Extended Data Fig. 5: Rear actomyosin contraction is essential for collective durotaxis.
From: Collective durotaxis along a self-generated stiffness gradient in vivo
a, Neural crest in vivo expressing fluorescently tagged myosin light chain II (MLC) and LifeAct. Note that a supracellular actomyosin cable exists at the edge of the neural crest cell group in vivo. Arrowheads indicate the actomyosin cable. Scale bar is 25 μm (a). b, Kymograph of the edge of the neural crest cluster in vivo. Green represents MLC, which is absent from cell-cell contacts, and red represents LifeAct. Note the in vivo contraction of the actomyosin cable. Cell-cell contact contraction is indicated with the black arrowheads. c, Neural crest ex vivo expressing fluorescently tagged myosin (MLC, myosin light chain), LifeAct and membrane marker. Corresponding low magnifications are shown in Fig.4a. Scale bar is 25 μm (c). d, Two time points from the edge of a neural crest cluster on a stiffness gradient. Yellow arrowheads mark cell-cell contacts. Note the contraction of the actomyosin cable and reduction in length between cell contacts. Scale bar is 10 μm (d). e, Heat map of an example actomyosin contraction at the edge of clusters in control, durotaxis and chemotaxis. Time point zero represents the start of actomyosin contraction. f, Quantification of actomyosin contraction by cable length. Note that the amplitude of contraction is the same in all conditions. g, h, An illustrative diagram showing rear contraction and front movement of a cluster (circle outline) at three time points (g), and a histogram representing the time at which front movement occurs relative to rear contraction (h). The dashed line indicates the rear contraction time point reference, t = 0. i, j, Tactic index (i) and speed (j) of clusters on shallow stiffness gradient gels exposed to the myosin II inhibitor, blebbistatin. k-m, Pictures of membrane and merge of LifeAct and membrane for the example ablation shown in Fig. 3c–g (k, top, middle) or for cytoplasmic ablation (k, bottom). Yellow arrowheads indicate location of ablation. Scale bar is 10 μm (k). l, Migration of neural crest clusters on physiological stiffness gradients. The dashed line (start of ablations) separates before and during laser ablation of the cytoplasm. m, Tactic index of clusters before and during actomyosin cable ablation in the front or rear portion of migrating cell groups, or in the cytoplasm. Thick bars (f, i, j, l, m) represent mean; error bars (f, i, j, l, m) represent s.d.; unpaired two-tailed t-test (i), two-tailed Mann-Whitney U test (j), Dunn’s test (m); ns, P>0.05, *P≤0.05, ****P≤0.0001; n = 20 (f), 15 (h), 30 (i, j), 6 (l, m) clusters. Statistics and reproducibility are in the source data and Methods
