Figure 6. Electron tomography of ultrastructural changes in lamellipodial actin networks.

(A) Transmission electron micrographs of representative wild-type (B16–F1) and EVM-KO cells showing distinct actin filament networks at the leading edge (left), and 2D projections of digital 3D tomograms showing either actin filament trajectories in green (middle), or barbed ends on grey filaments in red, and pointed ends in blue (right). Scale bar, 100 nm. (B) Quantification of filament length in wild-type and EVM-KO cells. ***p≤0.001 by Mann-Whitney U rank sum test. Whiskers indicate 10% and 90% confidence intervals. n indicates the number of filaments analyzed. Dashed black lines show median, red lines arithmetic mean. (C) Densities of filaments (upper panel), barbed and pointed ends (lower panel) in 106 nm-wide spatial bins throughout the lamellipodium. Error bars indicate SEM. 6 tomograms for each cell line were analyzed. (D) Histogram showing frequencies of filament angles to the leading edge (90° corresponding to filaments perpendicular to the leading edge). Dashed black lines are medians, and red lines arithmetic means.

Figure 6—video 1. Loss of Ena/VASP deteriorates lamellipodium architecture in B16-F1 cells, related to Figure 6.

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Animated visualization of the three-dimensional organization of lamellipodia of B16-F1 wild-type and EVM-KO cells resolved by electron tomography. A 3D model of the actin filament network corresponds to Figure 6A and was obtained by automatic tracking of filaments through the tomogram slices.