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

Abstract

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.

Fig. 1

Tracking of cAMP accumulation and PKA activity in living cells using CR-AKAR and YR-ICUE. (A) Schematic representation of the biosensors CR-AKAR and YR-ICUE. (B) Responses of HEK293T cells expressing CR-AKAR to 50 μM forskolin (Fsk) (n = 4, error bars represent standard deviation) and the pseudocolor images showing CR-AKAR response to Fsk in a HEK293T cell. Red fluorescence image (RFP) shows the distribution of the biosensor. (C) Responses of HEK293T cells expressing YR-ICUE to 50 μM Fsk (n = 4) and the pseudocolor images showing YR-ICUE response to Fsk in a HEK293T cell. Red fluorescence image (RFP) shows the distribution of the biosensor. Warmer color indicates an increase in PKA activity or cAMP level and cooler color indicates a decrease. Scale bar, 10 μm.

Fig. 2

Parallel tracking of CR-AKAR and YR-ICUE in HEK293T cells. (A) Responses of HEK293T cells co-expressing CR-AKAR and YR-ICUE treated with 50 μM Fsk followed by treatment with 10 μM H89 (n = 6). Fluorescence images showed distribution of biosensors in a single cell. (B) Responses of HEK293T cells co-expressing CR-AKAR and YR-ICUE treated with 50 μM Fsk in the presence of 20 μM H89 (n = 6). Fluorescence images showed distribution of biosensors in a single cell. Scale bar, 10 μm.

Fig. 3

Kinetic profiling of cAMP/PKA pathway agonists by parallel imaging of CR-AKAR and YR-ICUE responses. The response time courses of cAMP and PKA in HEK293T cells in response to (A) 50 μM Fsk (n = 9), (B) 10 μM prostaglandin E1 (PGE1) (n = 6), (C) 1 μM isoproterenol (ISO) (n = 6) and (D) 10 μM ritodrine (RITO) (n = 7). All data are presented as average ± STD unless otherwise noted.