Table 1.
Comparison of single FP-based indicators.
Figure 1.
Schematic representation of the domain structure of Flamindo2.
Citrine is a mutant of yellow fluorescent protein (YFP). cDNAs for yellow fluorescent cAMP indicators, Flamindo and Flamindo2, were created by insertion of DNA fragments encoding the cAMP-binding domain of mEPAC1 (199–358) and N- and C- terminus linker peptides including restriction sites. Nucleus-targeted Flamindo2 (nlsFlamindo2) was created by fusion of NLS (nuclear localization signal; MPKKKRKVEDVDP) to the N-terminus of Flamindo2.
Figure 2.
Spectral characterization of Flamindo2.
(A) Fluorescence spectra of Flamindo2 protein. Excitation and emission spectra were measured in the presence (dotted lines, +cAMP) or absence (solid lines, -cAMP) of 1 mM cAMP. Each fluorescence intensity (FI) was normalized to the peak of FI in the absence of cAMP. The representative excitation/emission spectra data from three independent experiments was shown in the graph. (B) Absorbance spectra of Flamindo2 protein in the presence (dotted line, +cAMP) or absence (solid line, -cAMP) of 1 mM cAMP. The representative absorption spectra data from three independent experiments was shown in the graph. (C) Titration curves of cAMP and cGMP. The peak of FI at each concentration of cAMP (closed circles) or cGMP (closed squares) was normalized to the peak of FI in the absence of cAMP or cGMP. The results are mean ± SD (n = 3) (D) Titration curves against pH. The peak of FI at each pH in the presence (closed circles) or absence (open circles) of 1 mM cAMP was normalized to the peak of FI at pH 9.0 in the absence of cAMP. The results are mean ± SD (n = 3).
Figure 3.
Live-cell imaging of cAMP in Flamindo2/nlsFlamindo2-expressing COS7 cells.
(A) Representative images showing changes in fluorescence intensity (FI) by 50 µM forskolin application in Flamindo2-expressing COS7 cells. (B) Time course of a normalized decrease in FI induced by reagents, which increase intracellular cAMP levels. Each reagent was applied at 2 min (dotted line). Three traces (black and gray lines) from single cells in three independent experiments are shown in all of the graphs. The black traces indicate representative data. (C) Representative images showing changes in FI by 10 mM bicarbonate application in nlsFlamindo2-expressing COS7 cells. (D) Different effects of bicarbonate and forskolin on cAMP dynamics in the nucleus. Bicarbonate or forskolin was applied at 2 min. Three traces (black and gray lines) from single cells in three independent experiments are shown in all of the graphs. The black trace indicates representative data.
Figure 4.
Dual-color imaging of cAMP and Ca2+ in Flamindo2 and R-GECO co-expressing HeLa cells.
(A) Images of changes in fluorescence of Flamindo2 and R-GECO induced by 100 µM noradrenaline. (B) Time course of changes in cAMP (solid line) and Ca2+ (dotted line) induced by 100 µM noradrenaline. Noradrenaline was applied at 120 s. Representative trace is shown in the graph.