Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: GEF-H1 and ZO-1 colocalisation after stimulation is cingulin dependent. (A) Cingulin-overexpressing (CGN-GFP) and control cells (GFP) were stained for ZO-1 (grey) and GEF-H1 (red) after stimulation with histamine (10 μg/ml) for 15 min. Nuclei were stained with DAPI (blue). Arrows highlight junctional localisation of GEF-H1. Scale bars: 10 μm. (B) The colocalisation of ZO-1 and GEF-H1 was quantified by determining junctional correlation coefficients. (C) Top, CGN-GFP (green) cells were stained for GEF-H1 (red) after 15 min of histamine (10 μg/ml) stimulation. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Immunofluorescence intensity was quantified along the white arrow. Middle, magnified view of indicated area in the top images. Scale bars: 2 μm. Bottom, immunofluorescence intensity graphs are shown for cingulin (green) and GEF-H1 (red) for the position denoted by the arrow. (D) Corresponding junctional correlation coefficients indicate the colocalisation of cingulin and GEF-H1 in CGN-GFP cells after 15 min stimulation with histamine (10 μg/ml), VEGF-A (50 ng/ml) or thrombin (0.5 U/ml). Data are presented as the mean±s.d. * P <0.05, ** P <0.01, *** P ≤0.001 (one-way ANOVA with Tukey's post-hoc test).

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Staining, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: The junctional colocalisation of GEF-H1 and cingulin after stimulation is dependent on S131, S134, and S149. (A) Left, cingulin (green) and GEF-H1 (red) are shown in CGN-overexpressing cells (CGN-GFP) and phospho-dead (CGN mut S>A) and phosphomimetic (CGN mut S>D) cingulin mutant cells after 15 min of histamine (10 μg/ml) stimulation. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. Middle, magnified view of indicated area in the images on the left. Scale bars: 2 μm. Right, Immunofluorescence intensity graphs for cingulin (green) and GEF-H1 (red) are shown for the position denoted by the white arrow. (B) Corresponding junctional correlation coefficients indicate the colocalisation of cingulin and GEF-H1 after 15 min stimulation. Data are presented as the mean±s.d. ( n =7–10). ** P <0.01, *** P ≤0.001 (one-way ANOVA with Tukey's post-hoc test).

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Mutagenesis, Staining, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells

doi: 10.1242/jcs.258557

Figure Lengend Snippet: Cingulin and GEF-H1 colocalise in inflamed small blood vessels of human skin samples. (A) Healthy and inflamed vessels of human skin tissues were stained for cingulin (green) and GEF-H1 (red). Nuclei were stained with DAPI (blue). Scale bars: 5 μm. (B) Immunofluorescence intensity graphs are shown for cingulin (green) and GEF-H1 (red) for the position denoted by the white arrow. (C) The colocalisation of cingulin and GEF-H1 was quantified based on the corresponding correlation coefficients. Datapoints for the same patient are denoted by use of the same colour ( n =12). Data are presented as the mean±s.d. *** P ≤0.001 (Student's t -test). (D) Schematic diagram of the proposed mechanism of the protective effect of cingulin (created using BioRender.com). AMPK-mediated phosphorylation of cingulin regulates its colocalisation with GEF-H1. Binding between GEF-H1 and cingulin leads to decreased MLC2 phosphorylation and reduced vascular permeability.

Article Snippet: The following primary antibodies were used: anti-cingulin (HPA027586 at 1:2000 dilution for immunofluorescence and HPA027657 at 1:500 for western blot; Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin (A2228, 1:5000; Sigma-Aldrich), anti-ZO-1 (610966, 1:100; BD Biosciences, San Jose, CA, USA), anti-ppMLC2 (Thr18/Ser19) (3674, 1:1000), anti-MLC2 (3672, 1:1000), anti-pAMPKα (Thr172) (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-phospho-AMPK Substrate Motif [LXRXX(pS/pT)] (5759, 1:1000; all from Cell Signaling Technology, Danvers, MA, USA), anti-GEF-H1 (HM2152, 1:100; Hycult, Uden, Netherlands), anti-VWF (A0082, 1:500; Dako, Glostrup, Denmark), anti-VE-cadherin (IM1597, clone TEA 1/31, Beckman Coulter Immunotech, Marseille, France) anti-PLVAP (RDI-PRO10705, clone PAL-E, 1:10; Research Diagnostics Inc., Flanders, NJ, USA), Alexa Fluor 488-conjugated anti-claudin-5 (352588, 1:100; Thermo Fischer Scientific, Waltham, MA, USA) and Alexa Fluor 647-conjugated anti-podoplanin (337008, 1:100; Biolegend, San Diego, CA, USA).

Techniques: Staining, Immunofluorescence, Binding Assay, Permeability