Two novel red-FRET ERK bosensors in the 670-720 nm range.

Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central node in the control of cell proliferation and differentiation. ERK typically cooperates with a network of other regulators, making it necessary 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 utility for multiplexing. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using the fluorescent proteins 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. In both polyclonal and clonal populations, REKAR67- or REKAR76-expressing cells displayed similar Signal-to-Noise ratios (SNR). Overall, the red-FRET ERK biosensors were highly consistent with existing CFP/YFP biosensors in reporting ERK activity. Both REKAR biosensors expand the available tools for measuring single-cell ERK activity by being spectrally compatible with other CFP/YFP FRET and cpGFP -based biosensors, allowing for multiplexed imaging.