Small GTPases of the Rho family regulate vital cellular processes by controlling actin polymerization. As is the case for other signalling molecules, the effects of these GTPases are very focused in space and time. Wouldn't it be useful, then, to quantify their spatio–temporal dynamics of activation? Klaus Hahn and colleagues now report in Science how to do this.

The authors expressed Rac tagged with green-fluorescent protein (GFP), and microinjected at the same time PDB — a domain of PAK1 that specifically binds to active Rac-GTP — labelled with the dye Alexa. The sites of attachment of the fluorophores were chosen so that, when Rac and PDB interacted, GFP and Alexa would be close enough for fluorescence resonance energy transfer (FRET) to occur. With this experimental set-up, the authors could quantify in real time, in the same moving cell, the changing localization of Rac (GFP–Rac) and of Rac activation (FRET), and found them remarkably uncorrelated.

The method was called FLAIR, for 'fluorescence activation indicator for Rho proteins'. And, as the name says, there is no reason why it should not work for other GTPases of the Rho family, if an appropriate binding partner is chosen instead of PDB. Knowing the when and where of actin polymerization should open many doors for the study of actin-dependent processes, such as cell motility, shape generation or phagocytosis.