Ratiometric Bioluminescence Indicators for Monitoring Cyclic

Oct 27, 2010 - ... in green (Brazilian Pyrearinus termitilluminans;(17, 18) Emerald Luc; .... Obtained images were analyzed using imaging software (Me...
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Anal. Chem. 2010, 82, 9306–9313

Ratiometric Bioluminescence Indicators for Monitoring Cyclic Adenosine 3′,5′-Monophosphate in Live Cells Based on Luciferase-Fragment Complementation Masaki Takeuchi,† Yasutaka Nagaoka,†,§ Toshimichi Yamada,† Hideo Takakura,† and Takeaki Ozawa*,†,‡ Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and PRESTO, Japan Science and Technology Agency, Tokyo, Japan Bioluminescent indicators for cyclic 3′,5′-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5′-triphosphate (ATP) and D-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with aminoterminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and D-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for timecourse and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects. Cyclic adenosine 3′,5′-monophosphate (cAMP) plays important roles as a second messenger in various biological processes such as metabolism, gene expression,1 cell division,2 and cell migration.3 Upon response to extracellular signals, cAMP is synthesized from adenosine 5′-triphosphate (ATP) by adenylate cyclases and activates its downstream receptor proteins including protein kinase A (PKA),4 an exchange protein activated by cAMP (Epac)5 and cyclic nucleotide-gated ion channels (CNGCs).6 Subsequently, * To whom correspondence should be addressed. E-mail: ozawa@ chem.s.u-tokyo.ac.jp. Phone: +81-3-5841-4351. Fax: +81-3-5802-2989. † The University of Tokyo. ‡ Japan Science and Technology Agency. § Current address: Institute for Molecular Science/CREST, Japan Science and Technology Agency. (1) Conkright, M. D.; Guzman, E.; Flechner, L.; Su, A. I.; Hogenesch, J. B.; Montminy, M. Mol. Cell 2003, 11, 1101–1108. (2) Prasad, K. N.; Cole, W. C.; Yan, X. D.; Nahreini, P.; Kumar, B.; Hanson, A.; Prasad, J. E. Apoptosis 2003, 8, 579–586. (3) McLeod, S. J.; Li, A. H. Y.; Lee, R. L.; Burgess, A. E.; Gold, M. R. J. Immunol. 2002, 169, 1365–1371.

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cAMP is degraded rapidly by phosphodiesterases to terminate the signals. The spatial and temporal production of cAMP is a central feature to govern a specific cellular activity. To investigate the molecular mechanisms of cAMP-mediated signaling and the composition of cAMP signaling complexes, it is necessary to quantify and obtain images of spatiotemporal patterns of cAMP in living cells and animals. For visualizing cAMP in living cells, several indicators have been developed based on Fo¨rster resonance energy transfer (FRET) using fluorescent proteins. The indicators comprise a cAMP-binding domain from PKA,7 Epac,8,9 or CNGCs10,11 flanking a pair of spectral mutants of green fluorescent protein. These indicators enable monitoring of rapid subcellular cAMP dynamics by ratiometric fluorescence detection of the two fluorescent proteins. The spatial dynamics of cellular cAMP under a stimulated condition was also revealed at a single-cell level. Although the FRET techniques have such strong points, the FRET-based indicators present several drawbacks. Autofluorescence from an intracellular organelle is induced by strong excitation light, which generates a higher background. In addition, physiological responses in light-sensitive tissues are perturbed by the excitation light. Moreover, irradiation of excitation light causes phototoxic damage to analyzed cells. In comparison to the fluorescent protein techniques, bioluminescence analysis and imaging using luciferases have important advantages: Luciferases require no high-energy excitation light and do not suffer from background noise and tissue damage in measurements.12-14 Taking advantage of these characteristics, we first demonstrated analysis of protein-protein interactions using (4) Kim, C.; Vigil, D.; Anand, G.; Taylor, S. S. Eur. J. Cell Biol. 2006, 85, 651– 654. (5) Bos, J. L. Nat. Rev. Mol. Cell Biol. 2003, 4, 733–738. (6) Zagotta, W. N.; Olivier, N. B.; Black, K. D.; Young, E. C.; Olson, R.; Gouaux, E. Nature 2003, 425, 200–205. (7) Zaccolo, M.; Pozzan, T. Science 2002, 295, 1711–1715. (8) Nikolaev, V. O.; Bunemann, M.; Hein, L.; Hannawacker, A.; Lohse, M. J. J. Biol. Chem. 2004, 279, 37215–37218. (9) Ponsioen, 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– 1180. (10) Rich, T. C.; Tsf, T. E.; Rohan, J. G.; Schaack, J.; Karpen, J. W. J. Gen. Physiol. 2001, 118, 63–77. (11) Fagan, K. A.; Schaack, J.; Zweifach, A.; Cooper, D. M. F. FEBS Lett. 2001, 500, 85–90. 10.1021/ac102692u  2010 American Chemical Society Published on Web 10/27/2010

luciferase-fragment complementation in intact cells.15,16 Because luciferases emit light from a chemical reaction, there is no need for external light. Consequently, high-sensitivity detection was realized. We also developed novel luciferase fragments from click beetles in green (Brazilian Pyrearinus termitilluminans;17,18 Emerald Luc; ELuc19) and in red (Caribbean Pyrophorus plagiophthalamus; CBR) to visualize dynamic protein-protein interactions in living Xenopus laevis embryos.20 The use of click beetle luciferase offers the important advantages of better brightness than firefly luciferase, in addition to pH independence. Recently we developed novel click beetle luciferase fragments with brighter luminescence and a higher signal-to-background ratio than the previous one. The fragments were applied to interaction of G-protein coupled receptor (GPCR) with β-arrestin.21 On the basis of split luciferase-fragment complementation, others have developed a luminescent indicator for cAMP using firefly luciferase.22 The indicator enables bioluminescence detection with high sensitivity and no background signal. However, the luminescent indicator presents an important disadvantage; the absolute photon count originated from the indicator fluctuates according to experimental conditions such as the expressed amount of the indicator in the cells and concentrations of D-luciferin and ATP. Herein, we describe a semiquantitative cAMP indicator with detection of dual-wavelength bioluminescence using two clickbeetle luciferases: ELuc and CBR. The indicator was designed to change the bioluminescence intensity from ELuc depending on the cAMP concentration, whereas the bioluminescence from CBR was used for monitoring its expression level and differences of ATP and D-luciferin as its internal control. We present the characteristics of the indicator in vitro and applications of the indicator to measure cAMP in living cells and animals. EXPERIMENTAL SECTION Materials. DNA-modifying enzymes were obtained from Takara Bio Inc. (Tokyo, Japan). A mammalian expression vector, pcDNA4/V5-His(B), was obtained from Invitrogen Corp. (Carlsbad, CA). cDNAs of ELuc, CBR, and PKA-RIIβ of humans were obtained from Toyobo Co. Ltd. (Japan), Promega Corp. (Madison, WI), and Kazusa DNA Research Institute (Chiba, Japan), respectively. The luciferase assay kit, Bright-Glo Luciferase Assay (12) Paulmurugan, R.; Umezawa, Y.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15608–15613. (13) Luker, K. E.; Smith, M. C. P.; Luker, G. D.; Gammon, S. T.; Piwnica-Worms, H.; Piwnica-Worms, D. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12288– 12293. (14) Kanno, A.; Yamanaka, Y.; Hirano, H.; Umezawa, Y.; Ozawa, T. Angew. Chem., Int. Ed. 2007, 46, 7595–7599. (15) Ozawa, T.; Kaihara, A.; Sato, M.; Tachihara, K.; Umezawa, Y. Anal. Chem. 2001, 73, 2516–2521. (16) Kim, S. B.; Kanno, A.; Ozawa, T.; Tao, H.; Umezawa, Y. ACS Chem. Biol. 2007, 2, 484–492. (17) Viviani, V. R.; Silva, A. C. R.; Perez, G. L. O.; Santelli, R. V.; Bechara, E. J. H.; Reinach, F. C. Photochem. Photobiol. 1999, 70, 254–260. (18) Neto, A. J. S.; Scorsato, V.; Arnoldi, F. G. C.; Viviani, V. R. Photochem. Photobiol. Sci. 2009, 8, 1748–1754. (19) Nakajima, Y.; Yamazaki, T.; Nishii, S.; Noguchi, T.; Hoshino, H.; Niwa, K.; Viviani, V. R.; Ohmiya, Y. PLoS One 2010, 5, e10011. (20) Hida, N.; Awais, M.; Takeuchi, M.; Ueno, N.; Tashiro, M.; Takagi, C.; Singh, T.; Hayashi, M.; Ohmiya, Y.; Ozawa, T. PLoS One 2009, 4, e5868. (21) Misawa, N.; Kafi, A. K. M.; Hattori, M.; Miura, K.; Masuda, K.; Ozawa, T. Anal. Chem. 2010, 82, 2552–2560. (22) Fan, F.; Binkowski, B. F.; Butler, B. L.; Stecha, P. F.; Lewils, M. K.; Wood, K. V. ACS Chem. Biol. 2008, 3, 346–351.

System, was purchased from Promega Corp. Fetal bovine serum (FBS), 0.05% (v/v) trypsin-ethylenediaminetetraacetic acid (EDTA), Hank’s balanced buffered saline (HBSS), penicillin and streptomycin solution, and Lipofectamine 2000 were obtained from Gibco BRL (Rockville, MD). cAMP, 8-bromoadenosine cyclic 3′,5′monophosphate (8-Br-cAMP), and cyclic guanosine 3′,5′-monophosphate (cGMP) were purchased from Sigma (St. Louis, MO). D-Luciferin potassium salt was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Sodium nitroprusside (SNP) and isoproterenol (ISO) were from Sigma. Construction of Plasmids for Mammalian Cell Expression. An Escherichia coli strain, DH5R, was used as a bacterial host for construction of all plasmids. To express genetically encoded proteins in mammalian cells, we used pcDNA4/V5His(B), which has a human cytomegalovirus immediate early promoter. The constructed plasmids were named pcELucN-PKAMcLuc1, pcELucN-PKA-McLuc1-CBRN, and pcELucN-PKAMcLuc1-CBRmN. The sequences of all plasmids were verified by sequencing with a genetic analyzer (ABI prism 310; Applied Biosystems, Tokyo, Japan). Cell Culture and Transfection. HEK293 and COS-7 cells were cultured in Dulbecco’s modified medium supplemented with 10% (v/v) heat-inactivated FBS, 100 unit/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5% (v/v) CO2. Cells were seeded onto 6-well or 12-well culture plates, transfected with cDNA plasmids in the presence of Lipofectamine 2000, and left at 37 °C in an atmosphere of 5% (v/v) CO2. Spectral Analysis. COS-7 cells seeded onto 6-well plates were transfected with plasmids and incubated for 24-36 h. Then, the culture medium was removed, and 250 µL of Bright-Glo reagent was added to each well. The cells were incubated for 2 min at 37 °C, and 200 µL of suspension was taken into a small tube. The tube was set into a luminescence spectrometer (LumiFLSpectrocapture; Atto Corp., Tokyo, Japan). The bioluminescence spectra were recorded under different conditions: First, the bioluminescence spectra were measured directly in the absence of 8-Br-cAMP. Next, 20 µL of 10 µM 8-Br-cAMP water solution was added to the tube, and the spectra were measured 15 min after addition of 8-Br-cAMP. The scan speed was set to 400 nm/ min. Bioluminescence Assays in Vitro. The HEK293 cells seeded onto 12-well plates were transfected with the plasmids and incubated for 24-36 h. Then, the culture medium was taken away, and 350 µL of Bright-Glo reagent was added to each well. After incubation for 3 min at 37 °C, cells were well suspended and 100 µL of the suspension was removed into each well of a 96-well plate. The luciferase activities were measured using a 96-well microtiter plate reader (TriStar; Berthold GmbH & Co. KG) under two conditions: One was a measurement with no stimulation to obtain a background luminescence. The other was a measurement in the presence of cAMP or cGMP. The time for measuring each luciferase activity was set to 5 s/well in all measurements. For measuring bioluminescence intensities of ratiometric indicators, emission band-pass filters (525 ± 25 or 630 ± 30 nm, respectively) were used for selection of wavelength. All measurements were made in triplicate. Results are presented as an average with standard deviations (n ) 3). Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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For evaluating the effects of ATP and D-luciferin concentrations on the reporter, the HEK293 cells harboring the indicator were scraped from a 10 cm dish using 3 mL of phosphate-buffered saline (PBS; 0.137 M sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogenphosphate, and 1.8 mM potassium dihydrogenphosphate, pH 7.4). The cells were centrifuged at 400g for 5 min at 4 °C; the precipitated cells were recovered. The cells were lysed in 0.8 mL of lysis buffer containing 5× Passive Lysis Buffer (Promega Corp. Madison, WI), 25 mM MgCl2, and 1 × HBSS. After gentle mixing for 15 min at room temperature, the cell debris was removed from the lysates by centrifugation at 400g for 5 min at 4 °C and the supernatant was collected in a test tube. Each assay was performed using 100 µL of a reaction solution containing the cell lysates, ATP, and D-luciferin as one assay unit. Data were obtained using a 96-well plate reader (TriStar; Berthold GmbH & Co. KG). Bioluminescence Assays Using Living Cells. The HEK293 cells seeded onto 35 mm dish were transfected with the plasmids and incubated for 24-36 h. The culture medium was removed from the dish and 1 mL of HBSS including 500 µM D-luciferin potassium was added. After setting the dish on a luminometer (Kronos; Atto Corp., Japan) at 37 °C, temporal changes of luciferase activities were measured for 20 s without filters and with a 600 nm long pass filter (R60 filter; Hoya Corp. Tokyo, Japan) at 1 min intervals. To evaluate responses of the indicator, the cells were stimulated with 10 µL of HBSS, 500 µM SNP, or 1 mM ISO at a different time in a time-course measurement. The independent luciferase activities of ELucN-McLuc1 complementation and McLuc1-CBRmN complementation were calculated according to

[ ] [

1 1 F0 ) kG F2 R60 kRR60

][ ] G R

in which F0 is the total luciferase activity measured without the optical filter, F2 is the luciferase activity through the R60 filter, κGR60 and κRR60, respectively, represent the transmission coefficients of the ELucN-McLuc1 and McLuc1-CBRmN luciferase activities through the R60 filters, and G and R, respectively, signify luciferase activities of ELucN-McLuc1 complementation and McLuc1-CBRmN complementation. Imaging of cAMP in Living Cells. The HEK293 cells were transfected with the plasmids onto a 35 mm dish and incubated for 24-36 h. Before taking bioluminescence images, the culture medium was replaced by 2 mL of HBSS containing 1 mM D-luciferin potassium. The cells were incubated at 37 °C for 30-120 min. Bioluminescence and bright field images of live cells were taken with an upright fluorescence and bioluminescence microscope (BX61; Olympus Corp.), which was used with a 20× dipping objective (0.40 NA) and emission band-pass filters (536 ± 10 and 624 ± 25 nm). Digital images were acquired using a cooled (set at -80 °C) electron multiplying charge-coupled device (EM-CCD) camera (ImagEM-1K; Hamamatsu Photonics KK). The filters and camera control were adjusted automatically using software (Meta Morph; Molecular Devices, Inc., CA.). Stray light was cut off by turning off the electric system and covering it tightly with foil. Bioluminescence images were acquired every 2 min, in which the interval bright field image was taken for 100 ms. Images of ELucNMcLuc1 or McLuc1-CBRmN were obtained using the filters (536 9308

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± 10 or 624 ± 25 nm, respectively). Obtained images were analyzed using imaging software (Meta Morph Offline; Molecular Devices, Inc.). Imaging of cAMP in Living Mice. COS7 cells were transfected with the ELucN-PKA-McLuc1-CBRmN indicator and then incubated for 24-48 h. The cells were harvested with rubber scrapers and suspended in a phosphate-buffered saline (PBS) buffer containing D-luciferin (600 mg/kg of body weight), and an aliquot of 5.0 × 106 cells was implanted on the back of BALB/ c-nude mouse (female, 5 week old, 17-20 g of body weight). After implantation of the cells, 150 µL of 20 mM ISO was intraperitoneally (i.p.) injected, and the mouse was imaged with a cooled CCD camera (Versarray 1300B, Princeton Instruments Inc.) with or without a filter (BP(ELuc); 520 ± 25 nm, BP(CBR); 630 ± 37.5 nm, Chroma Technology Corp.). Image processing was performed using an imaging software (SlideBook 4.1; Intelligent Imaging Innovation Inc.). RESULTS AND DISCUSSION Design of Dual-Bioluminescence Ratiometric Indicators for cAMP. We earlier developed a new carboxy- (C-) terminal fragment (McLuc1) engineered from click beetle luciferase, which has the unique ability to complement multiple amino- (N-) terminal fragments of luciferases with different spectral characteristics.20 We also demonstrated that the complementation of McLuc1 with the N-terminal luciferase fragments were reversible reactions in live cells.20 In the present study, we used the McLuc1, N-terminal fragments of a click beetle luciferase from Brazil (ELucN,19 Emmax ) 538 nm) and one from Jamaica (CBRN, Emmax ) 613 nm) for development of a dual-wavelength luminescent indicator. The indicator comprised one PKA domain and three luciferase fragments, from the N-terminus, the ELucN, a cAMP binding domain of PKA (PKA-BD), McLuc1, and the CBRN (Figure 1b). The components were connected with different peptide linkers to allow flexible dynamic motion. In the absence of cAMP, McLuc1 is complementary only to the CBRN, resulting in the constant production of red color bioluminescence. Upon an increase in the cAMP concentration, it binds to PKA-BD, which undergoes a conformational change. Consequently, McLuc1 dissociates from CBRN and interacts with ELucN, which emits green bioluminescence (Figure 1a). The green bioluminescence is separated from the red one using specific filters. Then, their intensities are measured using a CCD camera or photon counters. Development and Characterization of the Ratiometric Bioluminescent Indicator for cAMP. We first examined whether the structural change of PKA-BD induces complementation of McLuc1 with ELucN. The cDNA of PKA-BD was connected with the cDNAs of ELucN and McLuc1 (pcELucN-PKA-McLuc1), which was inserted in a mammalian expression vector (Figure 1b). The vector was transiently transfected in HEK293 cells; then, the cells were incubated for 48 h. The cells were harvested, and the bioluminescence intensities were measured after addition of cAMP. The bioluminescence intensity increased 3-fold upon addition of 10 µM cAMP (Supporting Figure S1, Supporting Information). The background bioluminescence was relatively strong in the absence of cAMP. The background may originate from a spontaneous complementation between ELucN and McLuc1; it has been shown that the two fragments have a character of week affinity and the bioluminescence activity is recovered upon

Figure 1. Basic principle of cAMP indicator. (a) Scheme of the cAMP indicator. In the absence of cAMP, McLuc1 is complementary to the CBR-N, generating red color bioluminescence. Upon increasing the cAMP concentration, it binds to PKA-BD and the structural change occurs, which allows complementation between ELucN and McLuc1. Consequently, the green bioluminescence is observed. The ratios of red and green bioluminescence intensities show the quantitative concentration of cAMP. (b) Schematic structures of cDNA constructs. ELuc-N, the N-terminal fragment of ELuc (1-408 aa); PKA-BD, a cAMP binding domain of PKARIIβ (269-410 aa); McLuc1, the multiple-complement luciferase fragment; CBR-N, the N-terminal fragment of CBR (1-414 aa). In fact, pcELucN-PKA-McLuc1-CBRmN is a variant of pcELucN-PKAMcLuc1-CBRN including three point amino acid mutations, R215S, V234R, and Q348A in the CBR-N.

overexpression of the fragments in live cells.20 The bioluminescence spectrum exhibited a major peak at approximately 540 nm, which was almost consistent with a spectral property of a fulllength larval click beetle luciferase from Brazil (Supporting Figure S1, Supporting Information).17–19 In the control experiments, McLuc1 was removed from pcELucN-PKA-McLuc1 to construct pcELucN-PKA. The vector was transiently transfected in HEK293 cells, and the protein was expressed. The cells were harvested, and the bioluminescence intensity was measured before and after addition of cAMP. No bioluminescence was observed in both conditions, confirming that complementation of McLuc1 with ELucN was indispensable for generating bioluminescence (Supporting Figure S1, Supporting Information). Taken together, these results indicate that the fragments of McLuc1 and ELucN were reconstituted into the active form upon binding of cAMP to PKABD. Next, we constructed a ratiometric bioluminescent indicator for cAMP. The C-terminal end of the above construct, ELucNPKA-McLuc1, was connected with the N-terminal fragment of CBR, which was named ELucN-PKA-McLuc1-CBRN (Figure 1b). The fusion protein was expressed transiently in HEK293 cells. The indicator was characterized using a luminometer equipped

with separate band-pass filters (525 ± 25 and 630 ± 30 nm). The bioluminescence intensity taken using the filter for ELucNMcLuc1 complementation (525 ± 25 nm) was found to increase upon injection of cAMP, although the intensity taken by the filter for CBRN-McLuc1 complementation (630 ± 30 nm) showed no difference between the presence and absence of cAMP (Figure 2a). The absolute photon count through the filter at 630 ± 30 nm is 20-fold higher than that through the filter at 525 ± 25 nm. The ratios of 525 ± 25 to 630 ± 30 nm emissions revealed a large difference upon addition of 10 µM cAMP. The indicator also showed a dose-dependent response to cAMP ranging from 1.0 × 10-7 to 1.0 × 10-4 M (Figure 2b), although the standard deviation is large. In the control experiments, the expression vectors of fulllength ELuc and CBR were separately or simultaneously transfected in the cells and the proteins were expressed. The cells were harvested, and the bioluminescence intensity was measured upon addition of cAMP. In the cell lysates harboring either ELuc or CBR, there was no difference in the bioluminescence intensity between the presence and absence of cAMP (Supporting Figure 2a,b, Supporting Information). In the cell lysates harboring both ELuc and CBR, the bioluminescence ratio of ELuc to CBR also showed no difference upon addition of cAMP (Supporting Figure 2c, Supporting Information), confirming that the bioluminescence intensities of ELuc and CBR were not affected by cAMP. From these results, we concluded that the ELucN-PKA-McLuc1CBRN indicator enabled for ratiometric bioluminescence detection of cAMP. The large standard deviation of the indicator might have originated from weak bioluminescence from ELucN-McLuc1 complementation, which was buried by the strong bioluminescence from CBRN-McLuc1 complementation. To investigate that possibility, we measured the bioluminescence spectrum of the indicator in the presence of cAMP. It displayed only a single major peak at approximately 610 nm; no peak was detected near 540 nm (Supporting Figure 3a, Supporting Information), indicating that the red color bioluminescence signal was so strong that it was impossible to discriminate the red bioluminescence signal from the green one from ELucN-McLuc1 complementation. To improve the standard deviation of bioluminescence ratios, the red and green bioluminescence intensities were made comparable by reducing only the red bioluminescence intensities. Several amino acid residues in the substrate binding region of CBR were selected and mutated using site-directed mutagenesis to maintain the ability to complement with McLuc1. The products were connected directly with the C-terminal end of ELucN-PKAMcLuc1, and the fusion constructs were expressed in COS-7 cells. Among the mutant proteins that were examined, an N-terminal fragment of CBR including three point mutations, R215S, V234R, and Q348A, demonstrated the most appropriate properties: a bioluminescence intensity near 540 nm appeared to increase upon addition of 8-Br-cAMP, whereas the bioluminescence intensity at 610 nm was almost constant (Supporting Figure 3b, Supporting Information). This mutant, named ELucN-PKA-McLuc1-CBRmN, was used for additional experiments. We characterized the ELucN-PKA-McLuc1-CBRmN indicator by expressing it in HEK293 cells. When the lysates of the cells were analyzed, bioluminescence intensity through the filter of 525 ± 25 nm showed a marked increase in the presence of cAMP Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Figure 2. Characterization of ratiometric cAMP indicators. (a) Photon counts of ELucN-PKA-McLuc1-CBRN in the absence and presence of 10 µM cAMP. The band-pass filter used for color separation is presented on each bar graph. (b) Concentration dependence of cAMP for ELucNPKA-McLuc1-CBRN. Mean bioluminescence intensities and the standard deviations (n ) 3) were determined at each cAMP concentration. (c) Photon counts of ELucN-PKA-McLuc1-CBRmN in the absence and presence of 10 µM cAMP. (d) Concentration dependence of cAMP (black circles) and cGMP (open circles) for ELucN-PKA-McLuc1-CBRmN. Mean bioluminescence intensities and the standard deviations (n ) 3) were determined at each cAMP concentration.

(Figure 2c). In contrast, a little change in the bioluminescence intensity through the filter of 630 ± 30 nm was obtained between the presence and absence of cAMP. The ratios of 525 ± 25 to 630 ± 30 nm emissions showed a dose-response sigmoidal curve with significantly improved standard deviations in comparison to the indicator: ELucN-PKA-McLuc1-CBRN (Figure 2d). In addition, the indicator was quite selective for cAMP over cGMP at concentrations of 1.0 × 10-7 to 1.0 × 10-4 M. It has been reported that cellular cAMP concentration was maintained in the range of 9310

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1.0 × 10-7 to 1.0 × 10-5 M under physiological conditions.23 The concentration range in live cells was consistent with the above dynamic range of the indicator, demonstrating that the indicator of ELucN-PKA-McLuc1-CBRmN enabled selective detection for cAMP over cGMP at the physiological concentration range. (23) Jiang, L. I.; Collins, J.; Davis, R.; Lin, K.; DeCamp, D.; Roach, T.; Hsueh, R.; Rebres, R. A.; Ross, E. M.; Taussig, R.; Fraser, I.; Sternweis, P. C. J. Biol. Chem 2007, 282, 10576–10584.

Figure 3. Effects of ATP and D-luciferin concentrations on the indicator. Bioluminescence intensities of the indicator were measured using 10 µM cAMP and different concentrations of ATP and D-luciferin. (a) Concentration dependence of ATP on the photon count (left) and bioluminescence ratio (right). The concentration of D-luciferin was fixed as 2 mM. (b) Concentration dependence of D-luciferin on the photon count (left) and bioluminescence ratio (right). The concentration of ATP was fixed as 2 mM (open triangles, 525 ( 25 nm filter; black squares, 630 ( 30 nm filter; open circles, bioluminescence ratios of 525 ( 25 nm/630 ( 30 nm)

Effects of D-Luciferin and ATP Concentrations on the Ratiometric Bioluminescence Indicator. The bioluminescence intensity of luciferase is well-known to be linearly proportional to the concentrations of D-luciferin and ATP.24,25 Fluctuation of the D-luciferin and ATP concentration causes misunderstanding of luciferase activities. To validate the effects of D-luciferin and ATP on the present indicator, the indicator was expressed in HEK293 cells. The cell lysates were used for dual color bioluminescence assays with different concentrations of D-luciferin and ATP in the presence of 10 µM cAMP. Results showed that the ratio of bioluminescence intensities (525 to 630 nm) was almost constant (Figure 3). In contrast, absolute photon counts of the indicator increased linearly with increased concentrations of ATP and D-luciferin. These results confirmed that changes of D-luciferin or ATP concentrations did not affect ratiometric analysis of the indicator. (24) Gajewski, C. D.; Yang, L. C.; Schon, E. A.; Manfredi, G. Mol. Biol. Cell 2003, 14, 3628–3635. (25) Berovic, N.; Parker, D. J.; Smith, M. D. Eur. Biophys. J. 2009, 38, 427– 435.

Real-Time Ratiometric Bioluminescence Analysis of cAMP in Live Cells. To show applicability of the indicator in live cells, we transiently expressed pcELucN-PKA-McLuc1-CBRmN in HEK293 cells and obtained the bioluminescence with green and red long-pass filters (LP560 and LP600 nm). The bioluminescence intensities through each filter were used to calculate independent bioluminescence intensities of ELucN-McLuc1 and McLuc1CBRmN complementation. No change in the bioluminescence was obtained upon stimulation with either HBSS buffer or 5 µM SNP, which is an inducer of cGMP synthesis (Figure 4a). In contrast, addition of 10 µM ISO induced a transient increase in the bioluminescence from ELucN-McLuc1 complementation, although no change was apparent in the bioluminescence from CBRmNMcLuc1 complementation. The ratios of green to red bioluminescence showed almost the same response as those of the ELucNMcLuc1 complementation (Figure 4b). Furthermore, ISO was added to the cells in the presence of a phosphodiesterase type 4 (PDE4) inhibitor, rolipram: an inhibitor of cAMP-specific hydrolysis enzyme (Figure 4c,d). Ratiometric analysis revealed that the signal was increased and sustained to the high ratio value, indicating that the response for ISO had indeed originated from the production of cAMP inside the cells. We further examined responses of the indicator for another stimulative, forskolin, which is an activator of adenylate cyclase. Addition of 100 µM forskolin strongly elevated the ratio value for 60 min (Figure 4e), demonstrating that the indicator is used for monitoring endogenous cAMP in subpopulation of living cells. Imaging of cAMP in Single Live Cells. We examined the capability of the indicator for imaging cAMP in living cells. The indicator was expressed transiently in the cytosol of HEK293 cells. Time-lapse images of bioluminescence taken with bandpass filters (536 ± 10 and 624 ± 25 nm) and bright-field images were obtained under a bioluminescence microscope. Upon stimulation of the cells with 100 µM forskolin, a rapid increase in the bioluminescence through the filter (536 ± 10 nm) was observed in the cytoplasm of the cells (Figure 5a and Supporting Information movie ac102692u_si_002.avi). In contrast, no change in the bioluminescence through the filter (624 ± 25 nm) was obtained (Figure 5b and Supporting Information movie ac102692u_si_003.avi). The ratiometric images were calculated from the images through the filters (Figure 5c and Supporting Information movie ac102692u_si_004.avi), indicating a rapid increase in the cAMP upon injection of forskolin. It reached a plateau thereafter, which indicates that the indicator is used for spatial and temporal analysis of endogenous cAMP in living cells. Imaging of cAMP in Living Mice. To demonstrate a further application of the indicator, we applied the indicator to imaging of cAMP in living mice. We expressed transiently the indicator in COS7 cells and implanted the cells into the back of mice. Timelapse images of bioluminescence taken with band-pass filters (520 ± 25 and 630 ± 37.5 nm) were obtained using a cooled CCD camera. The mice were intraperitoneally injected with 150 µL of 20 mM ISO to stimulate endogenous cAMP production. Ten minutes after stimulation with ISO, a weak but significant increase in the bioluminescence through the filter (520 ± 25 nm) was observed (Figure 6a,b). In contrast, no change in the bioluminescence through the filter (630 ± 37.5 nm) was obtained upon Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Figure 4. Time-dependent response in bioluminescence. (a) HEK 293 cells including ELucN-PKA-McLuc1-CBRmN indicator were stimulated with HBSS, 5 µM SNP, and 10 µM ISO. Open triangles and solid squares, respectively, represent ELucN-McLuc1 and McLuc1-CBRmN complementation. (b) The ratio of bioluminescence intensities acquired in (a). ELuc and CBR, respectively, show photon counts of ELucNMcLuc1 and McLuc1-CBRmN complementation. (c) Time-course analysis of photon count in the presence of rolipram. The HEK293 cells were cultured in 100 nM rolipram and stimulated with 10 µM ISO. (d) Ratio of bioluminescence intensities acquired in (c). (e) Time-course analysis of bioluminescence ratios upon stimulation of 100 µM forskolin.

Figure 5. Live cell imaging of cAMP. HEK293 cells harboring ELucN-PKA-McLuc1-CBRmN indicator were treated with 100 µM forskolin. The injection time was defined at 0 min. Bioluminescence images were acquired every 2 min with a band-pass filter (536 ( 10 nm) for ELucNMcLuc1 complementation (a) and with a band-pass filter (624 ( 25 nm) for McLuc1-CBRmN complementation (b). The ratio images of bioluminescence (536 ( 10 nm/624 ( 25 nm) were visualized and overlaid on bright field images in (c). Bar: 50 µm.

stimulation with ISO (Figure 6c,d). To evaluate the effect of ISO on the endogenous cAMP level quantitatively, absolute photon counts and the bioluminescence ratio (520 nm/630 nm) were calculated. The bioluminescence ratio increased 2-fold upon stimulation with ISO (Figure 6e). When we consider the fact that the bioluminescence ratio is not affected by the 9312

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expression level of the indicator or fluctuations of substrate concentrations, the results demonstrate that the increase in bioluminescence ratio was originated from endogenous cAMP production in living mice. We developed a novel ratiometric bioluminescence indicator for cAMP. This indicator is based on complementation of two

Figure 6. In vivo imaging of cAMP in living mice. Bioluminescence images of the mice implanted with COS7 cells harboring ELucN-PKAMcLuc1-CBRmN indicator were taken before and after stimulation with 20 mM ISO. (a) A bioluminescence image taken with a band-pass filter (520 ( 25 nm) before stimulation with ISO. (b) A bioluminescence image taken with a band-pass filter (520 ( 25 nm) 10 min after stimulation with ISO. (c) A bioluminescence image taken with a band-pass filter (630 ( 37.5 nm) before stimulation with ISO. (d) A bioluminescence image taken with a band-pass filter (630 ( 37.5 nm) 10 min after stimulation with ISO. (e) Comparison of the bioluminescence ratios before and after stimulation with ISO. Bioluminescence intensities of the implant areas (orange outlined areas) were quantified to calculate the bioluminescence ratio (520 nm/630 nm). Data represent the mean ( standard deviations of three mice.

luciferase N-terminal fragments with one shared C-terminal fragment. Obtained results show that the indicator enabled fluorescent-background-free, accurate, and selective detection, which is a great advantage for monitoring the cellular cAMP level in live cells. The ratiometric bioluminescence analysis has overcome a previous disadvantage of the photon counting analysis of bioluminescence with a single wavelength, which irregularly alters the concentrations of cofactors such as ATP and D-luciferin, making them fluctuate. To date, the analysis of cAMP in living cells has depended mostly on FRET-based techniques in combination with the fluorescence microscopy and computer-driven imaging system. Those systems offer only semiquantitative information because it is difficult in each cell to set uniform initial values of FRET ratios. That difficulty often hampers the accurate analysis of cAMP levels. Furthermore, precision of the FRET signals is not high because the dynamic range of the signals is narrow and the number of the cells examined under a fluorescence microscope is limited. In contrast, the present method using the ratiometric bioluminescent indicator makes it possible to analyze more than 104 cells at once, which is sufficient for precise evaluation of the average extent of cAMP in living cells. The ratiometric bioluminescence indicator enabled imaging of cAMP in living mammals. The indicator can avoid the difficulties such as low transmittance of excitation light and high background autofluorescence of tissues of mammals. The unique properties of the indicator will allow for pathological analyses such as evaluating the effective concentration of metabolized chemicals

in particular tissues or organs of living mammals. In addition, the indicator will also provide versatile applications to pharmacological and toxicological analyses, including assessment of novel anticancer drugs and the evaluation of risk factors of toxic chemical compounds on living mammals. Thus, the indicator will have broad applicability in in vivo experiments using mammals. Finally, the basic scheme of the present detection method is applicable to creating other ratiometric bioluminescent indicators by replacing the PKA-BD with a ligand-binding domain of interest. Development of novel ratiometric bioluminescent indicators for other biological molecules will open new avenues for in vivo imaging analyses in living mammals. ACKNOWLEDGMENT This work was supported by the Japan Society for the Promotion of Science (JSPS), Japan Science and Technology Corporation (JST), and New Energy and Industrial Technology Development Organization (NEDO) and in part by the Global COE Program and a grant (S0801035) from MEXT, Japan. SUPPORTING INFORMATION AVAILABLE Figures S1-S3 and Movies ac102692u_si_002-004.avi. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review July 8, 2010. Accepted October 15, 2010. AC102692U

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