Extended Data Fig. 6: Diffusive behavior of Wave2-ΔWCA, FMNL2, Sra1-AD after LatA treatment.
a, Super-resolution intensity image of mEos2-Wave2-ΔWCA in a MEF lamellipodium obtained by sptPALM (50 Hz, duration: 150 s) (left) (inset: α-actinin-GFP fluorescence image). Trajectories are color-coded according to their diffusion modes: diffusive (gray), confined (yellow), immobile (red) (right). b, Same as a for mEos2-Wave2-ΔWCA in a MEF treated with LatA (1 μM). c, Same as a for mEos2-Sra1-AD in a MEF treated with LatA (1 μM). d, Same as a for FMNL2-mEos2 in a MEF treated with LatA (1 μM). e, Distribution of Log(D) at the Tip versus Outside (mean for cells). f, Fraction of diffusive, confined and immobile populations at the tip versus outside. Data are presented as mean ± s.e.m. for cells. Light, mid and dark shading refer to diffusive, confined and immobile populations, respectively. g, Diffusion coefficients (D) for free diffusive trajectories at Tip versus Outside, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). Images in a-d are representative of 3, 2, 4 and 3 independent experiments that yielded similar results. e-f, (for inside and outside), data pooled from independent experiments: mEos3.2-Sra1-AD, n = 7 cells (pooled over 4 experiments); mEos3.2-Sra1-AD treated with 1 µM of LatA, n = 7 cells (over 2 experiments); mEos2-Wave2, n = 15 cells (over 6 experiments); mEos2-Wave2-ΔWCA, n = 8 cells (over 3 experiments); mEos2-Wave2-ΔWCA treated with 1 µM of LatA, n = 4 cells (over 2 experiments); FMNL2-mEos2, n = 19 cells (over 5 experiments) and FMNL2-mEos2 treated with 1 µM of LatA, n = 6 cells (over 3 experiments). k, Data pooled from cells in i-j, n is the number of trajectories: mEos3.2-Sra1-AD, nTip = 398, nOutside = 2971; mEos3.2-Sra1-AD treated with 1 µM of LatA, nTip = 202, nOutside = 738; mEos2-Wave2, nTip = 1936, nOutside = 6885; mEos2-Wave2-ΔWCA, nTip = 1033, nOutside = 8281; mEos2-Wave2-ΔWCA treated with 1 µM of LatA, nTip = 69, nOutside = 255; FMNL2-mEos2, nTip = 3388, nOutside = 19567 and FMNL2-mEos2 treated with 1 µM of LatA, nTip = 743, nOutside = 3245. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (f) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (g). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (f,g). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (f,g). Also see Supplementary Table 1. Numerical source data are provided in Source data.
