Extended Data Fig. 8: Retrograde actin flow and shape changes in adhesion-free confinement.
Related to Fig. 4h. a–c, Primary T cells expressing Lifeact-GFP confined on PLL-PEG. n = 51 cells from three independent experiments: n = 22 cells for 100 nM CCL19, n = 16 cells for 10 nM CCL19 and n = 13 cells for 1 nM CCL19. a, Representative snapshots (right, t = 20, 25 and 30 s) and kymographs (left), of primary T cells expressing Lifeact-GFP confined on PLL-PEG, showing actin retrograde flow (red arrows). Scale bar, 5 μm. b, Representative snapshots (right, t = 20, 25 and 30 s) of the cell outline and the colour-coded curvature. Kymograph analysis (left) of the migrating T cell shows retrograde movement of cell body deformations (right, red arrowhead). c, Top, scheme of the travelling cell body deformations. Bottom, retrograde actin flow versus curvature flow velocities in primary T cells confined on an inert substrate and exposed to the indicated chemokine (CCL19) concentrations. n = 51 cells from three independent experiments: n = 22 cells for 100 nM CCL19, n = 16 cells for 10 nM CCL19 and n = 13 cells for 1 nM CCL19. Pearson's rank correlation coefficient r = 0.5542. d, e, Cell curvature versus cortical actin retrograde flow analysis in channels (obtained from three cells in three experiments). A scheme of the segmentation used to measure channel-dependent cell curvature and cortical actin retrograde flow velocity used in e is shown (d). Fluorescent images were binarized and the local curvature was calculated from a spline fit to the cell outline. Measurement of channel-dependent cell curvature versus cortical actin retrograde flow shows no correlation (Pearson's correlation coefficient c ≈ −0.006425) (e). The green line shows mean ± s.d.
