Fig. 5: Claudin segregation is conserved in the extracellular loops and does not depend on cholesterol or the binding to ZO-1.
From: Nanoscale segregation of channel and barrier claudins enables paracellular ion flux
a Representative STED images, immunoblot analysis, strand length, and Pearson correlation analysis of TJ-like meshwork formed by SNAP-Cldn2 (yellow; BG-Atto590) and YFP-Cldn10a (magenta; α-GFP-NB-Atto647N) with different expression ratios (3:1, 1:3) in COS-7 cells. Blotting was performed against SNAP tag and YFP tag. HSP70 (70 kDa) served as loading control. For the strand and Pearson analysis the same TJ-meshworks were used. Data represent the mean ± SD. For the strand length analysis every n represents the mean of 40 strands of Cldn2 and Cldn10a per meshwork from 12 TJ-like meshworks (n = 12); one-way ANOVA Tukey’s multiple comparison test; ***P ≤ 0.0001, **P ≤ 0.01, ns (not significant); from 3 independent experiments; for the Pearson analysis every n represents one TJ-like meshwork (n = 12); Mann-Whitney test, two-tailed; ns (not significant) (P = 0.8623) from 3 independent experiments. b Representative STED images of control and cholesterol-depleted COS-7 cells expressing SNAP-Cldn2 (yellow; BG-Atto590) and YFP-Cldn10a (magenta; α-GFP-NB-Atto647N). White arrows point at claudin-containing vesicles that were increasingly formed under cholesterol depletion conditions. c Schematic representation of a claudin in the plasma membrane. The sites for the ECL exchange of Cldn2ECL10a and the PDZ- and C-term deletion mutants (ΔPDZ and ΔCT) are pointed out with red lines. d Representative STED images of COS-7 cells co-expressing SNAP-Cldn2ΔPDZ/-Cldn3ΔPDZ (yellow; BG-JF646) with YFP-Cldn10aΔPDZ/-Cldn15ΔPDZ (magenta; α-GFP 2nd-AF594) or SNAP-Cldn2ΔCT/-Cldn3ΔCT (yellow; BG-JF646) with YFP-Cldn10aΔCT/-Cldn15ΔCT (magenta; α-GFP 2nd-AF594). e Pearson analysis of co-expressed mutants from (d). Data represent the mean ± SD. Every n represents the Pearson of one TJ-like meshwork. n(Cldn10aΔPDZ + Cldn10aΔPDZ) = 27; n(Cldn2ΔPDZ + Cldn10aΔPDZ) = 15; n(Cldn10aΔCT + Cldn10aΔCT) = 15; n(Cldn2ΔCT + Cldn10aΔCT) = 15; n(Cldn15ΔPDZ + Cldn15ΔPDZ) = 16; n(Cldn3ΔPDZ + Cldn15ΔPDZ) = 16; n(Cldn15ΔCT + Cldn15ΔCT) = 15; n(Cldn3ΔCT + Cldn15ΔCT) = 16; from 3 independent experiments; one-way ANOVA Tukey’s multiple comparison test; ***P ≤ 0.001. f Representative STED images of COS-7 cells co-expressing cysteine mutant SNAP-Cldn2I66C (yellow; BG-Atto590) and YFP-Cldn10a (magenta; α-GFP-NB-Atto647N) in presence of channel blocking agent MTSET or DMSO as control. g Representative STED images of COS-7 cells co-expressing SNAP-Cldn3 or SNAP-Cldn10b (both yellow; BG-Atto590) with YFP-Cldn10bN48K (magenta; α-GFP-NB-Atto647N). h Representative STED images of co-expressed SNAP-Cldn2 (yellow; BG-JF646) with YFP-Cldn2 or YFP-Cldn10a (magenta; α-GFP 2nd-AF594) and SNAP-Cldn2ECL10a (yellow; BG-JF646) with YFP-Cldn2 or YFP-Cldn10a (magenta; α-GFP 2nd-AF594) in COS-7 cells. i Pearson analysis of co-expressed combinations from (h). Data represent the mean ± SD. Every n represents the Pearson of one TJ-like meshwork. n(Cldn2 + Cldn2) = 37; n(Cldn2 + Cldn10a) = 35; n(Cldn2ECL10a + Cldn10a) = 34; n(Cldn2ECL10a + Cldn2) = 29; from 4 independent experiments; one-way ANOVA with Dunnett’s multiple comparison test; ***P ≤ 0.001, ns (not significant). All representative images derive from 3 independent experiments. Scale bars, 200 nm (a, b, d, f–h). Source data are provided as a Source data file.
