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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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Stable suppression of tricellulin expression in Eph4 cells. (A) Immunoblot analysis using anti-tricellulin pAb. Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). Equal amounts of total proteins were analyzed by SDS-PAGE and immunoblotting. Tric, tricellulin. (B) Immunofluorescence staining of wild-type (WT) and tricellulin RNAi knockdown (KD-1 and KD-2) cells with anti-tricellulin pAb (red) and anti–E-cadherin mAb (green). Bar, 10 μm. (C) Immunofluorescence staining of WT, KD-1, and KD-2 cells with anti-occludin mAb. In KD-1 and KD-2 cells, both ends of occludin-positive bTJs showed characteristic teardrop-like shapes, and the tricellular contacts were disorganized with gaps (arrows in top panels; enlarged in bottom panels). Furthermore, in these cells, the bTJs were significantly thinner than those in wild-type Eph4 cells (arrowheads in top panels). Bars (top), 10 μm; (bottom) 3 μm. (D) TER measurements of WT, KD-1, and KD-2 cells. Tricellulin knockdown significantly affected the development of the epithelial barrier (n = 10 for each cell line). (E) FITC–dextran flux measurements of WT, KD-1, and KD-2 cells. There was a significant difference in the flux of 4-kD (but not 250 kD) FITC–dextran across monolayers between wild-type Eph4 cells and KD-1/2 cells (n = 10 for each cell line).
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fig5: Stable suppression of tricellulin expression in Eph4 cells. (A) Immunoblot analysis using anti-tricellulin pAb. Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). Equal amounts of total proteins were analyzed by SDS-PAGE and immunoblotting. Tric, tricellulin. (B) Immunofluorescence staining of wild-type (WT) and tricellulin RNAi knockdown (KD-1 and KD-2) cells with anti-tricellulin pAb (red) and anti–E-cadherin mAb (green). Bar, 10 μm. (C) Immunofluorescence staining of WT, KD-1, and KD-2 cells with anti-occludin mAb. In KD-1 and KD-2 cells, both ends of occludin-positive bTJs showed characteristic teardrop-like shapes, and the tricellular contacts were disorganized with gaps (arrows in top panels; enlarged in bottom panels). Furthermore, in these cells, the bTJs were significantly thinner than those in wild-type Eph4 cells (arrowheads in top panels). Bars (top), 10 μm; (bottom) 3 μm. (D) TER measurements of WT, KD-1, and KD-2 cells. Tricellulin knockdown significantly affected the development of the epithelial barrier (n = 10 for each cell line). (E) FITC–dextran flux measurements of WT, KD-1, and KD-2 cells. There was a significant difference in the flux of 4-kD (but not 250 kD) FITC–dextran across monolayers between wild-type Eph4 cells and KD-1/2 cells (n = 10 for each cell line).

Mentions: What happens to the paracellular barrier when the expression of tricellulin is suppressed in epithelial cells? Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). In both clones, tricellulin protein expression was suppressed by >95% as determined by Western blot analysis (Fig. 5 A), and immunofluorescence microscopy did not detect any tricellulin signals at tricellular contacts (Fig. 5 B). Under confluent conditions, KD-1 and KD-2 cells showed a typical cobblestone-like appearance, and there was no significant difference discerned between parental wild-type Eph4 and KD-1/2 cells with regard to the size/shape of individual cells and the distribution of cadherins (Fig. 5 B). However, when KD-1 and KD-2 cells were stained with anti-occludin mAb (Fig. 5 C) or anti–claudin-3 pAb (not depicted), tTJs as well as bTJs showed remarkable structural changes in that both ends of occludin-positive bTJs became thickened into a teardrop shape. At the tricellular contacts, three or more teardrop-shaped ends coalesced, but in most contacts, there remained gaps between these ends. These findings indicated that for a cellular sheet of KD-1/2 cells, the continuity of the TJ network was not maintained in the absence of tricellulin. Furthermore, as shown in Fig. 5 C, in KD-1/2 cells, bTJs themselves appeared to be poorly developed and appeared to be thinner and discontinuous as compared with those in wild-type Eph4 cells. These findings were confirmed by freeze-fracture replica electron microscopy (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). The epithelial barrier function was then compared between wild-type Eph4 and KD-1/2 cells by measuring transepithelial electric resistance (TER; Fig. 5 D) and the flux of membrane-impermeable paracellular tracers (FITC–dextran, 4 and 250 kD; Fig. 5 E). Clearly, when tricellulin was significantly reduced by RNAi, the epithelial barrier was compromised. The TER value did not increase under confluent conditions. Moreover, when challenged with 4 and 250 kD tracers, the paracellular barrier was severely affected in a size-selective manner. These findings have clearly demonstrated that the tricellular contacts play a crucial role in the epithelial barrier and that tricellulin is directly involved in this novel epithelial barrier mechanism.


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)

Stable suppression of tricellulin expression in Eph4 cells. (A) Immunoblot analysis using anti-tricellulin pAb. Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). Equal amounts of total proteins were analyzed by SDS-PAGE and immunoblotting. Tric, tricellulin. (B) Immunofluorescence staining of wild-type (WT) and tricellulin RNAi knockdown (KD-1 and KD-2) cells with anti-tricellulin pAb (red) and anti–E-cadherin mAb (green). Bar, 10 μm. (C) Immunofluorescence staining of WT, KD-1, and KD-2 cells with anti-occludin mAb. In KD-1 and KD-2 cells, both ends of occludin-positive bTJs showed characteristic teardrop-like shapes, and the tricellular contacts were disorganized with gaps (arrows in top panels; enlarged in bottom panels). Furthermore, in these cells, the bTJs were significantly thinner than those in wild-type Eph4 cells (arrowheads in top panels). Bars (top), 10 μm; (bottom) 3 μm. (D) TER measurements of WT, KD-1, and KD-2 cells. Tricellulin knockdown significantly affected the development of the epithelial barrier (n = 10 for each cell line). (E) FITC–dextran flux measurements of WT, KD-1, and KD-2 cells. There was a significant difference in the flux of 4-kD (but not 250 kD) FITC–dextran across monolayers between wild-type Eph4 cells and KD-1/2 cells (n = 10 for each cell line).
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Related In: Results  -  Collection

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fig5: Stable suppression of tricellulin expression in Eph4 cells. (A) Immunoblot analysis using anti-tricellulin pAb. Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). Equal amounts of total proteins were analyzed by SDS-PAGE and immunoblotting. Tric, tricellulin. (B) Immunofluorescence staining of wild-type (WT) and tricellulin RNAi knockdown (KD-1 and KD-2) cells with anti-tricellulin pAb (red) and anti–E-cadherin mAb (green). Bar, 10 μm. (C) Immunofluorescence staining of WT, KD-1, and KD-2 cells with anti-occludin mAb. In KD-1 and KD-2 cells, both ends of occludin-positive bTJs showed characteristic teardrop-like shapes, and the tricellular contacts were disorganized with gaps (arrows in top panels; enlarged in bottom panels). Furthermore, in these cells, the bTJs were significantly thinner than those in wild-type Eph4 cells (arrowheads in top panels). Bars (top), 10 μm; (bottom) 3 μm. (D) TER measurements of WT, KD-1, and KD-2 cells. Tricellulin knockdown significantly affected the development of the epithelial barrier (n = 10 for each cell line). (E) FITC–dextran flux measurements of WT, KD-1, and KD-2 cells. There was a significant difference in the flux of 4-kD (but not 250 kD) FITC–dextran across monolayers between wild-type Eph4 cells and KD-1/2 cells (n = 10 for each cell line).
Mentions: What happens to the paracellular barrier when the expression of tricellulin is suppressed in epithelial cells? Two independent Eph4 cell clones with suppressed tricellulin expression (KD-1 and KD-2) were established by stably expressing two distinct short interfering RNAs (Brummelkamp et al., 2002). In both clones, tricellulin protein expression was suppressed by >95% as determined by Western blot analysis (Fig. 5 A), and immunofluorescence microscopy did not detect any tricellulin signals at tricellular contacts (Fig. 5 B). Under confluent conditions, KD-1 and KD-2 cells showed a typical cobblestone-like appearance, and there was no significant difference discerned between parental wild-type Eph4 and KD-1/2 cells with regard to the size/shape of individual cells and the distribution of cadherins (Fig. 5 B). However, when KD-1 and KD-2 cells were stained with anti-occludin mAb (Fig. 5 C) or anti–claudin-3 pAb (not depicted), tTJs as well as bTJs showed remarkable structural changes in that both ends of occludin-positive bTJs became thickened into a teardrop shape. At the tricellular contacts, three or more teardrop-shaped ends coalesced, but in most contacts, there remained gaps between these ends. These findings indicated that for a cellular sheet of KD-1/2 cells, the continuity of the TJ network was not maintained in the absence of tricellulin. Furthermore, as shown in Fig. 5 C, in KD-1/2 cells, bTJs themselves appeared to be poorly developed and appeared to be thinner and discontinuous as compared with those in wild-type Eph4 cells. These findings were confirmed by freeze-fracture replica electron microscopy (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200510043/DC1). The epithelial barrier function was then compared between wild-type Eph4 and KD-1/2 cells by measuring transepithelial electric resistance (TER; Fig. 5 D) and the flux of membrane-impermeable paracellular tracers (FITC–dextran, 4 and 250 kD; Fig. 5 E). Clearly, when tricellulin was significantly reduced by RNAi, the epithelial barrier was compromised. The TER value did not increase under confluent conditions. Moreover, when challenged with 4 and 250 kD tracers, the paracellular barrier was severely affected in a size-selective manner. These findings have clearly demonstrated that the tricellular contacts play a crucial role in the epithelial barrier and that tricellulin is directly involved in this novel epithelial barrier mechanism.

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
Related in: MedlinePlus