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Updated: May 6, 2026
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
Two Novel Red-FRET ERK Biosensors in the 670-720nm Range
Nicholaus L DeCuzzi1, Jason Y Hu1, Florene Xu1
1Department of Molecular and Cellular Biology, University of California, Davis.
Abstract:
Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central regulator. However, ERK is rarely the only signaling pathway involved, creating a need to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their ability to be multiplexed. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. Mixed populations of REKAR67 or REKAR76 displayed similar Signal-to-Noise ratio (SNR), but in clonal cell populations, REKAR76 had a significantly higher SNR. Overall, our red-FRET ERK biosensors were highly consistent with existing ERK FRET biosensors and in reporting ERK activity and are spectrally compatible with CFP/YFP FRET and cpGFP -based biosensors. Both REKAR biosensors expand the available methods for measuring single-cell ERK activity.
Insights
Researchers developed novel red-Fluorescence Resonance Energy Transfer (FRET) biosensors for Extracellular signal-Regulated Kinase (ERK) to enable simultaneous single-cell pathway analysis. These new tools overcome spectral overlap issues, improving multiplexing capabilities for studying cell fate decisions.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Cell fate decisions are governed by complex signaling networks, with Extracellular signal-Regulated Kinase (ERK) playing a pivotal role.
- Studying multiple signaling pathways simultaneously at the single-cell level is crucial due to pathway crosstalk.
- Existing fluorescent biosensors often exhibit spectral overlap, hindering multiplexed measurements.
Purpose of the Study:
- To develop novel red-FRET biosensors for ERK activity measurement.
- To address the spectral overlap limitations of current biosensors for multiplexed single-cell analysis.
- To enhance the capacity for simultaneous monitoring of ERK signaling and other pathways.
Main Methods:
- Development of two red-FRET ERK biosensors, REKAR67 and REKAR76, utilizing miRFP670nano3 and miRFP720 fluorophores operating in the 670-720 nm range.
- Characterization of biosensor performance, including dynamic range, signal variance, and Signal-to-Noise ratio (SNR) in mixed and clonal cell populations.
- Assessment of spectral compatibility with existing biosensor technologies like CFP/YFP FRET and cpGFP-based sensors.
Main Results:
- REKAR67 exhibited a higher dynamic range but also greater signal variance compared to REKAR76.
- REKAR76 demonstrated a significantly higher SNR in clonal cell populations.
- Both REKAR biosensors showed high consistency with existing ERK biosensors and were spectrally compatible with other FRET and cpGFP biosensors.
Conclusions:
- The novel red-FRET ERK biosensors, REKAR67 and REKAR76, provide advanced tools for single-cell ERK activity measurement.
- These biosensors overcome spectral limitations, facilitating multiplexed analysis of signaling networks.
- The developed biosensors expand the methodological options for studying ERK signaling dynamics in cellular processes.
