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• W2032451415 abstract "Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT1A receptor. Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT1A receptor. Several constructs of fluorescent proteins have been developed to measure cyclic adenosine monophosphate concentration ([cAMP]) in living cells (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 2Zaccolo M. Pozzan T. Science. 2002; 295: 1711-1715Crossref PubMed Scopus (700) Google Scholar). In these biosensors, Epac1 or Epac2 (exchange proteins directly activated by cAMP) (3de Rooij J. Rehmann H. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 4Ponimaskin E.G. Heine M. Zeug A. Voyno-Yasenetskaya T. Salonikidis P.S. Chattopadhyay A. Serotonin Receptors in Neurobiology. CRC Press, Inc., Boca Raton, FL2007: 19-40Google Scholar) or their cAMP binding domains are used as backbones (5DiPilato L.M. Cheng X. Zhang J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16513-16518Crossref PubMed Scopus (387) Google Scholar, 6Nikolaev V.O. Bünemann M. Hein L. Hannawacker A. Lohse M.J. J. Biol. Chem. 2004; 279: 37215-37218Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 7Ponsioen B. Zhao J. Riedl J. Zwartkruis F. van der Krogt G. Zaccolo M. Moolenaar W.H. Bos J.L. Jalink K. EMBO Rep. 2004; 5: 1176-1180Crossref PubMed Scopus (358) Google Scholar) to which fluorescent proteins are tethered. The binding of cAMP to the biosensors leads to a conformational change, which alters the relative distance and/or orientation between the FRET pair, thus changing the efficiency of energy transfer and donor and acceptor fluorescence emission. For such biosensors with fixed donor-acceptor stoichiometry, common ratiometric fluorescence measurements can be performed to identify changes in FRET. These changes can be calibrated with reference ligand solutions using a single fluorescence excitation wavelength (e.g. see Ref. 8Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar). In contrast to multi-excitation wavelength approaches like the EfDA/γ analysis (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 9Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) or lux-FRET (10Wlodarczyk J. Woehler A. Kobe F. Ponimaskin E. Zeug A. Neher E. Biophys. J. 2008; 94: 986-1000Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), which require more complex algorithms and calibrations, single excitation wavelength measurements profit from a higher time resolution and reduced bleaching of the fluorophores. Ratiometric analysis also gives a better signal-to-noise ratio than the more complex approaches (11Woehler A. Wlodarczyk J. Neher E. Biophys. J. 2010; 99: 2344-2354Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). cAMP biosensors using the conventional FRET pair (enhanced cyan fluorescence protein (eCFP) 2The abbreviations used are: eCFPenhanced cyan fluorescence protein5-HT1ARserotonin receptor subtype 1AWAY 100635selective 5-HT1AR antagonistEPAC*cAMP-detecting FRET biosensor CFP–Epac(δDEP-CD)–YFPCEPAC*cAMP-detecting FRET biosensor mCerulean–Epac(δDEP-CD)–mCitrineeYFPenhanced yellow fluorescence proteinN1E-115mouse neuroblastoma cell linemCeruleanmonomeric CFP with S72A/Y145A/H148D/A206K point mutationsmCitrinemonomeric YFP with Q69M/A206K point mutationsMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. and enhanced yellow fluorescence protein (eYFP)) (12Patterson G. Day R.N. Piston D. J. Cell Sci. 2001; 114: 837-838Crossref PubMed Google Scholar) might be critical when ratiometric cAMP measurements are performed with single-channel fluorescence excitation, as demonstrated in a previous work (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Such ratiometric FRET analysis of the intensity ratio between eCFP and eYFP emission of the Epac1 biosensor CFP-Epac(δDEP-CD)–YFP (Ponsioen et al. (7Ponsioen B. Zhao J. Riedl J. Zwartkruis F. van der Krogt G. Zaccolo M. Moolenaar W.H. Bos J.L. Jalink K. EMBO Rep. 2004; 5: 1176-1180Crossref PubMed Scopus (358) Google Scholar)) is susceptible to eYFP fluorescence intensity changes whenever ion concentrations fluctuate (e.g. [Cl−] (13Jayaraman S. Haggie P. Wachter R.M. Remington S.J. Verkman A.S. J. Biol. Chem. 2000; 275: 6047-6050Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar)). Considering that ion concentrations change significantly during physiologically cellular activities (e.g. [H+] and [Cl−] change with normal synaptic interactions of neurons) (e.g. see Refs. 14Ballanyi K. Mückenhoff K. Bellingham M.C. Okada Y. Scheid P. Richter D.W. Neuroreport. 1994; 6: 33-36Crossref PubMed Scopus (26) Google Scholar and 15Duebel J. Haverkamp S. Schleich W. Feng G. Augustine G.J. Kuner T. Euler T. Neuron. 2006; 49: 81-94Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), FRET signals can be affected by secondary effects and may not reliably monitor the real [cAMP]. The condition becomes worse under pathologic conditions, when ionic homeostasis is unbalanced, further falsifying [cAMP] readouts. During hypoxia, for example, intracellular pH decreases, and chloride ions accumulate within neurons, whereas calcium influx is increased or calcium is released from intracellular stores (16Müller M. Neuroscience. 2000; 97: 33-45Crossref PubMed Scopus (35) Google Scholar, 17Yao H. Haddad G.G. Cell Calcium. 2004; 36: 247-255Crossref PubMed Scopus (93) Google Scholar). Exchange of the FRET pair had already been used to gain a higher FRET efficiency of the traditional cAMP biosensor (18van der Krogt G.N. Ogink J. Ponsioen B. Jalink K. PLoS One. 2008; 3: e1916Crossref PubMed Scopus (139) Google Scholar); no special focus, however, was directed on ion sensitivity. Particularly for quantitative ratiometric FRET analysis in vivo, it seemed necessary, therefore, to exchange the conventional FRET pair eCFP/eYFP for other fluorophores that are less sensitive to changes in these ion concentrations. From a variety of improved CFP and YFP derivatives, we have chosen mCerulean and mCitrine, which are less sensitive to pH changes and show only insignificant halide sensitivity. mCitrine also has a much higher photostability as compared with eYFP and has an improved folding efficiency (19Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). mCerulean is a better FRET donor than eCFP when combined with mCitrine (20Rizzo M.A. Springer G. Segawa K. Zipfel W.R. Piston D.W. Microsc. Microanal. 2006; 12: 238-254Crossref PubMed Scopus (112) Google Scholar, 21Rizzo M.A. Springer G.H. Granada B. Piston D.W. Nat. Biotechnol. 2004; 22: 445-449Crossref PubMed Scopus (908) Google Scholar). Förster distance R0 of the mCerulean/mCitrine FRET pair is almost 10% larger than for eCFP/mCitrine as calculated by a 68% larger quantum yield of mCerulean. Another important advantage also is that the monomeric versions (22Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1791) Google Scholar) of Cerulean and Citrine show a much lower tendency to oligomerization as compared with the eCFP/eYFP pair, which may cause additional artifacts. enhanced cyan fluorescence protein serotonin receptor subtype 1A selective 5-HT1AR antagonist cAMP-detecting FRET biosensor CFP–Epac(δDEP-CD)–YFP cAMP-detecting FRET biosensor mCerulean–Epac(δDEP-CD)–mCitrine enhanced yellow fluorescence protein mouse neuroblastoma cell line monomeric CFP with S72A/Y145A/H148D/A206K point mutations monomeric YFP with Q69M/A206K point mutations 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. Here, we report a reduced ion sensitivity (to pH and Cl− and indirectly also Ca2+) of quantitative radiometric measurements by exchanging the eCFP/eYFP FRET pair of the biosensor for mCerulean/mCitrine. In addition, we report an improvement of the biosensor aggregation behavior. Plasmids encoding mCerulean and mCitrine were obtained from Addgene, and their coding sequences were amplified by PCR, introducing recombinant recognition sites for restriction enzymes using the primers mCerulean-NotI-for (5′-GCGGCCGC aat ggt gag caa ggg cga gga g-3′), mCerulean-EcoRV-rev (5′-GATATC gag atc tga gtc cgg act tgt aca gct cgt cca tgc c-3′), mCitrine-NheI-for (5′-GCTAGC gag ctc atg gtg agc aag ggc gag gag-3′), and mCitrine-EcoRI-rev (5′-GAATTC ctt gta cag ctc gtc cat gcc-3′). Resultant PCR products were subcloned into a mammalian expression vector pTarget (Promega), which served for positive controls in FRET measurements. mCerulean and mCitrine were isolated from the vectors with the restriction enzyme pairs NotI/EcoRV and NheI/EcoRI (New England Biolabs) and cloned into corresponding sites in the vector pcDNA3.1-CFP-Epac(δDEP-CD)–YFP (7Ponsioen B. Zhao J. Riedl J. Zwartkruis F. van der Krogt G. Zaccolo M. Moolenaar W.H. Bos J.L. Jalink K. EMBO Rep. 2004; 5: 1176-1180Crossref PubMed Scopus (358) Google Scholar) (encoding the protein denoted EPAC*) to replace previous fluorophores. The cloning provided the vector pcDNA3.1-mCerulean-Epac(δDEP-CD)–mCitrine (encoding the protein denoted CEPAC*). Neuroblastoma cells (N1E-115) were transfected with cDNA encoding for (a) enhanced cyan fluorescence protein (pECFP-N1, Clontech), (b) enhanced yellow fluorescence protein (pEYFP-N1, Clontech), (c) mCerulean, (d) mCitrine, (e) pcDNA3.1/CAT (Invitrogen), (f) EPAC*, (g) CEPAC*, or (h) a co-transfection of 5-HT1AR (HA-tagged 5-HT1A-receptor cloned into the pcDNA3.1 plasmid (23Papoucheva E. Dumuis A. Sebben M. Richter D.W. Ponimaskin E.G. J. Biol. Chem. 2004; 279: 3280-3291Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar)) together with CEPAC* and EPAC*, respectively. Mouse N1E-115 neuroblastoma cells from the American Type Culture collection (LGC Promochem, Wesel, Germany) were grown at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Neuroblastoma cells were seeded at low density (1 × 106 cells) either in 60-mm dishes (for fluorescence spectroscopy measurements) or in 10-mm dishes, including glass coverslips on the bottom (for microscopic measurements). After 24 h, cells were transfected with appropriate vectors using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's instructions. 3 h after transfection, cells were serum-starved overnight and then used in the experiment. We had to avoid longer incubation periods, because EPAC* proteins aggregated, which impedes direct comparison with the biosensor CEPAC*. Primary cultures of hippocampal neurons were prepared according to Dityatev et al. (24Dityatev A. Dityateva G. Schachner M. Neuron. 2000; 26: 207-217Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Hippocampi from postnatal (P1 or P2) NMRI mice were isolated, and cells were dissociated with trypsin (6 mg/2 ml) and centrifuged (100 × g, 2 × 15 min, 4 °C,) and then were planted on 10-mm glass coverslips (cell density 700 cells/mm2) coated with 100 g/ml poly-l-lysine (Sigma-Aldrich) and 20 μg/ml laminin (Roche Applied Science). From the first day, cells were incubated in minimum essential Eagle's medium containing glucose (25.2 mm), transferrin (1.3 mm), insulin (25 μg/ml), Glutamax I (2 mm), gentamicin (0.5 μl/ml), and horse serum (0.1 ml/ml) at 37 °C and 5% CO2. After 4 days, primary cells were transfected with CEPAC* or EPAC* using an optimized protocol of 1 μg of DNA and 1 μl of Lipofectamine2000 per coverslip in 500 μl of serum-free medium. The transfection mix was removed after 1 h, and cells were incubated for the following days in Neurobasal-A medium containing l-glutamine (0.5 mm), basic fibroblast growth factor (125 ng/ml), B-27 supplement (20 μl/ml), penicillin/streptomycin (10 μl/ml), and cytosine arabinoside (5 μm) at 37 °C and 5% CO2. To change the intracellular pH (pHi) of N1E cells, we used a modified protocol described previously (25Flögel U. Willker W. Leibfritz D. NMR Biomed. 1994; 7: 157-166Crossref PubMed Scopus (41) Google Scholar), using extracellular buffer exchange with different pH values to affect intracellular conditions. Extracellular buffer (150 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm sodium-d-glucose) was prepared with pH values ranging from pH 6.5 to pH 8.0 and given to the N1E cells, immediately producing a pHi shift from 7.05 to 7.4, respectively. Under normal culture conditions at an extracellular pH of 7.4, N1E neuroblastoma cells exhibit a pHi of 7.35. The pHi shift was verified by pH indicator dye SNARF-5F (Invitrogen), which was also used to calibrate the pH range. The experiments with cAMP biosensor transfected cells were performed in an upright fluorescent microscope, whereas the experiments with SNARF-5F were performed in a laser-scanning microscope with a spectral resolving emission unit in order to measure the full emission spectrum of SNARF-5F. N1E-115 cells co-transfected with 5-HT1AR and either cAMP biosensor were kept in an extracellular solution of 150 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm sodium-d-glucose (pH 7.4), and 100 nm forskolin. 5-HT1AR agonist and antagonist were applied by exchanging the standard bath solution for a solution containing or 1 μm serotonin or WAY 100635 (Sigma-Aldrich), respectively. Experiments were performed in an upright fluorescence microscope. To test the viability of cells transfected with either of the two constructs, the MTS cell proliferation assay (Promega) was used. Approximately 5 × 103 N1E cells were seeded into 96-well plates and allowed to attach overnight. Transfection with a mock control, CEPAC*, or EPAC* was carried out with Lipofectamine2000 according to the manufacturer's instructions. After 4, 8, 12, 16, and 20 h, absorbance at 485 nm of triplicates of each transfection was measured in a SkanIt plate reader (Thermo Scientific). MTS is reduced by cells into a formazan product, and the quantity of formazan product as measured by the amount of absorbance is directly proportional to the number of living cells in culture. Normalized absorbance was plotted against time after transfection. Primary neurons transfected with the cAMP biosensors were kept in an extracellular solution of 128 mm NaCl, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm sodium-d-glucose (pH 7.4). Dendritic spines could be captured using an inverse laser-scanning microscope. The sample preparation was obtained as described previously (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and only modifications are mentioned here. For [cAMP] measurements, transfected N1E-115 cells were suspended in a buffer and lysed by ultrasonic treatment (DIGITAL Sonifier S-450D, Branson (Danbury, CT)). The homogenate was centrifuged for 1.5 h at 16,000 × g and 4 °C to extract the cytosol. For [cAMP] measurements, the lysis buffer contained 140 mm KCl, 5 mm NaCl, 1 mm MgCl2, and 10 mm HEPES at pH 7.2. For measuring the pH dependence, the solution contained 140 mm KCl, 5 mm NaCl, 1 mm MgCl2, and 10 mm HEPES at pH 7.2. For measuring the Ca2+ dependence, a calcium-free lysis buffer was used containing 140 mm KCl, 5 mm NaCl, 1 mm MgCl2, 1 mm EGTA, and 10 mm HEPES at pH 7.2. Such lysate contained a free [Ca2+] below 10 nm as a starting condition for the calcium titration as proven by calcium measurements with Fluo-5 (Invitrogen) (data not shown). The nominal free [Ca2+] was changed by titrating a defined amount of calcium to the cuvette and was estimated by the Max Chelator Ca-Mg-ATP-EGTA Calculator version 1 (26Patton C. Thompson S. Epel D. Cell Calcium. 2004; 35: 427-431Crossref PubMed Scopus (328) Google Scholar, 27Schoenmakers T.J. Visser G.J. Flik G. Theuvenet A.P. BioTechniques. 1992; 12 (876-879): 870-874PubMed Google Scholar) (available on the World Wide Web). Addition of high calcium concentrations led to a pH change due to proton release from EGTA. Therefore, the pH in the cuvette was adjusted during each test. For measuring the Cl− dependence, a chloride-free lysis buffer was used, containing 140 mm K+, 5 mm Na+, 1 mm Mg2+, 147 MeSO3−, and 10 mm HEPES at pH 7.2. A basal [Cl−] in the cell lysate of about 200 μm Cl− was determined with a chloride-selective electrode (DC235, Mettler Toledo, Greifensee, Switzerland). Spectroscopic measurements were performed as described previously (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) with a Fluorolog-322 (Horiba Jovbin Yvon, Munich, Germany). A donor excitation wavelength of 420/2 nm was chosen. A second excitation wavelength of 500/2 nm was necessary to apply the formalism of Equation 2. Reference emission spectra (FDref(λ) and FAref(λ)) of the donor and acceptor fluorophores used for the unmixing procedure (see Equation 1) were obtained at 420/2 nm excitation from cells transfected with donor or acceptor only. To estimate the spectral contributions of light scattering and autofluorescence of cells as an additional background component, reference spectra of pcDNA-transfected cells were used. A glass coverslip carrying the biosensor transfected or loaded N1E-115 cells or primary neurons was positioned in a perfused bath chamber of a microscope. Cells were perfused with an appropriate bath solution at room temperature. For sequential measurements with different solutions, total solution exchanges in the chamber were performed in a time range of 1 min. We used an upright epifluorescence microscope equipped with a water immersion objective (XLUMPlanFI, ×20, numerical aperture 0.95, Olympus, Germany). A 100-watt xenon lamp attached to a monochromator (Optoscan, Kinetic Imaging) served as an excitation light source and was coupled to the microscope via fiber optics. Emission intensities were measured at two excitation wavelengths using 420/10 nm (λ1) and 500/10 nm (λ2), frequently called donor and acceptor excitation, respectively. Fluorescence emission was separated from the excitation light by a dichroic mirror (505 nm). Using a DualView (Optical Insights, Tucson, AZ), the fluorescence emission signal was split by a dichroic mirror (515 nm) for the donor channel at 470/30 nm and the acceptor channel at 535/30 nm. This allowed us to acquire three images with an iXon camera DV887DCS (Andor Technology, South Windsor, CT): (a) the “donor image” at donor excitation and donor emission wavelength; (b) the “FRET image” at donor excitation and acceptor emission wavelength; (c) the “acceptor image” at acceptor excitation and acceptor emission wavelength. Due to the DualView, the donor image and the “FRET image” were obtained simultaneously, which required only two exposures. Exposure times (of about 3 s) were chosen according to the fluorescence intensity of the cells and were equal for all image series of an individual experiment. Special care was taken that bleaching can be ignored. For capturing dendritic spines and for recording intracellular pH in single cells with SNARF-5F, we used a confocal laser-scanning microscope LSM 510 META from Zeiss (Göttingen, Germany). CEPAC* in spines was observed at 458 nm excitation, and a bright emission bandwidth was observed from 464 to 603 nm using a C-Apochromat × 63/1.2 W corrected objective. For the pHi recording, the emission spectrum (562–646 nm) of SNARF-5F was detected by using the META DETECTOR with a resolution of 8 points (10 nm bandwidth) at an excitation of 543 nm and a Plan-Neofluar × 40/1.3 oil objective. Single images were taken with a wide field microscope at various times after transfection to measure protein aggregation seen as “granules” with the ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD). Protein aggregation was determined in each cell using a line scan through the entire cells, excluding obvious organelle structures, such as the nucleus. This line scan represents the fluorescence intensity at any given pixel. Calculating the S.D. value for each given fluorescence intensity plot gave a measure of the granularity of the cell because the line profile did not vary greatly in cells showing a uniform fluorophore expression, whereas in cells showing aggregates, the line profile changed between regions with high and low fluorescence. For the different time points, at minimum, six cells were analyzed, and the mean granularity (as the sum of S.D. values from different scans) was plotted over time after transfection. To image a single primary cell, multiple images were taken and stitched together using the ImageJ plugin MosaicJ (28Thévenaz P. Unser M. Microsc. Res. Tech. 2007; 70: 135-146Crossref PubMed Scopus (207) Google Scholar). Neurite length was then measured on the reconstructed image using the ImageJ plugin NeuronJ (29Meijering E. Jacob M. Sarria J.C. Steiner P. Hirling H. Unser M. Cytometry A. 2004; 58: 167-176Crossref PubMed Scopus (1104) Google Scholar). Captured z-stacks of dendritic spine pictures were projected on a single picture by using the maximal z-projection of the ImageJ software version 1.41. We calibrated the [cAMP] sensitivity of CEPAC* and EPAC* by the “sensitized emission” FRET signal using a fluorescence spectrometer (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) as described previously. Spectra were obtained at two excitation wavelengths (λi): at donor excitation wavelength (λ1), where mainly the donor is excited, and at the acceptor excitation wavelength (λ2), where the donor must not be excited (9Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). For each excitation wavelength, the CEPAC* as well as the EPAC* fluorescence signals Fi(λ) were fitted with a linear combination of the donor D and acceptor A reference spectra.Fi(λ)=[Di]FDref(λ)+[Ai]FAref(λ)Eq. 1 The apparent donor concentration, [Di], and the apparent acceptor concentration, [Ai], were used as fitting factors. The donor FDRef and the acceptor FARef reference spectra for both excitation wavelengths λi were obtained in a reference measurement of cells containing only donor and acceptor, respectively. The apparent acceptor concentrations [Ai] at both excitation wavelengths were used to calculate the following term,EfDA/γ=[A1]-α[A2]α[A2]Eq. 2 where α represents the relative acceptor emission intensity ratio used for the two excitations obtained in a separate experiment using an “acceptor only” sample, γ is the relative acceptor/donor extinction ratio, and fDA is the fraction of biosensor proteins in FRET state. For calibration of the FRET biosensors CEPAC* and EPAC*, the cAMP dependence of EfDA/γ was fitted by an adapted Hill equation,EfDA/γ=(pmax-p0)⋅[cAMP]nH(EC50)nH-[cAMP]nH+poEq. 3 where nH is the Hill coefficient, indicating the amount of cAMP-binding places; po and pmax are offset and maximum amplitude parameters, respectively; and EC50 is [cAMP] when 50% of the cAMP binding sites are occupied. Also the cAMP dependence of the acceptor/donor ratio as well as the ion dependences were fitted by a similar Hill equation. The apparent FRET efficiency (EfDA) was calculated from the donor fluorescence signal in quenched (FD,FRET) and non-quenched (FD,NON-FRET) state (30Lakowicz R.J. Principles of Fluorescence Spectroscopy. 3rd Ed. Springer, New York2006: 443-476Crossref Google Scholar).EfDA=1-FD,FRETFD,NON-FRETEq. 4 [cAMP] calibration curves of both biosensors were measured to ensure similar binding properties and to compare the dynamic range of the FRET signals. We exposed cell lysates from CEPAC* or EPAC*-transfected cells to various [cAMP] values, ranging from nominal 0 to 1 mm, and measured the biosensor FRET signal acceptor/donor ratio or EfDA/γ as a function of [cAMP]. Fluorescence spectra were recorded, as described previously (1Salonikidis P.S. Zeug A. Kobe F. Ponimaskin E. Richter D.W. Biophys. J. 2008; 95: 5412-5423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and the contributions of the donor and acceptor reference spectra were calculated as shown in Fig. 1, A and B. Both biosensors revealed a similar behavior regarding the contribution of reference spectra; the donor emission signal was decreased, and the acceptor emission signal increased with a rise of [cAMP], as expected for FRET changes. During donor excitation, photon counts for the donor and acceptor component of CEPAC* reached similar values in contrast to EPAC*, which is beneficial for the signal/noise ratio of the ratiometric FRET analysis. For both biosensors, we compared the FRET signal acceptor/donor channel ratio as the ratio between the acceptor and donor intensities (here at 420 nm excitation; Fig. 1, C and D). The intensities of the acceptor and donor channels were corrected for spectral bleed-through and background, whereas the channel bandwidth was adapted to that used in the microscope. Due to the higher energy transfer efficiency of the FRET pair mCerulean/mCitrine compared with eCFP/eYFP, the [cAMP] calibration curves of the acceptor/donor channel ratio of CEPAC* started at a higher value (0.94 ± 0.03) than for EPAC* (0.43 ± 0.005). For maximal [cAMP], the acceptor/donor channel ratio dropped to approximately half in both biosensors (to 0.53 ± 0.06 for CEPAC* and 0.23 ± 0.01 for EPAC*), indicating a similar dynamic range of the acceptor/donor channel ratio. The calibration curves were fitted by the Hill equation (Equation 3) to compare the binding affinity of cAMP to the biosensors. Both biosensors exhibited a Hill coefficient for the binding of cAMP of about 1 (nH for CEPAC* = 1.13 ± 0.6; nH for EPAC* = 0.96 ± 0.18). The range of [cAMP] sensing was also comparable, as seen in similar EC50 values for CEPAC* and EPAC* (23.6 ± 12.2 and 30.8 ± 6.9 μm)" @default.
• W2032451415 created "2016-06-24" @default.
• W2032451415 creator A5025492340 @default.
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• W2032451415 creator A5029095407 @default.
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• W2032451415 date "2011-07-01" @default.
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• W2032451415 title "An Ion-insensitive cAMP Biosensor for Long Term Quantitative Ratiometric Fluorescence Resonance Energy Transfer (FRET) Measurements under Variable Physiological Conditions" @default.
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