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Claudin-2 knockout by TALEN-mediated gene targeting in MDCK cells: claudin-2 independently determines the leaky property of tight junctions in MDCK cells.

Tokuda S, Furuse M - PLoS ONE (2015)

Bottom Line: Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells.Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout.Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

ABSTRACT
Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000-4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

No MeSH data available.


Related in: MedlinePlus

Effects of claudin-2 knockout on the localization of other claudins.(A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 μm. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.
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pone.0119869.g004: Effects of claudin-2 knockout on the localization of other claudins.(A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 μm. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.

Mentions: Claudins are the major constituents of TJ strands and the expression pattern of claudins is thought to determine the permeability of TJs [7,8]. Because MDCK II cells express claudin-1, -3, -4, and -7 as well as claudin-2 [13,19], we investigated the effects of claudin-2 knockout on the localization and expression levels of the other claudins. Immunofluorescence analysis revealed that claudin-1 and -7 showed a tendency to be more clearly localized in cell-cell contacts at the TJ level in claudin-2 knockout cells compared with control cells (Fig. 3A). In contrast, the localization of ZO-1 (scaffold protein in TJs), occludin (integral membrane protein in TJs), F-actin, and myosin was similar between control and claudin-2 knockout cells (Fig. 3A). To confirm the effects of claudin-2 knockout on the localization of other claudins, claudin-2 knockout clones 1 and 2 were co-cultured with control cells, and observed by immunofluorescence microscopy (Fig. 4A and S4 Fig.). Claudin-1, -3, -4, and -7 had clearer and stronger signals at cell-cell contacts at the TJ level in claudin-2 knockout clones compared with control cells. In z-axis plane, the signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells (Fig. 5 and S5 Fig.). To evaluate the degree of claudin localization at TJs, the signal intensity of claudins overlapping with ZO-3 signals (scaffold protein in TJs) was quantified in control and claudin-2 knockout cells as described by Yu et al. [28]. The signal intensity of claudin-1, -3, -4, and -7 at TJs was significantly higher in claudin-2 knockout cells than in control cells (Fig. 4B). On the other hand, the protein expression levels of claudins other than claudin-2 were similar between control cells and claudin-2 knockout clones (Fig. 3B). These results indicate that claudin-2 knockout increases the localization of other claudins at TJs without a significant effect on protein expression levels.


Claudin-2 knockout by TALEN-mediated gene targeting in MDCK cells: claudin-2 independently determines the leaky property of tight junctions in MDCK cells.

Tokuda S, Furuse M - PLoS ONE (2015)

Effects of claudin-2 knockout on the localization of other claudins.(A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 μm. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363821&req=5

pone.0119869.g004: Effects of claudin-2 knockout on the localization of other claudins.(A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 μm. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in Materials and Methods, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * p < 0.05, ** p < 0.01 compared with control.
Mentions: Claudins are the major constituents of TJ strands and the expression pattern of claudins is thought to determine the permeability of TJs [7,8]. Because MDCK II cells express claudin-1, -3, -4, and -7 as well as claudin-2 [13,19], we investigated the effects of claudin-2 knockout on the localization and expression levels of the other claudins. Immunofluorescence analysis revealed that claudin-1 and -7 showed a tendency to be more clearly localized in cell-cell contacts at the TJ level in claudin-2 knockout cells compared with control cells (Fig. 3A). In contrast, the localization of ZO-1 (scaffold protein in TJs), occludin (integral membrane protein in TJs), F-actin, and myosin was similar between control and claudin-2 knockout cells (Fig. 3A). To confirm the effects of claudin-2 knockout on the localization of other claudins, claudin-2 knockout clones 1 and 2 were co-cultured with control cells, and observed by immunofluorescence microscopy (Fig. 4A and S4 Fig.). Claudin-1, -3, -4, and -7 had clearer and stronger signals at cell-cell contacts at the TJ level in claudin-2 knockout clones compared with control cells. In z-axis plane, the signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells (Fig. 5 and S5 Fig.). To evaluate the degree of claudin localization at TJs, the signal intensity of claudins overlapping with ZO-3 signals (scaffold protein in TJs) was quantified in control and claudin-2 knockout cells as described by Yu et al. [28]. The signal intensity of claudin-1, -3, -4, and -7 at TJs was significantly higher in claudin-2 knockout cells than in control cells (Fig. 4B). On the other hand, the protein expression levels of claudins other than claudin-2 were similar between control cells and claudin-2 knockout clones (Fig. 3B). These results indicate that claudin-2 knockout increases the localization of other claudins at TJs without a significant effect on protein expression levels.

Bottom Line: Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells.Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout.Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

ABSTRACT
Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000-4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

No MeSH data available.


Related in: MedlinePlus