Improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Abstract

Akt is a key node in a range of signal transduction cascades and plays a critical role in diseases such as cancer and diabetes. Fluorescently-tagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt. Here we observed that fusion of eGFP to Akt impaired both insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt over eGFP-Akt enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt in the presence of insulin with a consistent periodicity of 2 minutes. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.