Single-cell imaging of ERK and Akt activation dynamics and heterogeneity induced by G-protein-coupled receptors

Kinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters.ThekinaseswereactivatedwithGPCRligands.Weobserved ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast,Giwas necessary forERKand Akt activationin response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERKresponse dynamicstoS1Pshowedhighheterogeneity,whichwas reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.