title: Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.
abstract: Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.
authors: Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore; Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
authors: Haruki
authors: H
authors: Odaka
authors: Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore.
authors: Satoshi
authors: S
authors: Arai
authors: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
authors: Takafumi
authors: T
authors: Inoue
authors: Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore.
authors: Tetsuya
authors: T
authors: Kitaguchi
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism*; D005456:Fluorescent Dyes; D015870:Gene Expression; D023061:Gene Order; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D010957:Plasmids / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000242:Cyclic AMP; D002118:Calcium