Development of a red fluorescent protein-based cGMP indicator applicable for live-cell imaging.

Cyclic guanosine 3', 5'-monophosphate (cGMP) is a second messenger that regulates a variety of physiological processes. Here, we develop a red fluorescent protein-based cGMP indicator, "Red cGull". The fluorescence intensity of Red cGull increase more than sixfold in response to cGMP. The features of this indicator include an EC 50 of 0.33 μM for cGMP, an excitation and emission peak at 567 nm and 591 nm, respectively. Live-cell imaging analysis reveal the utility of Red cGull for dual-colour imaging and its ability to be used in conjunction with optogenetics tools. Using enteroendocrine cell lines, Red cGull detects an increase in cGMP following the application of L-arginine. An increase in intracellular cGMP is found to be inhibited by Ca 2+ , and L-arginine-mediated hormone secretion is not potentiated. We propose that Red cGull will facilitate future research in cell signalling in relation to cGMP and its interplay with other signalling molecules.