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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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Immunofluorescence localization of tricellulin. (A) Immunofluorescence staining of frozen sections of mouse small intestine, stomach, and kidney doubly with anti-tricellulin pAb (red) and anti-occludin mAb (green). Individual renal tubules in the kidney are represented as dotted lines. Tricellulin was predominantly concentrated at the tricellular contacts, where three occludin-positive bTJs converged. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. Bar, 20 μm. (B) Immunostaining of mouse Eph4 cells. Tricellulin (red) was concentrated at each tricellular contact, whereas occludin (green) was distributed along bTJs. The right bottom panel shows vertical sectional images generated from confocal microscopic images. Arrow and arrowheads represent bTJs and tTJs, respectively. White arrows are equivalent in length to the thickness of the cellular sheet. Bar, 10 μm.
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fig3: Immunofluorescence localization of tricellulin. (A) Immunofluorescence staining of frozen sections of mouse small intestine, stomach, and kidney doubly with anti-tricellulin pAb (red) and anti-occludin mAb (green). Individual renal tubules in the kidney are represented as dotted lines. Tricellulin was predominantly concentrated at the tricellular contacts, where three occludin-positive bTJs converged. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. Bar, 20 μm. (B) Immunostaining of mouse Eph4 cells. Tricellulin (red) was concentrated at each tricellular contact, whereas occludin (green) was distributed along bTJs. The right bottom panel shows vertical sectional images generated from confocal microscopic images. Arrow and arrowheads represent bTJs and tTJs, respectively. White arrows are equivalent in length to the thickness of the cellular sheet. Bar, 10 μm.

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)

Immunofluorescence localization of tricellulin. (A) Immunofluorescence staining of frozen sections of mouse small intestine, stomach, and kidney doubly with anti-tricellulin pAb (red) and anti-occludin mAb (green). Individual renal tubules in the kidney are represented as dotted lines. Tricellulin was predominantly concentrated at the tricellular contacts, where three occludin-positive bTJs converged. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. Bar, 20 μm. (B) Immunostaining of mouse Eph4 cells. Tricellulin (red) was concentrated at each tricellular contact, whereas occludin (green) was distributed along bTJs. The right bottom panel shows vertical sectional images generated from confocal microscopic images. Arrow and arrowheads represent bTJs and tTJs, respectively. White arrows are equivalent in length to the thickness of the cellular sheet. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171318&req=5

fig3: Immunofluorescence localization of tricellulin. (A) Immunofluorescence staining of frozen sections of mouse small intestine, stomach, and kidney doubly with anti-tricellulin pAb (red) and anti-occludin mAb (green). Individual renal tubules in the kidney are represented as dotted lines. Tricellulin was predominantly concentrated at the tricellular contacts, where three occludin-positive bTJs converged. There are also weaker signals at bTJs between two adjacent cells, especially in the small intestine. Bar, 20 μm. (B) Immunostaining of mouse Eph4 cells. Tricellulin (red) was concentrated at each tricellular contact, whereas occludin (green) was distributed along bTJs. The right bottom panel shows vertical sectional images generated from confocal microscopic images. Arrow and arrowheads represent bTJs and tTJs, respectively. White arrows are equivalent in length to the thickness of the cellular sheet. Bar, 10 μm.
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