Fig. 4: The α-catenin oligomers are adhesion- and myosin II-independent.

a Western blot (probed for GFP, marked as in Fig. 1e) of GFP-αCat-C805/871-expressing cells cross-linked after culturing in standard media (Ctrl), in low calcium media for 3 h (LowCa), in presence of function blocking E-cadherin antibody (SHE), in low calcium media or with SHE antibody in combination with LnB (LowCa+LnB, SHE+LnB), and as in lane LowCa+LnB but 15 min after LnB removal (LowCa-postLnB). b Western blot of GFP-αCatTCS-C805/871 cells cultured in control (Ctrl) or low calcium media (LowCa). The cells were processed with thrombin as in Fig. 3e. c Projections of all x-y optical slices of wt A431 cells stained before permeabilization for E-cadherin ectodomain (Ecad, red) and after permeabilization for F-actin (actin, green). Bar, 20 µm. The zoomed area (dashed box) of a single optical z slice passed through a middle of the cells is presented at the bottom. Bar, 10 µm. The area shown by the arrow is further magnified in the insets. The optical z-cross-sections along the dashed lines are shown on the right. Bar, 10 µm. Note clear co-localization between surface-exposed E-cadherin and F-actin. d Projections of all x-y slices of GFP-αCat-C805/871 cells stained for GFP (GFP, green) and for F-actin (actin, red). Only green channel is shown. Bar, 20 µm. The areas in dashed boxes are zoomed and shown in individual staining on the bottom. The arrows mark some of numerous actin-enriched AJs. Bar, 10 µm. e Western blot probed for GFP of GFP-αCatC805/871-expressing cells cross-linked after culturing in standard media (Ctrl), with Y-27632 (Y), blebbistatin (Bleb), CK666 in combination with Y-27632 (Y + CK) or blebbistatin (Bleb+CK). f, g The parallel cultures of cells shown in (d) were stained for GFP and for vinculin or FBLIM1. f Quantification of peak intensities of vinculin and FBLIM1 in AJs (n = 15). The means +/- SD are indicated by bars. Statistical significance was calculated using a two tailed Student’s t-test. ****P < 0.0001. g Vinculin and FBLIM1 staining of the cells used for quantifications present in (f). Only isolated cell-cell contacts are shown. Bar, 10 μm.