The Ser/Thr kinase Akt is implicated in many cellular processes, such as cell growth, metabolism, cell survival and apoptosis. A critical event for Akt activation is its recruitment to the plasma membrane and subsequent phosphorylation. The recruitment of Akt is strongly promoted by growth factor stimulation. Previous work relied on GFP-tagged fusion proteins to study plasma membrane localisation and activation of Akt. In this issue (p. 2757), James Burchfield and colleagues describe an improved Akt reporter for which Akt was fused to a monomeric photostable RFP variant (TagRFP-T). The authors noticed that plasma membrane recruitment upon insulin stimulation and phosphorylation of Akt2 are impaired when using a GFP-based fusion protein. In contrast, TagRFP-T−Akt2 localises to the plasma membrane in a way that is similar to that of the endogenous protein; moreover, it is insulin-responsive and highly phosphorylated. Interestingly, the authors noticed certain subcellular properties of Akt2 when using the TagRFP-T tag, i.e. the protein localises in a polarised fashion and, every 2 minutes, self-organising oscillations at the plasma membrane were observed in adipocytes. In summary, this work demonstrates the importance of choosing the right fluorophore that recapitulates the endogenous behaviour of a protein, and uncovers details on the subcellular recruitment of Akt signalling kinases.
