Fig. 4: Channel-forming claudins form segregated meshworks in non-polarized cells, epithelial cells and the murine proximal tubule.
From: Nanoscale segregation of channel and barrier claudins enables paracellular ion flux
a Representative STED image of TJ-like meshwork formed by SNAP-Cldn2 (yellow; BG-Atto590) co-expressed with YFP-tagged Cldn2, Cldn1, Cldn10a, Cldn10b, and Cldn15 (all magenta; α-GFP-NB-Atto647N) in COS-7 cells. b Pearson correlation analysis of Cldn2 co-expressed with Cldn2 (yellow), Cldn1 (gray) and Cldn10a, Cldn10b, Cldn15 (magenta). Data represent the mean ± SD. Every n represents the Pearson of one TJ-like meshwork. n(Cldn2 + Cldn2) = 21; n(Cldn2 + Cldn1) = 29; n(Cldn2 + Cldn10a) = 35; n(Cldn2 + Cldn10b) = 28; n(Cldn2 + Cldn15) = 22; from 3 to 5 independent experiments; one-way ANOVA with Dunnett’s multiple comparison test; ***P ≤ 0.001, ns (not significant). c Spectral FRET analysis of Trq2-Cldn2 (blank; negative control) or co-expressed with YFP-tagged Cldn2 (yellow; positive control) and Cldn10a (magenta) in HEK cells. Data represent the mean ± SD. Every n represents one cell–cell contact. n(negative control) = 126, from one experiment; n(Cldn2 + Cldn2) = 405; n(Cldn2 + Cldn10a) = 115; from 4 to 10 independent experiments; one-way ANOVA with Dunnett’s multiple comparison test; ***P ≤ 0.001, ns (not significant). d Representative STED image of segregating SNAP-Cldn2 (yellow; BG-JF646) and Halo-Cldn10a (magenta; CA-Atto590) in living COS-7 cells. Image noise was removed for visualization using noise2Void70. e Representative STED image of segregation of SNAP-Cldn2 (yellow; BG-Atto590) and YFP-Cldn10a (magenta; α-GFP-NB-Atto647N) localized in an overlap region of two transfected MDCKC7 cells. f Pearson correlation analysis of Cldn2 co-expressed with Cldn2 (yellow) and Cldn10a (magenta) in MDCKC7. Data represent the mean ± SD. Every n represents the Pearson of one TJ-like meshwork. n(Cldn2 + Cldn2) = 17; n(Cldn2 + Cldn10a) = 16; from 3 independent experiments; Mann-Whitney test, two-tailed; ****P ≤ 0.0001. g Representative STED image of the TJ in mouse proximal tubule immunostained for Cldn2 (yellow; 2nd-Atto647N) and Cldn10a (magenta; 2nd-AF594). The white rectangle indicates the area of the magnification. h Pearson correlation analysis of Cldn2 and Cldn10a in three mouse proximal tubules. Data represent the mean ± SD. Every n represents the Pearson of one STED image of the tubular TJ. n(Tubule I) = 9; n(Tubule II) = 12; n(Tubule III) = 11; one-way ANOVA Tukey’s multiple comparison test; ns (non-significant). i Strand length analysis of Cldn2 and Cldn10a strands in three proximal tubules (Tub I-III). Data represent the mean ± SD; n = 4, mean of the measured strands for each tubule; strands Tubule I: 541 (Cldn2)/664 (Cldn10a), strands Tubule II: 747 (Cldn2)/791 (Cldn10a), strands Tubule III: 693 (Cldn2)/828 (Cldn10a); one-way ANOVA with Tukey’s multiple comparison test; ***P ≤ 0.001, ns (non-significant). All representative images derive from 3 independent experiments. Scale bars, 500 nm (g), 200 nm (a, d, e, magnification in g). Source data are provided as a Source data file.
