Figure 1.
Construction of TALENs and ZO-1 gene knockout in MDCK I and II cells.
(A) TALEN binding sites in the ZO-1 gene. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK II cells transfected with TALEN constructs for ZO-1 gene knockout. After transfection, cells were subcultured on filter inserts for 4 d before analysis. At the boundary of control and ZO-1 knockout cells, characteristic convex curves of cell–cell junctions are observed (arrows). (C) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK I cells transfected with TALEN constructs for ZO-1 gene knockout. Similar morphological changes of cell–cell junctions at the boundary of control and ZO-1 knockout cells were observed in MDCK I cells. Staining of ZO-3 was reduced in ZO-1 knockout cells (arrowheads). Scale bars, 10 µm.
Figure 2.
Effects of ZO-1 knockout on the localization of ZO-2 and ZO-3.
(A) Effects of ZO-1 knockout on the localization of ZO-2 in MDCK II cells. The images in Figure 1 were used for the analysis. Signal intensity of ZO-2 and ZO-1 on lines shown in confocal microscopic image (arrows) were analyzed. ZO-2 fluorescent signal at TJs was reduced but was increased in the cytoplasm of ZO-1 knockout cells. (B) Effects of ZO-1 knockout on the localization of ZO-3 in MDCK II cells. ZO-3 fluorescent signal at TJs was slightly reduced but was increased in the cytoplasm of ZO-1 knockout cells. (C) Effects of ZO-1 knockout on the localization of ZO-2 in MDCK I cells. ZO-2 fluorescent signal was not altered in ZO-1 knockout MDCK I cells. (D) Effects of ZO-1 knockout on the localization of ZO-3 in MDCK I cells. ZO-3 fluorescent signal at TJs was markedly reduced in ZO-1 knockout MDCK I cells. Scale bar, 10 µm.
Figure 3.
Knockout of ZO-2 or ZO-3 in MDCK II cells.
(A) TALEN binding sites in ZO-2 and ZO-3 genes. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO proteins in MDCK II cells transfected with TALEN constructs for ZO-2 or ZO-3 gene knockout. The shape of cell–cell junctions was unchanged by ZO-2 knockout (upper panels) and ZO-3 knockout (lower panels). Scale bar, 10 µm.
Figure 4.
Establishment of ZO-1 knockout clones in MDCK II cells.
(A) Immunofluorescence microscopic analysis of ZO-1 in control (CTL) MDCK II cells and ZO-1 knockout clones (KO 1–3). ZO-1 staining was completely lost in ZO-1 knockout clones. Scale bar, 10 µm. (B) Immunoblots of ZO-1 and E-cadherin (E-cad) in control MDCK II cells and ZO-1 knockout clones. Knockout clones showed no detectable bands of ZO-1. (C) DNA sequences of TALEN targeting sites in each allele of ZO-1 knockout clones. One type of mutation was present in the alleles of ZO-1 knockout clone 1 (KO 1) and two types of mutations in the alleles of clones 2 and 3 (KO 2 and 3). Dashes indicate loss of nucleotides and green letters indicate additional nucleotides. Loss of initiating codon or frameshift were confirmed in all alleles. (D) Genomic PCR analysis of control and ZO-1 knockout clones using primers for TALENs and ZO-1 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs were detected in ZO-1 knockout clones.
Figure 5.
Effects of ZO-1 knockout on the shape of cell–cell junctions and cytoskeleton.
(A) Immunofluorescence microscopic analysis of ZO-1, ZO-3, F-actin and myosin heavy chain II-B (MHC-B) in control MDCK II cells and ZO-1 knockout clone. ZO-1 knockout induced striking changes in MHC-B organization. Scale bar, 10 µm. (B) Immunofluorescence microscopic analysis of MHC-B in control MDCK II cells and ZO-1 knockout clones. Similar punctate staining of MHC-B at cell–cell contacts was observed in all three ZO-1 knockout clones. Scale bar, 10 µm. (C) Immunofluorescence microscopic analysis of MHC-B, occludin and 1-phosphomyosin light chain (1p-MLC) at high magnification. Coimmunolocalization of MHC-B and occludin revealed punctate staining of MHC-B was arranged in two rows along the cell–cell junctions across the staining of occludin. 1p-MLC was concentrated at cell–cell contacts in ZO-1 knockout cells. Scale bar, 5 µm.
Figure 6.
Effects of ZO-1 knockout on the localization of ZO-2, ZO-3 and occludin in the stable ZO-1 knockout clones.
(A) Immunofluorescence microscopic analysis and line scanning of ZO-1 and ZO-2 in ZO-1 knockout clones 1 (KO 1) and 3 (KO 3) co-cultured with control cells. ZO-2 fluorescent signal at TJs was reduced in KO 1 but was slightly increased in KO 3. (B) Immunofluorescence microscopic analysis and line scanning of ZO-1 and ZO-3. ZO-3 signal at TJs was reduced but was increased in the cytoplasm in both of ZO-1 knockout clones compared to co-cultured control cells. (C) Immunofluorescence microscopic analysis and line scanning of ZO-1 and occludin. Occludin signal at TJs was reduced in both of ZO-1 knockout clones compared to co-cultured control cells. Scale bar, 10 µm. (D) Immunoblots of ZO-2, ZO-3 and occludin in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of ZO-2, ZO-3 and occludin were observed in control cells and knockout clones.
Figure 7.
Effects of blebbistatin on the shape of cell-cell junctions in ZO-1 knockout cells.
Effects of blebbistatin on the shape of cell-cell junctions in the co-culture of control and ZO-1 knockout cells. Cells were treated with 50 µM blebbistatin for 2 h and analyzed by immunofluorescence microscopy. Blebbistatin treatment attenuated the curves of cell-cell junctions at the boundary of the control and ZO-1 knockout cells, and the shape of cell-cell junctions in the control and ZO-1 knockout cells were almost indistinguishable in blebbistatin treated cells. Scale bar, 10 µm.
Figure 8.
Effects of the amount of ZO-1 expression on the shape of cell–cell junctions.
(A) Immunoblots of ZO-1 and E-cadherin in control MDCK II cells and ZO-1 knockout clones expressing exogenous ZO-1. FLAG-tagged ZO-1 was transfected into ZO-1 knockout cells, and clones expressing a nominal amount of ZO-1 (Lo 1 and 2) and an excessive amount of ZO-1 (Hi 1 and 2) were established. The bands of ZO-1 from Lo 1 and 2 clones were detected at very low levels in the enhanced image of immunoblots incubated with anti-ZO-1 antibody. (B) Immunofluorescence microscopic analysis of ZO-1 and ZO-3 in clones Lo 1 and Hi 1. Intensive zigzag shape of cell–cell junctions was observed in Hi 1 clone. Scale bar, 10 µm. (C) Effect of ZO-1 expression levels on the shape of cell–cell junctions. The degree of zigzag of cell–cell junctions was quantified in each clone and presented as zigzag index. The zigzag index was significantly lower in ZO-1 knockout clones and Lo 1 and 2 clones, and was significantly higher in Hi 1 and 2 clones compared with control cells. Data are shown as means ± S.E. N = 3–4 for each clone. *, p<0.05 compared with control. (D) Immunofluorescence microscopic analysis of ZO-1 and FLAG-tagged ZO-1. Control MDCK II cells and Hi 1 cells were co-cultured on filter inserts. Hi 1 cells expressing FLAG-tagged ZO-1 have an intensive zigzag cell–cell contact shape. Scale bar, 10 µm.
Figure 9.
Effect of ZO-1 expression levels on the cytoskeleton.
(A) Immunofluorescence microscopic analysis of F-actin and MHC-B in Lo 1 and Hi 1 clones. The organization of MHC-B is recovered in Lo 1 clone. The assembly of F-actin and MHC-B at cell–cell contacts was observed in the Hi 1 clone. Scale bar, 10 µm. (B and C) Immunofluorescence microscopic analysis of ZO-1, F-actin and MHC-B in the co-culture of control MDCK II cells and Hi 1 cells. Assemblies of F-actin and MHC-B at cell–cell contacts were observed in cells expressing an excess amount of ZO-1. Scale bar, 10 µm. (D) Immunofluorescence microscopic analysis of MHC-B, occludin and 1-pMLC at high magnification in Lo 1 and Hi 1 clones. Coimmunolocalization of MHC-B and occludin revealed an overlap of MHC-B and occludin staining. 1p-MLC was assembled at cell–cell contacts in Hi 1 cells. Scale bar, 5 µm.
Figure 10.
Time-lapse imaging of Venus-ZO-1 expressing MDCK II cells.
(A) ZO-1 and E-cadherin in control and Venus-ZO-1 expressing MDCK II cells. ZO-1 tagged with fluorescent protein Venus was transfected into control MDCK II cells, and stably expressing clones were established. Expression of excessive levels of Venus-ZO-1 was detected as a band with higher molecular weight in addition to endogenous ZO-1 in this clone. (B and C) Time lapse-imaging of Venus-ZO-1 expressing MDCK cells. Cells were cultured on the reverse side of filter inserts for 3 d, and time-lapse images were collected every 30 min for 24 h. The shape of cell–cell junctions showed gradually increasing degree of zigzag with continual changes in shape. Scale bar, 10 µm.
Figure 11.
The localization of claudins in ZO-1 knockout, Lo 1 and Hi 1 clones.
Immunofluorescence microscopic analysis of claudin-1, -2, -3, -4 and -7 in control cells and ZO-1 knockout, Lo 1 and Hi 1 clones. Claudin-2 staining at TJs was reduced but claudin-1 and claudin-7 staining at TJs were increased in ZO-1 knockout cells, and a nominal expression of ZO-1 (Lo 1) restored these changes. Claudin-3, -4 and -7 staining were similar in control, ZO-1 knockout, Lo 1 and Hi 1 cells. Scale bar, 10 µm.
Figure 12.
Effects of ZO-1 knockout on the localization and expression levels of claudins.
(A) Effects of ZO-1 knockout on the localization of claudins. Control and ZO-1 knockout cells of clone 1 were co-cultured on filter inserts. Signal intensity of claudins on lines shown in confocal microscopic images (arrows) were analyzed. Claudin-2 fluorescent signal at TJs was increased but claudin-1 and -7 signals at TJs were reduced in ZO-1 knockout cells. Scale bar, 10 µm. (B) Immunoblots of claudins in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of claudins were observed in control and knockout cells.
Figure 13.
Effects of ZO-1 knockout on the barrier properties of TJs.
(A) Time course of TER and TER values 6 days after the seeding in control cells and ZO-1 knockout and rescue clones. ZO-1 overexpressing clones (Hi 1 and 2) showed higher TER values on the next day of the seeding on filter inserts than other clones, but TER showed steady values after 4 days of seeding in all clones. TER values 6 days after the seeding was not changed in ZO-1 knockout clone 1 (KO 1) but increased in clones 2 (KO 2) and 3 (KO 3) compared with control cells. (B) Charge selectivity (the ratio of PNa and PCl: PNa/PCl) in control cells and ZO-1 knockout and rescue clones. PNa/PCl was similar in ZO-1 knockout clone 1 whereas were decreased clones 2 and 3. (C) PNa and PCl in control cells and ZO-1 knockout and rescue clones. PNa was decreased in ZO-1 knockout clones 2 and 3 and PCl was increased in clone 3. (D) The flux of 4 kDa FITC-dextran in control cells and ZO-1 knockout and rescue clones. ZO-1 knockout clone 1 showed small, but significant, increase in the flux of 4 kDa dextran compared with control cells. In contrast, the flux of 4 kDa dextran in clone 2 and 3 were more than 10 times higher than control cells. Data are shown as means ± S.D. N = 4 for each experiment.