A genetically encoded fluorescent sensor of ERK activity.

Area of Science:

• Molecular Biology
• Cell Signaling
• Neuroscience

Background:

• Extracellular signal-regulated kinase (ERK) activity exhibits complex spatiotemporal dynamics crucial for cellular function.
• Existing biochemical methods lack the resolution to measure ERK signaling with fine spatiotemporal detail.
• Understanding ERK dynamics is vital for deciphering specific downstream cellular effects.

Purpose of the Study:

• To develop a novel biosensor for measuring ERK activity with high spatiotemporal resolution.
• To validate the sensor's specificity and performance in various cellular contexts.
• To enable real-time monitoring of ERK signaling in living cells and neuronal tissues.

Main Methods:

• Genetically encoded, Förster Resonance Energy Transfer (FRET)-based sensor development (EKAR).
• Optimization for signal-to-noise ratio and fluorescence lifetime imaging.
• Validation in HEK293 cells with epidermal growth factor stimulation and in hippocampal neuron slices.

Main Results:

• EKAR selectively and reversibly reported ERK activation in HEK293 cells.
• EKAR signals correlated with ERK phosphorylation and required ERK activity, showing specificity against JNK and p38.
• EKAR successfully measured ERK activation in neuronal dendrites and nuclei in brain slices.

Conclusions:

• EKAR provides a powerful tool for measuring spatiotemporal ERK signaling dynamics.
• This sensor enables unprecedented insights into ERK function in living cells and complex neuronal environments.
• EKAR facilitates the study of ERK-mediated processes with high spatial and temporal precision.