Extended Data Fig. 3: Clusters lack front-back polarity during migration.

a. Montage from time-lapse of a myosin light chain (MLC)-GFP expressing A431 cell cluster. MLC-GFP intensity was measured in the peripheral cortical region (segmentation in magenta) around the perimeter of the cluster. See also Supplementary Video 3. At each time point, the relative cortical intensity was measured for different angles around the perimeter (blue bars) as well as the cluster trajectory (red arrow). Lower panel: The relative cortical intensity of myosin (mean ± SD), averaged over all time points for n=39 clusters, N=3 independent experiments. Scale bar: 25μm. p-value reflects Rayleigh test of uniformity. b. Fixed cell clusters plated on a collagen network and immunostained for Rac1 GTPase with DAPI (DNA) and Phalloidin (F-Actin). Shown are two representative examples from n=21 clusters and N=3 independent experiments. Scale bar: 25μm. c.Upper panels: Single time point from a live imaging experiment using Raichu-Rac1 showing YFP:CFP ratio. Middle panels: Micrographs of YFP:CFP ratio for two successive time points with segmentation of the cortical region (magenta lines). Lower panel: The cortical YFP:CFP ratio for Raichu-Rac1 (mean ± SD) with respect to the migration direction (red arrow), averaged over all time points for n=8 clusters, N=3 independent experiments. Scale bar: 25μm. d. Boxplots of mean instantaneous speed and coefficient of persistence for cluster migration on collagen network conditions with the Rac1 inhibitor NSC23766. Data represents n=114, 57, 37, 26, 65, 23, 24 clusters, N=6, 3, 2, 1, 3, 1, 1 independent experiments. Control data is the same data as presented in Fig. 1f. For left panel, one-way ANOVA p=9.58e-9. *p < 0.05, ***p < 0.001 for Tukey HSD post-hoc test (p=0.243, < 0.001, 0.893, 0.0858, 0.0508, 0.1155, 0.00143, 0.430, 0.00304, 0.00205, 0.00844, < 0.001, < 0.001, < 0.001, < 0.001, 0.0949, 0.0103, 0.0497, 0.238, 0.552, 0.549).