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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
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Two novel red-FRET ERK bosensors in the 670-720 nm range
Nicholaus L DeCuzzi1, Jason Y Hu1, Florene Xu1
1Department of Molecular and Cellular Biology, University of California Davis, CA, Davis, USA.
Journal of Biological Engineering
|November 13, 2025
Summary
Researchers developed novel red-FRET biosensors for Extracellular signal-Regulated Kinase (ERK) to enable simultaneous single-cell pathway analysis. These tools overcome spectral overlap issues, allowing for multiplexed imaging of cell signaling networks.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell fate decisions rely on complex signaling networks, with Extracellular signal-Regulated Kinase (ERK) playing a key role in cell proliferation and differentiation.
- Simultaneous single-cell analysis of multiple signaling pathways is crucial due to the interconnected nature of cellular regulation.
- Existing fluorescent biosensors often exhibit spectral overlap, hindering multiplexed imaging and simultaneous pathway analysis.
Purpose of the Study:
- To develop novel red-FRET biosensors for ERK activity measurement.
- To overcome spectral limitations of existing biosensors for multiplexed single-cell imaging.
- To provide new tools for studying ERK signaling in conjunction with other cellular pathways.
Main Methods:
- Development of two red-FRET ERK biosensors, REKAR67 and REKAR76, utilizing miRFP670nano3 and miRFP720 fluorescent proteins operating in the 670-720 nm range.
- Characterization of biosensor performance, including dynamic range, signal variance, and Signal-to-Noise ratio (SNR).
- Comparison of red-FRET biosensor activity with established CFP/YFP biosensors in both polyclonal and clonal cell populations.
Main Results:
- REKAR67 and REKAR76 biosensors successfully measured ERK activity in the red spectrum.
- REKAR67 showed a higher dynamic range, while REKAR76 exhibited lower signal variance.
- Both biosensors demonstrated high consistency with CFP/YFP biosensors and comparable SNR, indicating reliable ERK activity reporting.
- The red-FRET biosensors are spectrally compatible with CFP/YFP FRET and cpGFP-based biosensors.
Conclusions:
- The novel red-FRET ERK biosensors (REKAR67 and REKAR76) provide a valuable tool for single-cell ERK activity measurement.
- These biosensors enable multiplexed imaging by minimizing spectral overlap with existing biosensor systems.
- The developed tools expand capabilities for dissecting intricate cell signaling networks and understanding cell fate decisions.
