A Novel Single-Color FRET Sensor for Rho-Kinase Reveals Calcium-Dependent Activation of RhoA and ROCK.

Area of Science:

• Cell Biology
• Molecular Signaling

Background:

• Ras homolog family member A (RhoA) is crucial for cellular processes like cytoskeletal dynamics and migration.
• The spatiotemporal control of RhoA activity is well-established, but its downstream effectors' dynamics remain unclear.

Purpose of the Study:

• To develop and validate a novel biosensor for measuring Rho-associated kinase (ROCK) activity in real-time within live cells.
• To investigate the mechanisms regulating ROCK activation in mouse fibroblasts, particularly in response to intracellular calcium changes.

Main Methods:

• Development of a novel single-color Förster Resonance Energy Transfer (FRET) biosensor, Rho-Kinase Activity Reporter (RhoKAR).
• Validation of RhoKAR specificity for ROCK activity and its insensitivity to Protein Kinase A (PKA) activity.
• Assessment of calcium-dependent ROCK activation in mouse fibroblasts using ionomycin and EGTA treatments, and evaluation of signaling intermediates like calmodulin and CaMKII.

Main Results:

• The RhoKAR biosensor accurately measures ROCK activity with high spatiotemporal resolution in live cells.
• Increased intracellular calcium levels (via ionomycin) enhanced RhoKAR activity, while calcium depletion (via EGTA) decreased it.
• Blocking calmodulin or CaMKII inhibited calcium-dependent ROCK activation, indicating their role as upstream mediators.

Conclusions:

• ROCK activity is significantly increased by intracellular calcium in fibroblasts.
• Calcium-dependent ROCK activation occurs downstream of calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase II (CaMKII).
• The RhoKAR biosensor provides a valuable tool for studying ROCK signaling dynamics in cellular processes.