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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.

Show MeSH
Structure and expression of mouse tricellulin. (A) Membrane folding models for mouse tricellulin and occludin. Tricellulin and occludin share two structural characteristics in addition to bearing four transmembrane domains: (1) The first extracellular loop has a high content of tyrosine and glycine residues (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). (2) The COOH-terminal (C) ∼130 amino acids are 32% identical (boxed in red; Fig. S1 C). It should be noted that tricellulin bears a longer NH2-terminal (N) cytoplasmic domain (187 amino acids) as compared with occludin (61 amino acids). (B) Northern blotting. Both tricellulin (Tric) and occludin (Occ) are expressed in large amounts in epithelium-derived tissues. rRNA, ribosomal RNA. (C) Immunoblotting of cultured mouse epithelial cells. The total lysate of Eph4 cells exogenously expressing mouse Snail (Eph4-mSnail; Ikenouchi et al., 2003), Eph4 cells, CSG1 cells, and MTD1A cells were separated by SDS-PAGE followed by immunoblotting with anti-tricellulin pAb. Tricellulin was detected as multiple bands in Eph4, CSG1, and MTD1A cells, but after alkaline phosphatase treatment, there was only a single, smaller Mr band (Eph4 + AP). In Eph4-mSnail cells, tricellulin is undetectable.
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fig2: Structure and expression of mouse tricellulin. (A) Membrane folding models for mouse tricellulin and occludin. Tricellulin and occludin share two structural characteristics in addition to bearing four transmembrane domains: (1) The first extracellular loop has a high content of tyrosine and glycine residues (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). (2) The COOH-terminal (C) ∼130 amino acids are 32% identical (boxed in red; Fig. S1 C). It should be noted that tricellulin bears a longer NH2-terminal (N) cytoplasmic domain (187 amino acids) as compared with occludin (61 amino acids). (B) Northern blotting. Both tricellulin (Tric) and occludin (Occ) are expressed in large amounts in epithelium-derived tissues. rRNA, ribosomal RNA. (C) Immunoblotting of cultured mouse epithelial cells. The total lysate of Eph4 cells exogenously expressing mouse Snail (Eph4-mSnail; Ikenouchi et al., 2003), Eph4 cells, CSG1 cells, and MTD1A cells were separated by SDS-PAGE followed by immunoblotting with anti-tricellulin pAb. Tricellulin was detected as multiple bands in Eph4, CSG1, and MTD1A cells, but after alkaline phosphatase treatment, there was only a single, smaller Mr band (Eph4 + AP). In Eph4-mSnail cells, tricellulin is undetectable.

Mentions: Full-length cDNA encoding tricellulin was isolated from a mouse kidney cDNA library and has an ORF encoding a 555–amino acid polypeptide (63.6 kD) with four predicted transmembrane domains (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). Immunofluorescence staining of cultured cells suggested that both the NH2 and COOH termini of tricellulin were located in the cytoplasm, as depicted in Fig. 2 A. Interestingly, the COOH-terminal sequence (∼130 amino acids) was conserved between tricellulin and occludin; in occludin, this region was reported to be remarkably conserved among various vertebrate species (Ando-Akatsuka et al., 1996) and to contain a domain responsible for its association with ZO-1, a major TJ plaque protein (Furuse et al., 1994). Northern analysis showed that tricellulin mRNA was expressed in large amounts in epithelium-derived tissues (Fig. 2 B). When the whole cell lysate of mouse epithelial cell lines (Eph4, CSG1, and MTD1A) was resolved by electrophoresis and immunoblotted with anti-tricellulin pAb, tricellulin migrated as multiple bands in the Mr range of 66–72 kD, and the band of the lowest Mr was predominant (Fig. 2 C). On alkaline phosphatase treatment of the lysate of Eph4 cells, these multiple bands converged into the lowest Mr band, suggesting that, similar to occludin (Sakakibara et al., 1997), tricellulin is phosphorylated within cells. Furthermore, it was confirmed also at the protein level that the expression of tricellulin was repressed completely by Snail.


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)

Structure and expression of mouse tricellulin. (A) Membrane folding models for mouse tricellulin and occludin. Tricellulin and occludin share two structural characteristics in addition to bearing four transmembrane domains: (1) The first extracellular loop has a high content of tyrosine and glycine residues (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). (2) The COOH-terminal (C) ∼130 amino acids are 32% identical (boxed in red; Fig. S1 C). It should be noted that tricellulin bears a longer NH2-terminal (N) cytoplasmic domain (187 amino acids) as compared with occludin (61 amino acids). (B) Northern blotting. Both tricellulin (Tric) and occludin (Occ) are expressed in large amounts in epithelium-derived tissues. rRNA, ribosomal RNA. (C) Immunoblotting of cultured mouse epithelial cells. The total lysate of Eph4 cells exogenously expressing mouse Snail (Eph4-mSnail; Ikenouchi et al., 2003), Eph4 cells, CSG1 cells, and MTD1A cells were separated by SDS-PAGE followed by immunoblotting with anti-tricellulin pAb. Tricellulin was detected as multiple bands in Eph4, CSG1, and MTD1A cells, but after alkaline phosphatase treatment, there was only a single, smaller Mr band (Eph4 + AP). In Eph4-mSnail cells, tricellulin is undetectable.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Structure and expression of mouse tricellulin. (A) Membrane folding models for mouse tricellulin and occludin. Tricellulin and occludin share two structural characteristics in addition to bearing four transmembrane domains: (1) The first extracellular loop has a high content of tyrosine and glycine residues (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). (2) The COOH-terminal (C) ∼130 amino acids are 32% identical (boxed in red; Fig. S1 C). It should be noted that tricellulin bears a longer NH2-terminal (N) cytoplasmic domain (187 amino acids) as compared with occludin (61 amino acids). (B) Northern blotting. Both tricellulin (Tric) and occludin (Occ) are expressed in large amounts in epithelium-derived tissues. rRNA, ribosomal RNA. (C) Immunoblotting of cultured mouse epithelial cells. The total lysate of Eph4 cells exogenously expressing mouse Snail (Eph4-mSnail; Ikenouchi et al., 2003), Eph4 cells, CSG1 cells, and MTD1A cells were separated by SDS-PAGE followed by immunoblotting with anti-tricellulin pAb. Tricellulin was detected as multiple bands in Eph4, CSG1, and MTD1A cells, but after alkaline phosphatase treatment, there was only a single, smaller Mr band (Eph4 + AP). In Eph4-mSnail cells, tricellulin is undetectable.
Mentions: Full-length cDNA encoding tricellulin was isolated from a mouse kidney cDNA library and has an ORF encoding a 555–amino acid polypeptide (63.6 kD) with four predicted transmembrane domains (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). Immunofluorescence staining of cultured cells suggested that both the NH2 and COOH termini of tricellulin were located in the cytoplasm, as depicted in Fig. 2 A. Interestingly, the COOH-terminal sequence (∼130 amino acids) was conserved between tricellulin and occludin; in occludin, this region was reported to be remarkably conserved among various vertebrate species (Ando-Akatsuka et al., 1996) and to contain a domain responsible for its association with ZO-1, a major TJ plaque protein (Furuse et al., 1994). Northern analysis showed that tricellulin mRNA was expressed in large amounts in epithelium-derived tissues (Fig. 2 B). When the whole cell lysate of mouse epithelial cell lines (Eph4, CSG1, and MTD1A) was resolved by electrophoresis and immunoblotted with anti-tricellulin pAb, tricellulin migrated as multiple bands in the Mr range of 66–72 kD, and the band of the lowest Mr was predominant (Fig. 2 C). On alkaline phosphatase treatment of the lysate of Eph4 cells, these multiple bands converged into the lowest Mr band, suggesting that, similar to occludin (Sakakibara et al., 1997), tricellulin is phosphorylated within cells. Furthermore, it was confirmed also at the protein level that the expression of tricellulin was repressed completely by Snail.

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