Extended Data Fig. 3: WRC and FMNL2 lateral movements are driven by actin filaments elongation.
a, Fluorescence image of a MEF lamellipodium labeled with α-actinin-GFP (left). Right, mEos2-Abi1 kymographs generated from a sptPALM time-lapse sequence (2 Hz) perpendicular to the edge (left panel, dashed lines). mEos2-Abi1 motions highlighted (white lines) and corresponding velocities (nm.s-1). b, Distributions of anterograde and retrograde perpendicular movements (duration > 3 s) for mEos2-ArpC5A (n = 535 movements, pooled over 8 cells from 5 independent experiments); mEos2-Nap1 (n = 161 movements, over 6 cells from 3 experiments) and mEos2-Abi1 (n = 143 movements, over 3 cells from 2 experiments). Fractions of anterograde and retrograde movements superior to displacements measured in fixed cells are shown in the plot. c, Fluorescence image of a MEF lamellipodium labeled with α-actinin-GFP (left). Scale bar, 3 µm. Right, mEos2-Abi1 kymographs generated from a sptPALM time-lapse sequence (2 Hz) tangential to the edge (left panel, dashed lines). mEos2-Abi1 motions highlighted (white lines) and corresponding velocities (nm.s-1). d, Same as c for mEos2-Wave2. e, Same as c for mEos2-Abi1 in a fixed MEF. f, Speed of lateral movements (duration > 3 s) for mEos2-Wave2, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). g, Dwell times of lateral movements (duration > 1.5 s) for mEos2-Wave2, box plots displaying median (notch) and mean (square) ± percentiles (25-75%). f, g, Data pooled from independent experiments: mEos2-Wave2, nSpeed = 165 movements, nDwell-Time = 303 movements (pooled over 5 cells from 3 experiments). h, α-actinin-GFP images of a spreading MEF before (left) and after (middle left) acute treatment with LatA (200 nM). Same for LatA (1 µM) (before: middle right, after: right). i, Same as h for CytoD (100 nM, 1 µM). j, Same as c for mEos2-Abi1 in a live MEF treated with LatA (1 μM). k, Same as c for mEos3.2-Sra1 treated with LatA (200 nM). l, Same as c for mEos3.2-Sra1 treated with CytoD (100 nM). m, Same as c for mEos3.2-Sra1-AD treated with LatA (1 µM). n, Distribution of left and right lateral movements (duration > 3 s) for mEos2-Nap1 (n = 263 movements, pooled over 5 cells from 3 independent experiments);mEos2-Nap1 LatA 1 µM (n = 639 movements, over 6 cells from 3 experiments); mEos2-Abi1 (n = 342 movements, over 3 cells from 2 experiments); mEos2-Abi1 LatA 1 µM (n = 210 movements, over 4 cells from 2 experiments); mEos3.2-Sra1 (n = 164 movements, over 5 cells from 2 experiments); mEos3.2-Sra1 LatA 200 nM (n = 275 movements, over 5 cells from 2 experiments); mEos3.2-Sra1 LatA 1 µM (n = 562 movements, over 5 cells from 3 experiments); mEos3.2-Sra1 CytoD 100 nM (n = 327 movements, over 6 cells from 3 experiments); mEos3.2-Sra1 CytoD 1 µM (n = 361 movements, over 6 cells from 3 experiments); mEos3.2-Sra1-WCA* (n = 471 movements, over 6 cells from 3 experiments); mEos3.2-Sra1-AD (n = 62 movements, over 6 cells from 4 experiments); mEos3.2-Sra1-AD LatA 1 µM (n = 145 movements, over 6 cells from 2 experiments); mEos2-Wave2 (n = 165 movements, over 5 cells from 3 experiments); mEos2-Wave2-ΔWCA (n = 270 movements, over 3 cells from 2 experiments); mEos2-Wave2-ΔWCA LatA 1 µM (n = 155 movements, over 3 cells from 2 experiments); FMNL2-mEos2 (n = 479 movements, over 5 cells from 2 experiments); FMNL2-mEos2 LatA 1 µM (n = 206 movements, over 3 cells from 1 experiments); FMNL2-A272E-mEos2 (n = 488 movements, over 4 cells from 2 experiments); FMNL2-I704A-mEos2 (n = 200 movements, over 6 cells from 3 experiments) and fixed mEos2-Abi1 (n = 103 movements, over 2 cells from 1 experiment). Images in a, c-e and h-m are representative of 2, 2, 3, 1, 3, 3, 2, 3, 2, 3 and 3 independent experiments that yielded similar results. Fractions of left and right movements superior to displacements measured in fixed cells are shown in the plot. All results for each condition correspond to pooled data from several independent experiments. Also see Supplementary Table 2. Numerical source data are provided in Source data.
