Fig. 2: α-catenin undergoes a conformational change during AJ maturation.

a Representative FLIM images of differentiated MEKs expressing the force-insensitive F7-NF-based sensor (F7-NF-697). Note the increased fluorescence lifetimes in MA. b Quantification of live-cell FLIM measurements of differentiated MEKs expressing F7-NF-697. PA and MA are characterized by significantly different FRET efficiencies (N = 6 replicates, n = 105, 104 cells). c Live-cell FLIM measurements of differentiated MEKs stably expressing F7-NF-697. Cells were treated with either scrambled (ctrl) or an α-catenin targeting siRNA (siRNA), and classified into PA and MA (N = 3 replicates, n = 45, 45, 45, 45 cells). d FLIM data of differentiated MEKs co-expressing intermolecular FRET controls with either inactivated donor or acceptor chromophore (N = 3 replicates, n = 31, 29 cells). e MEKs expressing α-catenin expression constructs with C-terminal fusions of FL-TS or F7-NF display similar FRET efficiencies (N = 3 replicates, n = 45, 45 cells). f Representative anisotropy images of differentiated MEKs expressing the force-insensitive F7-NF-based sensor in which the acceptor was inactivated by a point mutation (F7-NF-697*). g Mean time-resolved fluorescence anisotropy shown with the standard error of the mean (s.e.m.); data are categorized into PA (green) and MA (blue). h, i Quantification of live-cell time-resolved fluorescence anisotropy imaging of differentiated MEKs expressing F7-NF-697*. PA and MA are characterized by different starting (h) and residual anisotropies (i). j The depolarization time is slightly reduced in MA but overall similar to PA. (N = 3 replicates, n = 45, 32 cells). k Representative images of fixed and immunostained MDCK-II cells; merged images display signals from the f-actin and α-catenin channels. l Representative FLIM images of MDCK-IIs expressing the force-insensitive F7-NF-based sensor (F7-NF-697). m Live-cell FLIM measurements of MDCK-II cells showing comparable variation in F7-NF-697 FRET efficiencies as seen in pooled MEK dataset (N = 6 replicates, n = 90, 90 cells). Boxplots show the median, the 25th and 75th percentiles and whiskers reaching to the last data point within 1.5× interquartile range. Scale bar: 10 μm. Two-sample KS test: ***p < 0.001, **p < 0.01, *p < 0.05, n.s. (not significant) p ≥ 0.05.