were identified between bTJ networks of two adjacent cells (1 and 2), were specifically labeled with anti-tricellulin pAb (arrowheads; see Fig.1). Asterisk indicates adherens junction. In the right panel, the central sealing elements between two adjacent cells (1 and 2) were enlarged, where many immunogold particles were detected (arrows). Bars, 200 nm.
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fig4: Tricellular localization of tricellulin. (A) Immunostaining of various epithelial cell lines—mouse MTD1A, mouse CSG1, and dog MDCK II cells—with anti-tricellulin pAb (red) and anti-occludin mAb (green). Tricellulin was concentrated at tricellular contacts. (B) Immunostaining of Eph4 cells expressing HA-tagged tricellulin with anti-HA mAb (red) and anti-occludin pAb (green). HA–tricellulin was recruited to the tricellular contacts in large amounts and also to occludin-positive bTJs in small amounts. (A and B) Bars, 10 μm. (C) Immunofreeze-fracture replica electron microscopy. Freeze-fracture replicas obtained from tricellular contacts of Eph4 cells were immunolabeled with anti-tricellulin pAb. In the left panel, the central sealing elements (arrow), which were identified between bTJ networks of two adjacent cells (1 and 2), were specifically labeled with anti-tricellulin pAb (arrowheads; see Fig.1). Asterisk indicates adherens junction. In the right panel, the central sealing elements between two adjacent cells (1 and 2) were enlarged, where many immunogold particles were detected (arrows). Bars, 200 nm.

Mentions: We then examined the subcellular distribution of tricellulin: immunofluorescently stained frozen sections of epithelial cellular sheets such as the small intestine, stomach, and kidney, doubly with anti-tricellulin pAb and anti-occludin mAb, showed a unique staining pattern (Fig. 3 A) predominantly localized to the tricellular contacts. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. When the confluent cellular sheets of the mouse epithelial cell line Eph4 were double stained with anti-tricellulin pAb and anti-occludin mAb, tricellulin was again characteristically concentrated at each tricellular contact as dots where three occludin-positive lines converged (Fig. 3 B). In vertical confocal microscopic images generated at the tricellular contacts, tricellulin-positive structures appear as vertically oriented short rods, whereas occludin was concentrated as dots only in the most apical region of these rods. A similar tricellular contact–specific concentration of tricellulin was observed in other epithelial cell lines such as mouse MTD1A cells, CSG1 cells, and dog MDCK II cells (Fig. 4 A). To confirm the specificity of the unique staining pattern with anti-tricellulin pAb, we then performed transfection experiments. When HA-tagged tricellulin was exogenously expressed in Eph4 cells followed by double staining with anti-HA pAb and anti-occludin mAb, HA-tagged tricellulin was predominantly recruited to the tricellular contacts, although it was also recruited in small amounts to bTJs (Fig. 4 B).


Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells.

Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S - J. Cell Biol. (2005)

Tricellular localization of tricellulin. (A) Immunostaining of various epithelial cell lines—mouse MTD1A, mouse CSG1, and dog MDCK II cells—with anti-tricellulin pAb (red) and anti-occludin mAb (green). Tricellulin was concentrated at tricellular contacts. (B) Immunostaining of Eph4 cells expressing HA-tagged tricellulin with anti-HA mAb (red) and anti-occludin pAb (green). HA–tricellulin was recruited to the tricellular contacts in large amounts and also to occludin-positive bTJs in small amounts. (A and B) Bars, 10 μm. (C) Immunofreeze-fracture replica electron microscopy. Freeze-fracture replicas obtained from tricellular contacts of Eph4 cells were immunolabeled with anti-tricellulin pAb. In the left panel, the central sealing elements (arrow), which were identified between bTJ networks of two adjacent cells (1 and 2), were specifically labeled with anti-tricellulin pAb (arrowheads; see Fig.1). Asterisk indicates adherens junction. In the right panel, the central sealing elements between two adjacent cells (1 and 2) were enlarged, where many immunogold particles were detected (arrows). Bars, 200 nm.
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fig4: Tricellular localization of tricellulin. (A) Immunostaining of various epithelial cell lines—mouse MTD1A, mouse CSG1, and dog MDCK II cells—with anti-tricellulin pAb (red) and anti-occludin mAb (green). Tricellulin was concentrated at tricellular contacts. (B) Immunostaining of Eph4 cells expressing HA-tagged tricellulin with anti-HA mAb (red) and anti-occludin pAb (green). HA–tricellulin was recruited to the tricellular contacts in large amounts and also to occludin-positive bTJs in small amounts. (A and B) Bars, 10 μm. (C) Immunofreeze-fracture replica electron microscopy. Freeze-fracture replicas obtained from tricellular contacts of Eph4 cells were immunolabeled with anti-tricellulin pAb. In the left panel, the central sealing elements (arrow), which were identified between bTJ networks of two adjacent cells (1 and 2), were specifically labeled with anti-tricellulin pAb (arrowheads; see Fig.1). Asterisk indicates adherens junction. In the right panel, the central sealing elements between two adjacent cells (1 and 2) were enlarged, where many immunogold particles were detected (arrows). Bars, 200 nm.
Mentions: We then examined the subcellular distribution of tricellulin: immunofluorescently stained frozen sections of epithelial cellular sheets such as the small intestine, stomach, and kidney, doubly with anti-tricellulin pAb and anti-occludin mAb, showed a unique staining pattern (Fig. 3 A) predominantly localized to the tricellular contacts. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. When the confluent cellular sheets of the mouse epithelial cell line Eph4 were double stained with anti-tricellulin pAb and anti-occludin mAb, tricellulin was again characteristically concentrated at each tricellular contact as dots where three occludin-positive lines converged (Fig. 3 B). In vertical confocal microscopic images generated at the tricellular contacts, tricellulin-positive structures appear as vertically oriented short rods, whereas occludin was concentrated as dots only in the most apical region of these rods. A similar tricellular contact–specific concentration of tricellulin was observed in other epithelial cell lines such as mouse MTD1A cells, CSG1 cells, and dog MDCK II cells (Fig. 4 A). To confirm the specificity of the unique staining pattern with anti-tricellulin pAb, we then performed transfection experiments. When HA-tagged tricellulin was exogenously expressed in Eph4 cells followed by double staining with anti-HA pAb and anti-occludin mAb, HA-tagged tricellulin was predominantly recruited to the tricellular contacts, although it was also recruited in small amounts to bTJs (Fig. 4 B).

Bottom Line: In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts.When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized.These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

View Article: PubMed Central - PubMed

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

ABSTRACT
For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

Show MeSH