Two Novel Red-FRET ERK Biosensors in the 670-720nm Range

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.