Parallel tracking of cAMP and PKA signaling dynamics in living cells with FRET-based fluorescent biosensors.

Proper regulation of cellular functions relies upon a network of intricately interwoven signaling cascades in which multiple components must be tightly coordinated both spatially and temporally. To better understand how this network operates within the cellular environment, it is important to define the parameters of various signaling activities and to reveal the characteristic activity structure of the signaling cascades. This task calls for molecular tools capable of parallelly tracking multiple activities in cellular time and space with high sensitivity and specificity. Here, we present new biosensors developed based on two conveniently co-imageable FRET pairs consisting of CFP-RFP and YFP-RFP, specifically Cerulean-mCherry and mVenus-mCherry, for parallel monitoring of PKA activity and cAMP dynamics in living cells. These biosensors provide orthogonal readouts in co-imaging experiments and display a comparable dynamic range to their cyan-yellow counterparts. Characterization of signaling responses induced by a panel of pathway activators using this co-imaging approach reveals distinct activity and kinetic patterns of cAMP and PKA dynamics arising from differential signal activation and processing. This technique is therefore useful for parallel monitoring of multiple signaling dynamics in single living cells and represents a promising approach towards a more precise characterization of the activity structure of the dynamic cellular signaling network.