Extended Data Fig. 9: Diffusive behavior of transmembrane-Sra1 versus controls.
a, Schematic representation of TM-Sra and the transmembrane control protein, TM-ctr. b, Super-resolution intensity image of TM-ctr 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). c, Same as b for TM-Sra1. 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%). d, e (for inside and outside), data pooled from independent experiments: mEos3.2-Sra1, n = 9 cells (pooled over 4 experiments); TM-Sra1, n = 9 cells (over 2 experiments) and mEos2-TM-ctr, n = 10 cells (over 2 experiments). f, Data pooled from cells in d-e and Fig. 5c,d, n is the number of trajectories: mEos2-Nap1, nTip = 2976, nOutside = 12493; TM-Nap1, nTip = 1820, nOutside = 6729; mEos3.2-Sra1, nTip = 921, nOutside = 3713; TM-Sra1, nTip = 629, nOutside = 3174 and mEos2-TM-ctr, nTip = 1893, nOutside = 16442. g, Western blotting of Nap1-deficient (Nap1-KO) and control B16-F1 cells expressing TM-Nap1 and using anti-Nap1 or anti-GFP antibodies, as indicated; tubulin expression was used as loading control. Blot in g is representative of 3 independent experiments that yielded similar results. h, Filamentous actin labeled with ATTO-594-labeled phalloidin in Sra1/PIR121-KO B16 cells (upper left and right). Arrowheads point to lamellipodia. GFP fluorescence images of TM-Sra1 (lower left) transfected into Sra1-KO B16-F1 melanoma cells or untransfected cells (lower right). i, Quantification of cells displaying lamellipodia in Sra1/PIR121-KO B16 cells transfected with TM-Sra1 (n = 80 cells pooled over 3 independent experiments) or untransfected (n = 55 cells pooled over 3 independent experiments) (mean ± s.e.m. for experiments). Images in b, c and h are representative of 2, 2 and 3 independent experiments that yielded similar results. Where indicated, statistical significances were obtained using two-tailed unpaired t-test for fractions of immobilization (e) or t-test for lamellipodia formation (i), 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. Unprocessed blot from g is available in the Source Data.
