An Improved Akt Reporter Reveals Intra- And Inter-Cellular Heterogeneity and Oscillations in Signal Transduction

J Cell Sci. 2017 Aug 15;130(16):2757-2766. doi: 10.1242/jcs.205369. Epub 2017 Jun 29.

Abstract

Akt is a key node in a range of signal transduction cascades and play 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 proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its 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-Akt2 over eGFP-Akt2 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 Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. 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.This article has an associated First Person interview with the first author of the paper.

Keywords: Adipocyte; Akt; Insulin; Oscillation; Recruitment; Signalling.

MeSH terms

  • 3T3-L1 Cells
  • Animals
  • Genes, Reporter*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mice
  • Molecular Imaging / methods
  • Molecular Imaging / standards
  • Protein Stability
  • Protein Transport
  • Proto-Oncogene Proteins c-akt / genetics*
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / genetics
  • Tissue Distribution

Substances

  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • AKT2 protein, human
  • Proto-Oncogene Proteins c-akt