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Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.

Furuse M, Furuse K, Sasaki H, Tsukita S - J. Cell Biol. (2001)

Bottom Line: Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease).Similar results were obtained in mouse epithelial cells, Eph4.These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

View Article: PubMed Central - PubMed

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

ABSTRACT
There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

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Claudins in MDCK I and II cells. (A) TER measurement of MDCK I and II clones used in this study. MDCK I or II cells (4 × 105 cells) were plated on 24-mm filters. In 6-d culture, the TER values of MDCK I and II cells reached the maximum levels, 12,992 ± 594 and 206 ± 35 Ωcm2, respectively (mean ± SD, n = 11). (B) Immunoblotting. Total lysates of MDCK I and II cells were separated by SDS-PAGE, followed by immunoblotting with pAbs for claudin-1, -2, and -4, and mAbs for occludin and ZO-1. In both MDCK I and II cells, claudin-1 and -4 were expressed, although their expression levels in MDCK II cells were significantly lower than those in MDCK I cells. Claudin-2 was expressed only in MDCK II cells. Occludin was expressed in larger amounts in MDCK I than MDCK II cells. (C) Immunofluorescence microscopy. In MDCK I cells, claudin-1, claudin-4, occludin, and ZO-1 were coconcentrated at TJs, where claudin-2 was undetectable. In contrast, in MDCK II cells, in addition to claudin-1, claudin-4, occludin, and ZO-1, claudin-2 was clearly detected at TJs. The claudin-4 signal was weak. Bar, 10 μm.
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Figure 1: Claudins in MDCK I and II cells. (A) TER measurement of MDCK I and II clones used in this study. MDCK I or II cells (4 × 105 cells) were plated on 24-mm filters. In 6-d culture, the TER values of MDCK I and II cells reached the maximum levels, 12,992 ± 594 and 206 ± 35 Ωcm2, respectively (mean ± SD, n = 11). (B) Immunoblotting. Total lysates of MDCK I and II cells were separated by SDS-PAGE, followed by immunoblotting with pAbs for claudin-1, -2, and -4, and mAbs for occludin and ZO-1. In both MDCK I and II cells, claudin-1 and -4 were expressed, although their expression levels in MDCK II cells were significantly lower than those in MDCK I cells. Claudin-2 was expressed only in MDCK II cells. Occludin was expressed in larger amounts in MDCK I than MDCK II cells. (C) Immunofluorescence microscopy. In MDCK I cells, claudin-1, claudin-4, occludin, and ZO-1 were coconcentrated at TJs, where claudin-2 was undetectable. In contrast, in MDCK II cells, in addition to claudin-1, claudin-4, occludin, and ZO-1, claudin-2 was clearly detected at TJs. The claudin-4 signal was weak. Bar, 10 μm.

Mentions: When MDCK I and II cells were grown on permeable filters for 6 d, confluent monolayers of MDCK I cells showed markedly higher TER values than those of MDCK II cells under the culture condition used in this study (Fig. 1 A). Furthermore, by measuring the flux of membrane-impermeable paracellular tracers (FITC-dextran, 40 K), it was confirmed that this TER disparity is not simply due to the disruption of the continuity of TJs (MDCK I, 1,157 ± 379 ng/ml; MDCK II, 3,826 ± 266 ng/ml; mean ± SD, n = 5). We then compared the expression of claudins between these two strains by immunoblotting and immunofluorescence microscopy. As reported previously (Sonoda et al. 1999), immunoblotting revealed that MDCK I cells primarily expressed claudin-1 and -4. In contrast, in MDCK II cells claudin-2 was detected abundantly in addition to claudin-1 and -4, the expression levels of which were significantly lower than those in MDCK I cells (Fig. 1 B). No differences were detected in the levels of ZO-1 expression between MDCK I and II cells, but occludin appeared to be expressed in larger amounts in MDCK I than in MDCK II cells.


Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.

Furuse M, Furuse K, Sasaki H, Tsukita S - J. Cell Biol. (2001)

Claudins in MDCK I and II cells. (A) TER measurement of MDCK I and II clones used in this study. MDCK I or II cells (4 × 105 cells) were plated on 24-mm filters. In 6-d culture, the TER values of MDCK I and II cells reached the maximum levels, 12,992 ± 594 and 206 ± 35 Ωcm2, respectively (mean ± SD, n = 11). (B) Immunoblotting. Total lysates of MDCK I and II cells were separated by SDS-PAGE, followed by immunoblotting with pAbs for claudin-1, -2, and -4, and mAbs for occludin and ZO-1. In both MDCK I and II cells, claudin-1 and -4 were expressed, although their expression levels in MDCK II cells were significantly lower than those in MDCK I cells. Claudin-2 was expressed only in MDCK II cells. Occludin was expressed in larger amounts in MDCK I than MDCK II cells. (C) Immunofluorescence microscopy. In MDCK I cells, claudin-1, claudin-4, occludin, and ZO-1 were coconcentrated at TJs, where claudin-2 was undetectable. In contrast, in MDCK II cells, in addition to claudin-1, claudin-4, occludin, and ZO-1, claudin-2 was clearly detected at TJs. The claudin-4 signal was weak. Bar, 10 μm.
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Figure 1: Claudins in MDCK I and II cells. (A) TER measurement of MDCK I and II clones used in this study. MDCK I or II cells (4 × 105 cells) were plated on 24-mm filters. In 6-d culture, the TER values of MDCK I and II cells reached the maximum levels, 12,992 ± 594 and 206 ± 35 Ωcm2, respectively (mean ± SD, n = 11). (B) Immunoblotting. Total lysates of MDCK I and II cells were separated by SDS-PAGE, followed by immunoblotting with pAbs for claudin-1, -2, and -4, and mAbs for occludin and ZO-1. In both MDCK I and II cells, claudin-1 and -4 were expressed, although their expression levels in MDCK II cells were significantly lower than those in MDCK I cells. Claudin-2 was expressed only in MDCK II cells. Occludin was expressed in larger amounts in MDCK I than MDCK II cells. (C) Immunofluorescence microscopy. In MDCK I cells, claudin-1, claudin-4, occludin, and ZO-1 were coconcentrated at TJs, where claudin-2 was undetectable. In contrast, in MDCK II cells, in addition to claudin-1, claudin-4, occludin, and ZO-1, claudin-2 was clearly detected at TJs. The claudin-4 signal was weak. Bar, 10 μm.
Mentions: When MDCK I and II cells were grown on permeable filters for 6 d, confluent monolayers of MDCK I cells showed markedly higher TER values than those of MDCK II cells under the culture condition used in this study (Fig. 1 A). Furthermore, by measuring the flux of membrane-impermeable paracellular tracers (FITC-dextran, 40 K), it was confirmed that this TER disparity is not simply due to the disruption of the continuity of TJs (MDCK I, 1,157 ± 379 ng/ml; MDCK II, 3,826 ± 266 ng/ml; mean ± SD, n = 5). We then compared the expression of claudins between these two strains by immunoblotting and immunofluorescence microscopy. As reported previously (Sonoda et al. 1999), immunoblotting revealed that MDCK I cells primarily expressed claudin-1 and -4. In contrast, in MDCK II cells claudin-2 was detected abundantly in addition to claudin-1 and -4, the expression levels of which were significantly lower than those in MDCK I cells (Fig. 1 B). No differences were detected in the levels of ZO-1 expression between MDCK I and II cells, but occludin appeared to be expressed in larger amounts in MDCK I than in MDCK II cells.

Bottom Line: Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease).Similar results were obtained in mouse epithelial cells, Eph4.These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

View Article: PubMed Central - PubMed

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

ABSTRACT
There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

Show MeSH
Related in: MedlinePlus