Evidence for noisy oscillations of cAMP under nutritional stress condition in budding yeast

The Ras/cAMP/PKA pathway is one of the best studied signalling pathway in the budding yeast that regulates cellular responses to nutrients availability and stress. The cAMP levels and the PKA activity are also subjected to a strong negative feedback that operates either through the activity of the phosphodiesterase Pde1 and also on the amount of Ras2-GTP. We have previously made and simulated a dynamic model of the whole pathway and our results suggest the existence of stable oscillatory states that depend on the activity of the RasGEF (Cdc25) and RasGAP (Ira proteins) (Pescini et al. Biotechnol Adv 30, 99-107, 2012). Stochastic oscillations related to activity of the pathway were reported by looking at the nuclear localization of the trascription factors Msn2 and Msn4 (Gamedia-Torres et al. Curr Biol 17, 1044-9, 2007). In particular Medvedik et al. (PloS Biol 5, 2330-41, 2007) reported stable oscillations of the nuclear accumulation of Msn2 in condition of limited glucose availabiliy. We were able to reproduce the periodic accumulation of Msn2-GFP protein in yeast cells under condition of limiting glucose, and we tried to detect also in the same condition oscillations of cAMP levels in single yeast cells. We used a specific Fluorescence Resonance Energy Transfer (FRET) sensor based on a fusion protein between CFP-EPAC-YFP expressed in yeast cells. The FRET between CFP and YFP is controlled by cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in single yeast cell for a relative long time and a peak of cAMP was normally detected after addition of glucose to derepressed cells (Colombo et al. Biochem Biophys Res Commun 487, 594-99, 2017). Using this method we were able to detect noisy oscillations of cAMP levels in single yeast cells under condition of nutritional stress caused by limiting glucose availability (0.1%). We used Spectral analysis to discriminate between true oscillations and random noise. The oscillations were characterized by period of about 4-5 min, close to that observed for Msn2-GFP oscillations.