Figure 6. Contractility inhibition leads to Rac activation and Rac-dependent dissemination.

(A) Inhibition of ROCK does not impair TGFβ-induced dissemination. TGFβ-treated cells were pre-treated for 2 hrs with 10 μM Y27632 ROCK inhibitor and then submitted to CIA in the presence of the inhibitor. Selected time points from a representative experiment are shown. See Supplementary Movie S3 for entire video sequence. Yellow arrows indicate broad lamellipodia-like protrusions frequently observed in TGFβ-treated cells in presence of Y27632. Scale bar, 100 μm. (B) Quantification of disseminating cells. The number of TGFβ-treated cells per field that in 2 days reached a distance >130 μm from the starting monolayer were counted, in absence or in presence of 10 μM Y27632, from three experiments (3 to 8 fields counted per experiment). (C) Rac1 activity is necessary for TGFβ-induced dissemination upon ROCK inhibition. 10 μM Y27632 ROCK inhibitor and 50 μM NCS237666 Rac1 inhibitor were added to TGFβ-treated cells as indicated. Number of cells TGFβ-treated n = 163, TGFβ/Y27632 treated n = 134, TGFβ/Y27632/NCS237666 treated n = 47, TGFβ/NCS237666 treated n = 42 from at least two experiments. (D) ROCK inhibition increases Rac1 activity at cellular protrusions. TGFβ-treated cells were transfected with a plasmid expressing Raichu-Rac1 (KRasCter) biosensor, treated with or without 10 μM Y27632 and visualized live by FRET microscopy the day after. Representative cells are shown. Scale bar 20 μm. (E) Quantification of whole-cell Rac1 activity. Number of untreated cells n = 54, Y27632 treated cells n = 33, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.