Figure 5.
CGN and CGNL1 are positioned between TJ and AJ scaffolding complexes, respectively, and NM2s. (A) Super-resolution STED analysis of ZO-1 (blue), CGN (green), and NM2B (red) in mCCD WT cells (merged in left panel, scale bar = 2 µm), and magnified (2.5×) images of rectangular inset detailing labeling for ZO-1 (blue), ZO-1+CGN, and merge image with arrow indicating linescan direction. (B) Linescan analysis with fluorescence intensities of the three channels as a function of distance from midline ZO-1 labeling. (C) Box plots showing measured distances of CGN (green) and NM2B (red) from midline (ZO-1 labeling; nm; n = 25 from two independent experiments). (D) IF microscopy analysis of the localization of the N- and C-termini (GFP-green and myc-red tags, respectively) of exogenous CGN (upper panels in D) and CGNL1 (bottom panels in D) and endogenous ZO-2 (blue) in double-KO (dKO) MDCK cells. High magnification panels correspond to highlighted white box in low magnification micrograph; scale bars = 10 μm (low magnification) and 1 μm (high magnification). (E and F) Linescan analysis of signal distribution (E) and box plots of distances of GFP and myc signal from ZO-2 signal (F; n = 24 from two independent experiments and data in quantifications are represented as mean ± SD). (G) IF microscopy analysis of the localization of the N- and C-termini (GFP-blue and myc-red tags, respectively) of exogenous CGN (upper panels in G) and CGNL1 (bottom panels in G) and endogenous NM2B (green) in double-KO MDCK cells. High magnification panels correspond to highlighted white box in low magnification micrograph; scale bars = 10 μm (low magnification) and 1 μm (high magnification). (H and I) Linescan analysis of signal distribution (H) and box plots of distances of NM2B and myc signal from GFP signal (I; n = 23 from two independent experiments and data in quantifications are represented as mean ± SD). (J–M) IB analysis, using anti-Flag tag antibodies, of preys (either Flag-NM2B or Flag-mCherry as negative control [J and L]) and third proteins (IB with anti-GFP in J and L), in trimolecular GST pulldowns using either GST (negative control) or GST-mZO-1CTerL (J) or GST (negative control) or GST-hPLEKHA7(351–820) (L) as baits, and either GFP or GFP-tagged full length or C-terminally truncated CGN (normalization in K), or either GFP-tagged full length or C-terminally truncated CGNL1 (normalization in M) as third proteins. Numbers on the left of IBs show migration of pre-stained markers. Baits are shown in Ponceau-S labeled blots. (N and O) Schematic models of the spatial organization and relative positions of the ZO-1–CGN–NM2B and PLEKHA7–CGNL1–NM2B complexes at TJ and AJ, respectively. The stoichiometries of interactions between the proteins of the complexes are not known. Source data are available for this figure: SourceData F5.