Extended Data Fig. 5: Diffusive behavior of WRC subunits after LatA treatment.
a, Super-resolution intensity image of mEos2-Nap1 in the lamellipodium of a MEF treated with LatA (1 μM) (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-Abi1 in a MEF treated with LatA (1 μM). c, Same as a for mEos2-CAAX in a MEF treated with LatA (1 μM). d, Distribution of Log(D) at the Tip versus Outside (mean for cells). e, 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. f, 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-c are representative of 3, 5 and 2 independent experiments that yielded similar results. d-e, (for inside and outside), data pooled from independent experiments: mEos2-Nap1, n = 22 cells (pooled over 6 experiments); mEos2-Nap1 treated with 1 µM of LatA, n = 6 cells (over 3 experiments); mEos2-Abi1, n = 11 cells (over 3 experiments); mEos2-Abi1 treated with 1 µM of LatA, n = 9 cells (over 5 experiments); mEos2-CAAX, n = 7 cells (over 6 experiments) and mEos2-CAAX treated with 1 µM of LatA, n = 4 cells (over 2 experiments). k, Data pooled from cells in d-e, n is the number of trajectories: mEos2-Nap1, nTip = 2976, nOutside = 12493; mEos2-Nap1 treated with 1 µM of LatA, nTip = 399, nOutside = 667; mEos2-Abi1, nTip = 4183, nOutside = 7233; mEos2-Abi1 treated with 1 µM of LatA, nTip = 383, nOutside = 1191; mEos2-CAAX, nTip = 455, nOutside = 4605 and mEos2-CAAX treated with 1 µM of LatA, nTip = 314, nOutside = 1671. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (e) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (f). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (e,f). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (e,f). Also see Supplementary Table 1. Numerical source data are provided in Source data.
