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First published online 28 September 2004
doi: 10.1242/jcs.01399

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Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

¹ Department of Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
² NCCR `Frontiers in Genetics', University of Geneva, 30 Quai Ernest-Ansermet, 1211 Genève 4, Switzerland
³ Department of Cell Physiology and Metabolism, CMU, University of Geneva, 1 Rue Michel Servet, 1211 Genève 4, Switzerland
⁴ Department of Biology, University of Padova, Via U. Bassi 58B, 35121 Padova, Italy

View larger version (38K): [in a new window]   Fig. 1. Targeting of cingulin alleles by homologous recombination in mouse ES cells. (A) Restriction maps of the cingulin genomic locus [wild-type (WT) allele] and the neomycin (Neo) and hygromycin (Hyg) targeting vectors. Putative exons 2-5 are represented by black boxes. A 1.98 kb KpnI-XbaI fragment beginning 775 bp upstream of the ATG translation start codon and ending 353 bp downstream of the 3' end of exon 2 was replaced by a neomycin-resistance cassette in the first targeted allele and by a hygromycin resistance cassette in the second targeted allele. In the targeting vectors, the antibiotic resistance cassettes were flanked on the 5' side by a 5.5 kb XhoI-KpnI intron fragment (left arm) and on the 3' side by a 2.4 kb XbaI-EcoRV fragment (right arm) containing intronic sequences and exons 3-5. Positions of the 5' and 3' probes, and the neomycin and hygromycin probes used for Southern-blot analyses are shown as thick lines. A, ApaI; E, EcoRI; E5, EcoRV; K, KpnI; Xb, XbaI; Xh, XhoI. (B-G) Southern-blot analysis. Restriction endonucleases used for digestions are indicated beneath each blot. Genotypes and probes are indicated above each blot. Sizes (kb) of hybridizing bands are indicated on the left.

View larger version (205K): [in a new window]   Fig. 3. Transmission electron microscopy reveals TJs in wild-type and mutant cystic EBs. Micrographs show junctional complexes between epithelial cells of wild-type (B), heterozygous (+/–) (A,C) and homozygous (–/–) (D) EBs. Images were obtained from cystic EBs sectioned through their centers in a near-equatorial position. The outer surface of the cyst is at the top of each frame. (A) Low-magnification micrograph showing an example of a cystic EB (+/–), comprising an outer layer of epithelial cells (corresponding to visceral endoderm, indicated by bracket) and an inner layer. The outer layer cells were polarized, with apical microvilli (mv) projecting towards the outer surface and phagocytic vesicles (ph) in the cytoplasm. The two layers were usually separated by an intercellular space and the inner layer was not consistently detected. The inner layer of cells is referred to in the literature as ectoderm (Doetschman et al., 1985; Ikeda et al., 1999; Saitou et al., 1998) or undifferentiated cells (Soudais et al., 1995). The same ultrastructure was observed in all wild-type and mutant EBs. (B-D) Higher-magnification micrographs showing the apico-lateral membranes of adjoining outer epithelial cells. Inserts show, at higher magnifications, the areas of junctional complexes. The typical morphology of TJs, with intimate membrane interconnections, is seen irrespective of the cells genotype (B-D, arrows). TJs are occasionally associated with desmosomes (De in D). Scale bars, 2 µm (A), 100 nm (B-D).

View larger version (81K): [in a new window]   Fig. 2. Levels of ZO-1, ZO-2, claudin-6, occludin and Lfc proteins are altered following cingulin mutation in EBs. Triton-soluble and Triton-insoluble fractions of wild-type (+/+), heterozygous (+/–) and homozygous (–/–) EBs (genotype and clone name indicated above each lane) were analysed by SDS-PAGE and western blotting using antibodies against the proteins indicated on the left. Anti--tubulin signal was used to normalize protein loading. Numbers on the right indicate size (kDa) of protein markers. FL, full-length cingulin (140 kDa); T, truncated cingulin (100 kDa). Densitometric analysis was performed to measure protein levels when marked differences were observed. The numbers below the cingulin, ZO-1, ZO-2, claudin-6 and occludin lanes represent the signal intensity (mean of at least three independent experiments, one of which is shown here) relative to an arbitrary level of 1, corresponding to a reference lane for each set. ND, not detected.

View larger version (128K): [in a new window]   Fig. 4. Freeze-fracture electron microscopy reveals assemblies of TJ fibrils in wild-type and mutant cystic EBs. (A) Low-magnification image of large portions of the plasma membrane of a cystic EB. TJ fibrils are seen arranged both as a continuous belt, separating the apical (am) and the basolateral (lm) membrane domains, and as spatially restricted focal arrays within the basolateral membrane. (B-D) Higher magnification views, showing similar TJ belts (three to five strands thick) in one wild-type (WT1) (B), one heterozygous (+/–) (1;166) (C) and one homozygous (–/–) (3;96) (D) EB clone. Similar results were obtained with other clones (Table 5). (E-G) Higher-magnification views, showing focal arrays of TJ fibrils, which segregate microdomains within the basolateral membrane in wild-type (E), heterozygous (+/–) (F) and homozygous (–/–) (G) EBs. The arrowheads point to microvilli. Scale bars, 0.5 µm (A), 225 nm (B-G).

View larger version (41K): [in a new window]   Fig. 5. Cingulin mutation does not influence the junctional localization of ZO-1, occludin or claudin-6, or the permeability of TJs to NHS-LC-Biotin in cystic EBs. (A-C) Immunofluorescence analysis of EBs that were fixed, sectioned and stained with antibodies against the antigens indicated above each set of images. Color coding corresponds to secondary antibodies fluorophores used (green, FITC-labeled anti-mouse antibody; red, Cy5-labeled anti-rabbit antibody), except in the case of the double cingulin-ZO-1 labeling, in which color was inverted (rabbit anti-cingulin antibody followed by Cy5-anti-rabbit antibody, and rat anti-ZO-1 antibody followed by FITC-anti-rat antibody were used). Monoclonal antibody against cingulin was used for co-localization with occludin (B). In `merge' images, the yellow color shows co-localization of proteins. Junctional cingulin labeling is not detected in homozygous (–/–) cystic EBs (A,B). (D) Biotin permeability assay. EBs were incubated in 1 mg ml–1 NHS-LC-Biotin, washed, fixed, sectioned and stained with FITC-conjugated avidin (green) and TRITC-phalloidin (red) to visualize NHS-LC-Biotin and actin filaments, respectively. Epithelial cells with functional TJs exclude NHS-LC-Biotin from intercellular spaces, resulting in labeling being restricted to apical surfaces. `Out' and `In' in the (+/+) merge image indicate spaces outside and inside the cavity of the EBs, respectively. Scale bar, 10 µm.