Fig. 5: Adhesion-independent durotaxis depends on retrograde actomyosin flow.

Still images and corresponding kymographs of Myosin-GPF Walker cells in channels with stiffness gradient (a) or uniform stiffness (b). Fluorescence signal shown as heatmap, dotted lines represent channel. Scale bars, 10 µm in still images, 5 µm (horizontal) and 10 min (vertical) in kymographs. c Still image and kymograph (red arrow) demonstrating retrograde myosin flow in migrating Walker cell. Scale bars, 10 µm in still image; 5 µm (horizontal) and 200 s (vertical) in kymograph. d Colour-coded projection of Lifeact-GPF Walker cells in uniform stiffness channels, in absence or presence of 30 µM Y-27632. Scale bars, 10 µm. e Normalised Myosin-GFP intensity along normalised front-rear axis in absence (black) or presence of 30 µM Y-27632 (purple). Solid lines with transparent areas depict mean ± s.d. of n = 6 cells from N = 3 independent experiments, each. f Blebbing vs non-blebbing cells under control or myosin inhibiting (30 µM Y-27632) conditions. Bars represent mean ± s.d. from N = 3 independent experiments. g Migration speed in channels of uniform stiffness. Box plot for n = 43 (control) and n = 47 (30 µM Y-27632) cells; two-tailed Mann–Whitney test; ****P ≤ 0.0001 (exact: P = 4.44e–16). Box bounds the IQR divided by the mean, and whiskers extend to a maximum of 1.5× IQR. h Colour-coded projections of Lifeact-GPF Walker cell in channel of uniform stiffness, showing cell reversal event. Scale bar, 10 µm. i Cell axis normalisation as shown in (k) and actomyosin intensity field (heatmap). Scale bar, 5 µm. j Actomyosin gradient strength versus cell speed. Data points obtained from individual frames of Supplement Video 8, as depicted in (i). k Actomyosin intensity peak along normalised cell axis (blue) versus cell displacement normalised over time (grey). Zero marks cell body centre in cell axis coordinate system. Positive values indicate forward movement, negative values reverse movement on displacement axis. Blue dashed line marks the time when the actomyosin peak crosses the cell axis centre, grey dashed line marks time when cell reverses direction. Source data are provided as Source Data file.