Fig. 3: Claudins form characteristic copolymers including segregation of channel-forming claudins from Cldn3.

a Scheme is illustrating predicted claudin-claudin organization patterns. Two claudins (yellow and magenta) that are compatible with each other tightly colocalize in the TJ (pink). Incompatible claudins separate into different strands or even larger claudin-specific parts. This separation might facilitate permeability of specific ions (cyan) over the TJ meshwork. b Representative STED images of TJ-like meshwork of the observed five organization patterns formed by the indicated co-expressed claudins (SNAP-tagged (yellow; BG-Atto590) and YFP-tagged (magenta; α-GFP-NB-Atto647N)) in fixed COS-7 cells are shown. Tight colocalization: intermixing of mesh-forming claudins, integration of non-mesh-forming with a mesh-forming claudin or induction (de novo mesh-forming of co-expressed non-mesh-forming claudins). Separated claudins: segregation and exclusion. c Pearson correlation analysis of Cldn3 co-expressed with barrier-forming (gray) or channel-forming (magenta) claudins. Co-expression of Cldn3 with Cldn3 (yellow) served as positive control. Data represent the mean ± SD. Every n represents the Pearson of one TJ-like meshwork. n(Cldn3 + Cldn3) = 55; n(Cldn3 + Cldn1) = 54; n(Cldn3 + Cldn5) = 20; n(Cldn3 + Cldn6) = 20; n(Cldn3 + Cldn7) = 17; n(Cldn3 + Cldn9) = 15; n(Cldn3 + Cldn14) = 19; n(Cldn3 + Cldn19b) = 25; n(Cldn3 + Cldn2) = 36; n(Cldn3 + Cldn10a) = 30; n(Cldn3 + Cldn10b) = 24; n(Cldn3 + Cldn15) = 42; from 3–6 independent experiments; one-way ANOVA with Dunnett’s multiple comparison test; ***P ≤ 0.001, ns (not significant). d Spectral FRET analysis of Trq2-Cldn3 alone (blank; negative control) or co-expressed with indicated YFP-tagged barrier Cldn3 (yellow; positive control) and channel-forming Cldn2 or Cldn15 (magenta) in HEK293 cells. Data represent the mean ± SD. Every n represents one cell–cell contact. n(negative control) = 57, from one experiment; n(Cldn3 + Cldn3) = 312; n(Cldn3 + Cldn2) = 162; n(Cldn3 + Cldn15) = 154; all from 4 to 5 independent experiments; one-way ANOVA with Dunnett’s multiple comparison test; ***P ≤ 0.001, ns (not significant). e Representative STED image of segregating SNAP-Cldn3 (yellow; BG-JF646) and Halo-Cldn15 (magenta; CA-Atto590) in living COS-7 cells. Image noise was removed for visualization using noise2Void⁷⁰. f Representative STED images of intermixing and segregation of SNAP-Cldn3 (yellow; BG-Atto590) with YFP-tagged Cldn1 and Cldn15 (both magenta; α-GFP-NB-Atto647N), respectively expressed in Caco-2 cells. g Representative STED images of the endogenous formed TJ in cryo-sections from mouse duodenum immunostained for Cldn3 (yellow; 2^nd-Atto647N) and Cldn15 (magenta; 2^nd-AF594). White arrows indicate regions in the TJ that shows the segregation of Cldn3 and Cldn15 strands. A Gaussian blur with a sigma of 20 nm was applied. All representative images derive from 3 independent experiments. Scale bars, 500 nm (g), 200 nm (b, e, f, magnification in g). Source data are provided as a Source data file.