biostudies-literatureMancini AENational Institutes of Health (NIH)NHLBI NIH HHSNational Institute of General Medical SciencesNIGMS NIH HHS6869https://www.ebi.ac.uk/biostudies/studies/S-EPMC11548655biostudies-literatureUnknown24(21)Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreaSensors (Basel, Switzerland)A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.PMC11548655R01HL1228275R01HL122827R01 HL122827T32GM008181T32 GM008181Mancini AERizzo MAfalseA Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decrea2024-01-01T00:00:00Z2024 Oct2026-07-12T03:21:14.461Z2025-04-03T23:23:38.068ZS-EPMC115486553951777010.3390/s24216869