A novel single-color FRET biosensor for Rho-kinase activity reveals calcium-dependent activation of RhoA and ROCK

Area of Science:

• Cellular biology
• Molecular signaling
• Biochemistry

Background:

• Ras homolog family member A (RhoA) is a key regulator of cellular processes.
• RhoA's spatiotemporal activity is critical, but its downstream effectors' dynamics are not well understood.

Purpose of the Study:

• To develop and validate a novel biosensor for measuring Rho-associated kinase (ROCK) activity in live cells.
• To investigate the spatiotemporal regulation of ROCK activity in response to intracellular calcium changes.

Main Methods:

• Development of a single-color Förster Resonance Energy Transfer (FRET) biosensor, Rho-Kinase Activity Reporter (RhoKAR).
• Validation of RhoKAR specificity for ROCK activity.
• Measurement of RhoKAR activity in mouse fibroblasts under varying intracellular calcium conditions (ionomycin and EGTA).
• Assessment of signaling intermediates including calmodulin and CaMKII.

Main Results:

• The RhoKAR biosensor accurately reports ROCK activity with high spatiotemporal resolution and is specific to ROCK.
• Increased intracellular calcium levels significantly enhanced RhoKAR activity.
• Decreased intracellular calcium levels reduced RhoKAR activity.
• Calcium-dependent ROCK activation was found to be downstream of calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase II (CaMKII).

Conclusions:

• ROCK activity is modulated by intracellular calcium levels in fibroblasts.
• The CaM/CaMKII pathway mediates calcium-dependent ROCK activation.
• This study provides a novel tool for studying ROCK signaling dynamics in live cells.