Extended Data Fig. 2: Diffusive behavior of WRC subunits at the lamellipodium tip and the membrane outside of the lamellipodium.
a, Super-resolution intensity image of mEos2-Abi1 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-Sra1. c, Same as a for mEos2-Wave2. d, Same as a for mEos2-Actin. e, Same as a for mEos2-CAAX. f, Same as a for cytosolic mEos2. g, Distribution of Log(D) at the Tip versus Outside (mean for cells). h, 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. i, 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-f are representative of 3, 3, 6, 8, 6 and 2 independent experiments that yielded similar results. g-h (for inside and outside), data pooled from independent experiments: mEos2-Nap1, n = 22 cells (pooled over 6 experiments); mEos2-Abi1, n = 11 cells (over 3 experiments); mEos2-Wave2, n = 15 cells (over 6 experiments); mEos2-Sra1, n = 11 cells (over 3 experiments) and cytosolic mEos2, n = 5 cells (over 2 experiments). i, Data pooled from cells in g-h and Fig. 1d,e, n is the number of trajectories: mEos2-ArpC5A, nTip = 1035, nOutside = 691; mEos2-Actin, nTip = 1948, nOutside = 6764; cytosolic mEos2, nTip = 26, nOutside = 111; mEos2-Nap1, nTip = 2976, nOutside = 12493; mEos2-Abi1, nTip = 4183, nOutside = 7233; mEos2-Wave2, nTip = 1936, nOutside = 6885; mEos2-Sra1, nTip = 1715, nOutside = 12564 and mEos2-CAAX, nTip = 455, nOutside = 4605. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (h) or non-parametric, two-tailed Mann-Whitney rank sum test for diffusion coefficient (i). Inside the lamellipodium tip and outside the lamellipodium, all the different conditions were compared (black P-values) (h-i). Inside the lamellipodium tip, each given condition was compared with the value obtained outside the lamellipodium (colored P-values) (h-i). Note that a significant fraction of the Wave2 protein displays a faster membrane free-diffusion which differs from all the other subunits of WRC that we tested in this study. This could correspond to a fraction of the Wave2 protein not incorporated in WRC12, which might diffuse as single proteins at the plasma membrane. Also see Supplementary Table 1. Numerical source data are provided in Source data.
