Extended Data Fig. 7: Actin-based force transmission in primary T cells.
a, Scheme (top and side view) of cells migrating under agarose on a non-adhesive, topography-baring substrate. b, Tracks of migrating cells analysed in c–f. n = 18; mean cell speed = 6.58 μm/min; mean actin retrograde flow speed = 6.53 μm/min. Starting points of tracks are shifted to the origin. c, Mean speed of cells in relation to their mean actin retrograde flow speed (in relation to the substrate). Pearson's rank correlation coefficient r = −0.6484. A drop in retrograde flow with increasing locomotion speed demonstrates that, as in the transmembrane clutch paradigm of force transmission, actin slippage is inversely related to locomotion. d, Snapshots of actin flow analysis on ridged surfaces of cells expressing Lifeact-GFP. Left, cell core segmented with Ilastik (pink); the yellow arrow indicates cell direction, the blue arrow indicates the mean optical flow direction and the green arrow indicates actin retrograde flow. Middle, optical flow analysis (obtained with a customized MATLAB code). Right, actin single-spot tracking (TrackMate). Representative of three independent experiments. e, Angular distribution of actin retrograde flow and forwards locomotion of cells migrating on nanoridges. Only frames where optical flow analysis was confirming automatic particle tracking (deviation of <30°; see d) were subjected to analysis. The histogram on the left shows predominant retrograde actin flow when aligned along the ridges (90°). Retrograde actin flow declines towards 0° (perpendicular to the ridges), while forwards locomotion (right histogram) steadily increases, indicating that retrograde actin flow couples to topographical barriers and drives locomotion. n = 422 events of actin flow and cell movements obtained in n = 18 cells from three independent experiments. f, Frame-to-frame speed of cells migrating on nanoridges increases when forwards locomotion aligns perpendicular to topographical barriers (90° → 0° ≃ 15% increase). The orientation-dependent increase of cell speed is paralleled by a steady decrease of retrograde actin flow (90° → 0° ≃ 55%), indicating that retrograde actin flow couples to topographical barriers and drives locomotion. Frame-to-frame cell speed and actin retrograde flow speed were recorded in 18 cells from 3 independent experiments, and pooled to n = 412 (cell speed) and n = 422 (actin retrograde flow speed) events. The centre shows the mean, and both s.e.m. (solid lines) and s.d. (thick transparent lines) are shown. Before pooling, cell speed was normalized to the mean speed of the tracked cell, and actin retrograde flow speed was normalized to the mean actin retrograde flow speed.
