Dual observation of the ATP-evoked small GTPase activation and Ca(2+) transient in astrocytes using a dark red fluorescent protein

Intracellular signal transduction involves a number of biochemical reactions, which largely consist of protein-protein interactions and protein conformational changes. Monitoring Förster resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy (FLIM), called FLIM-FRET, is one of the best ways to visualize such protein dynamics. Here, we attempted to apply dark red fluorescent proteins with significantly smaller quantum yields. Application of the dark mCherry mutants to single-molecule FRET sensors revealed that these dark mCherry mutants are a good acceptor in a pair with mRuby2. Because the FRET measurement between mRuby2 and dark mCherry requires only the red region of wavelengths, it facilitates dual observation with other signaling sensors such as genetically encoded Ca(2+) sensors. Taking advantage of this approach, we attempted dual observation of Ca(2+) and Rho GTPase (RhoA and Cdc42) activities in astrocytes and found that ATP triggers both RhoA and Cdc42 activation. In early phase, while Cdc42 activity is independent of Ca(2+) transient evoked by ATP, RhoA activity is Ca(2+) dependent. Moreover, the transient Ca(2+) upregulation triggers long-lasting Cdc42 and RhoA activities, thereby converting short-term Ca(2+) signaling to long-term signaling. Thus, the new FRET pair should be useful for dual observation of intracellular biochemical reactions.