Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various F？rster 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.
References
[1]
Sunahara RK, Taussig R (2002) Isoforms of mammalian adenylyl cyclase: multiplicities of signaling. Mol Interv 2: 168–184. doi: 10.1124/mi.2.3.168
[2]
Neves SR, Ram PT, Iyengar R (2002) G protein pathways. Science 296: 1636–1639. doi: 10.1126/science.1071550
[3]
Baillie GS (2009) Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J 276: 1790–1799. doi: 10.1111/j.1742-4658.2009.06926.x
[4]
Willoughby D, Cooper DM (2008) Live-cell imaging of cAMP dynamics. Nat Methods 5: 29–36. doi: 10.1038/nmeth1135
[5]
Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349: 694–697. doi: 10.1038/349694a0
[6]
Zaccolo M, Pozzan T (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295: 1711–1715. doi: 10.1126/science.1069982
[7]
DiPilato LM, Cheng X, Zhang J (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A 101: 16513–16518. doi: 10.1073/pnas.0405973101
[8]
Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279: 37215–37218. doi: 10.1074/jbc.c400302200
[9]
Ponsioen B, Zhao J, Riedl J, Zwartkruis F, van der Krogt G, et al. (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 5: 1176–1180. doi: 10.1038/sj.embor.7400290
[10]
Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol. 19: 137–141. doi: 10.1038/84397
[11]
Ohkura M, Sasaki T, Sadakari J, Gengyo-Ando K, Kagawa-Nagamura Y, et al. (2012) Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. PLoS One. 7: e51286. doi: 10.1371/journal.pone.0051286
[12]
Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A 98: 3197–3202. doi: 10.1073/pnas.051636098
[13]
Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, et al. (2011) An expanded palette of genetically encoded Ca2+ indicators. Science 333: 1888–1891. doi: 10.1126/science.1208592
[14]
Nausch LW, Ledoux J, Bonev AD, Nelson MT, Dostmann WR (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci U S A 105: 365–370. doi: 10.1073/pnas.0710387105
[15]
Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, et al. (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3: 281–286. doi: 10.1038/nmeth866
[16]
Kawai Y, Sato M, Umezawa Y (2004) Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics. Anal Chem 76: 6144–6149. doi: 10.1021/ac040037s
[17]
Berg J, Hung YP, Yellen G (2009) A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat Methods 6: 161–166. doi: 10.1038/nmeth.1288
[18]
Tantama M, Martínez-Fran？ois JR, Mongeon R, Yellen G (2013) Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. Nat Commun 4: 2550. doi: 10.1038/ncomms3550
[19]
Marvin JS, Borghuis BG, Tian L, Cichon J, Harnett MT, et al. (2013) An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10: 162–170. doi: 10.1038/nmeth.2333
[20]
Kitaguchi T, Oya M, Wada Y, Tsuboi T, Miyawaki A (2013) Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells. Biochem J 450: 365–373. doi: 10.1042/bj20121022
[21]
Ghosh I, Hamilton AD, Regan L (2000) Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. J Am Chem Soc 122: 5658–5659. doi: 10.1021/ja994421w
[22]
Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394: 192–195. doi: 10.1038/28190
[23]
Rizzuto R, Pozzan T (2006) Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev 86: 369–408. doi: 10.1152/physrev.00004.2005
[24]
Zippin JH, Farrell J, Huron D, Kamenetsky M, Hess KC, et al. (2004) Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain. J Cell Biol 164: 527–534. doi: 10.1083/jcb.200311119
[25]
Buck J, Sinclair ML, Schapal L, Cann MJ, Levin LR (1999) Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci U S A 96: 79–84. doi: 10.1073/pnas.96.1.79
[26]
Tallman JF, Smith CC, Henneberry RC (1977) Induction of functional beta-adrenergic receptors in HeLa cells. Proc Natl Acad Sci U S A 74: 873–877. doi: 10.1073/pnas.74.3.873
[27]
Li BY, Fu B, Zhao YL, Li WH (1999) Effects of berbamine on intracellular calcium concentration in cultured HeLa cells. Zhongguo. Yao Li Xue Bao 20: 1011–1014.
[28]
Zippin JH, Chen Y, Nahirney P, Kamenetsky M, Wuttke MS, et al. (2003) Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains. FASEB J 17: 82–84. doi: 10.1096/fj.02-0598fje
[29]
Schwinn DA, Page SO, Middleton JP, Lorenz W, Liggett SB, et al. (1991) The alpha 1C-adrenergic receptor characterization of signal transduction pathways and mammalian tissue heterogeneity. Mol Pharmacol 40: 619–626.
[30]
Kasai H, Petersen OH (1994) Spatial dynamics of second messengers: IP3 and cAMP as long-range and associative messengers. Trends Neurosci 17: 95–101. doi: 10.1016/0166-2236(94)90112-0
