Supplementary Figure 4

(a) Time lapse images of cells stably expressing GFP-tagged FL-Kank2 (green) and Paxillin-TagRFP (Pxn-TagRFP; red) during isotropic spreading on FN. Arrow heads indicate Kank2 recruitment to the proximal FA border. (b) Normalized Pxn-TagRFP intensities during FA disassembly behind lamella in cells expressing FL-Kank2 (red) or Kank2ΔKN (blue) (mean ± s.d.; n = 8 cells for FL-Kank2, and n = 10 cells for Kank2ΔKN pooled from 4 independent experiments). (c) FA disassembly rates calculated from (b) as rate constants in a one-phase exponential decay (mean ± s.d.). (d) Ten overlaid, sequential time lapse images of Vinculin-mCherry in the cell centre temporally colour-coded using spectrum look up tables (LUT). Corresponding time lapse images of GFP are shown as projection of average intensity. Scale bar, 5 μm. (e) Percentage of adhesion sites with sliding velocity above 0.3 μm min⁻¹ in indicated cell lines (mean ± s.d., n = 5 cells from 3 independent experiments; P values were calculated using one-way ANOVA Tukey test). (f) 2-D random migration of indicated cells on FN (box plot with median, 1st–3rd quantile box, minimal to maximal whiskers; shScr, n = 52 cells; shKank2#1, n = 58 cells; shKank2#2, n = 60 cells; data aggregated from 4 independent experiments; P values were calculated using Student t-test). (g) Cell spreading area of indicated cells at different time points (n ≥ 50 cells for each condition). (h) Percentage of adhesion sites with sliding velocity above 0.3 μm min⁻¹ in Kank2-depleted fibroblasts stably expressing Paxillin-TagRFP and Kank2-GFP on FN treated with or without 50 μM broad spectrum MMP inhibitor Gm6001 for 5h (mean ± s.d., n = 5 cells from 3 independent experiments).