Fig. 2: Kindlin-2 undergoes lateral free diffusion along the plasma membrane.

Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2 (a) and talin-1-tdEos (b) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of talin-1-tdEos (yellow) and mEos2‐kindlin-2 (blue) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s⁻¹ corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of mEos2‐kindlin-2 (blue) and talin-1-tdEos (yellow) undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Super-resolution PALM intensity image of mEos2-kindlin-2 (f) and talin-1-tdEos (g) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. h Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in f and g. i Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs for kindlin-2 and talin-1. j Distribution of rearward speed (mean ± s.d. for cells) for kindlin-2 and talin-1. k Fraction of rearward speed equal or superior to 2 nm.s⁻¹ (mean ± s.d. for cells) for kindlin-2 and talin-1. Corresponding data points are overlaid on the bar chart as dot plots. Results for mEos2‐kindlin-2 (a–e: 13 cells; f–k: 1127 immobilization events in 12 cells) correspond to pooled data from three independent experiments. Results for β1-WT-mEos2 (dark gray) and β3-WT-mEos2 (light gray) corresponds to data shown in Fig. 1 and Supplementary Fig. 1. Results for talin-1-tdEos (a–e: 8 cells; f–k: 584 immobilization events in 7 cells) correspond to pooled data from two independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2 condition Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.