Fig. 3

Actin flow dynamics, orientation, and relation to GFP-LFA-1 anisotropy in migrating T cells. a Representative frames of structured illumination microscopy movies (above) of the actin cytoskeleton visualized with lifeact-mNeonGreen in Jurkat T cells migrating on ICAM-1 (10 µg/ml), anti-CD43 IgG (10 µg/ml), or their mixture (10 µg/ml each). Optical actin flow vector maps of the same cells are shown below with zoom insets of representative areas. Vectors encode flow direction by color (circular keys in the direction from center of circle to perimeter) and velocity by length. Scale bars encode dimensions in the micrograph (white, 5 µm) and velocity (yellow, 30 nm/s). b Actin flow direction relative to tangent of leading edge membrane. Bins of 15° are shown with Gaussian fit and mean ± SEM. ICAM-1, N = 62; αCD43, N = 63; ICAM-1 + αCD43, N = 67. ROI (N) came from six to eight cells. c Leading edge actin flow velocity from optical flow analysis. Plots show data on each cell. Bars show mean ± s.d. Two-tailed Mann–Whitney tests all show p < 0.0001 (****). ICAM-1, N = 39; αCD43 N = 38; and ICAM-1 + αCD43, N = 25. d, e Jurkat T cells migrating on ICAM-1 (10 µg/ml) expressing αL-T-GFP were treated with DMSO as control, blebbistatin (100 µM), or cytochalasin D (100 nM) and fixed. d Whole-cell emission anisotropy analyzed as in Fig. 2e with error bars showing SEM of three independent experiments. Mann–Whitney test for DMSO (N = 37) vs. blebbistatin (N = 9) was 0.130 and for DMSO vs. cytochalasin D (N = 14) was 0.003. **p < 0.01. e Representative total fluorescence intensity (I _|| + 2I _⊥, left) and anisotropy (right). Scale bars: 5 µm