Abstract
Collective cell migration (CCM) is a significant driver of cancer metastasis and invasion. Ras homolog family member A (RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the tight control over RhoA activity in space and time, it is unknown whether the downstream targets share similar activation patterns. To measure Rho-associated kinase (ROCK) activity in migrating cells, we developed a new FRET-based biosensor. Here we report the validation of a FLuorescence Anisotropy Reporter (FLARE)-type ROCK sensor. Activation of the ROCK-FLARE sensor was also specific to ROCK activity and insensitive to activation of the PKA pathway. We then assessed the mechanism of ROCK activation in collectively migrating fibroblasts. Increased cytoplasmic activated the ROCK sensor. Further, depletion of calcium levels with 100 µM EGTA decreased ROCK activity and impaired CCM. Inhibition of ROCK with Y27362 also blunted CCM behavior in fibroblasts. Thus, ROCK activity in collectively migrating fibroblasts is downstream of calcium release, likely from intracellular stores.
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