Supplementary Figure 6: Inhibition of rho-ROCK or myosin II activity increases cell branch complexity but has no effect on myosin II localization to the cortex.

(A) Cells were treated with blebbistatin, Y-27632, or C-3 and branch path complexity determined as described in Fig. 1. Note control and blebbistatin data is re-plotted from Fig. 1 here for comparison. (B) Comparison of drug treatments or mutations (R702C) on cortical localization of myosin IIA (left) compared to myosin IIB (right). Plot shows mean curvature as a function of myosin IIA or IIB intensity percentile. Solid lines indicate mean and dashed lines indicate bootstrapped 95% confidence intervals. (C) An AEC expressing myosin IIA-GFP (green) in a 3D collagen gel was fixed and phospho-serine-19 myosin II regulatory light chain was immunolocalized (purple). Maximal intensity projection of a 3D reconstruction, inset: Co-localization plot of fluorescence intensities. Bar equals 10 μm. (D–F) Computation of a cell center reference that is robust against cell morphological variations. (D) The Euclidean distance transform (blue) measures distance from the cell surface (grey). (E) The maximum of the distance transform (yellow) defines the centermost point in the cell volume (F), which deviates significantly from the actual centroid (blue). (G) Autocorrelation of cell center speed over time (blue line). Dashed line shows 99% confidence bounds. N = 8 cells for 240 time points.