Figure 10.

CGNL1 binding to NM2s is required to prevent the fragmentation of AJ protein complexes into discrete puncta. (A and B) IF microscopy analysis of the localization of PLEKHA7 (A) and profile of labeling intensity of PLEKHA7 (B) of the localization of PLEKHA7 in mixed cultures of WT and CGNL1-KO Eph4 cells. Insets on the right of images in A show high magnification details of areas outlined in square boxes. White arrows indicate linear junctional labeling. White arrowheads indicate gaps in junctional labeling. Yellow arrows indicate PLEKHA7 puncta in CGNL1-KO cells. (C–G) U-ExM analysis (C–E) and measured distances (F and G) of the localization of PLEKHA7 (green) and E-cadherin (red) in Eph4 WT cells (C) or CGNL1-KO (D and E) cells. Insets on the right of images in C and D show high magnification details of areas outlined in square boxes. Yellow arrows indicate punctate PLEKHA7/E-cadherin signal. (E) High magnification of area outlined in the inset of D showing the different distances/length quantified in F. (F) Quantification of the inter-puncta distance (red), gap between two puncta (light blue), and length of the puncta (purple) as depicted in E. Dots show replicates (n = 82–100) and bars represent mean ± SD. (G) Comparison of inter-puncta distances calculated on conventional and U-ExM images. Dots show replicates (n = 82–131) and bars represent mean ± SD. (H and I) IF microscopy analysis of the localization of PLEKHA7 (green), NM2A (red), and ZO-1 (white) in expanded (U-ExM) Eph4 WT (H) or CGNL1-KO (I) cells. Insets on the right of images show high magnification details of areas outlined in square boxes (double color channel, indicated on the right). (J–L′) IF microscopy analysis and quantification of PLEKHA7 dots in CGNL1-KO Eph4 cells transfected with GFP-tagged full-length (FL) CGNL1 (J and J′), CGNL1 lacking the NM2-binding site (Δ881-1297; K and K′) or GFP alone (L and L′). Insets on the left of images show high magnification details of areas outlined in square boxes. Asterisks indicate transfected cells and yellow arrows indicate punctate PLEKHA7 signal. (J′–L′) Dots show replicates (n = 64–132) and bars represent mean ± SD. (M and N) IF microscopy analysis (M) and quantification of number of PLEKHA7 puncta (N) in CGNL1-KO Eph4 cells, either untreated (control) or treated with solvent (DMSO) or treated with drugs that affect the contractility and polymerization of the actomyosin cytoskeleton. Note that control and DMSO-treated cells show punctate PLEKHA7 labeling, (yellow arrows) which is attenuated after treatment with the drugs. Dots show replicates (n = 41–180) and bars represent mean ± SD. Unless otherwise indicated on the micrographs (A, C–E, and H–M), scale bar = 10 µm. Data in J′–L′ and N are represented as mean ± SD. Statistical significance was determined by unpaired Mann–Whitney test (J′–L′) and Kruskal–Wallis test followed by Dunn’s multiple comparison (N), *P < 0.05, ****P < 0.0001.