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. Author manuscript; available in PMC: 2021 Mar 17.
Published in final edited form as: Methods Mol Biol. 2020;2173:201–216. doi: 10.1007/978-1-0716-0755-8_14

Dual-activation of cAMP production through photo-stimulation or chemical stimulation

Nyla Naim 1,2,3, Jeff M Reece 1,4, Xuefeng Zhang 1, Daniel L Altschuler 5
PMCID: PMC7968876  NIHMSID: NIHMS1671895  PMID: 32651920

Abstract

cAMP is a crucial mediator of multiple cell signaling pathways. This cyclic nucleotide requires strict spatio-temporal control for effective function. Light-activated proteins have become a powerful tool to study signaling kinetics due to having quick on/off rates and minimal off-target effects. The photoactivated adenylyl cyclase from Beggiatoa (bPAC) produces cAMP rapidly upon stimulation with blue light. However, light delivery is not always feasible, especially in vivo. Hence, we created a luminescence-activated cyclase by fusing bPAC with Nanoluciferace (nLuc) to allow chemical activation of cAMP activity. This dual-activated adenylyl cyclase can be stimulated using short bursts of light or long-term chemical activation with furimazine and other related luciferins. Together these can be used to mimic transient, chronic, and oscillating patterns of cAMP signaling. Moreover, when coupled to compartment-specific targeting domains, these reagents provide a new powerful tool for cAMP spatiotemporal dynamic studies. Here, we describe detailed methods for working with bPAC-nLuc in mammalian cells, stimulating cAMP production with light and luciferins, and measuring total cAMP accumulation.

Keywords: cAMP, adenylyl cyclase, optogenetics, bPAC, nanoluciferase

1. Introduction

cAMP (cyclic adenosine monophosphate) is a ubiquitous signaling molecule found in all prokaryotic and eukaryotic cells. It was first identified as a second messenger in G-protein coupled receptor (GPCR) signaling and is now recognized as a common node in multiple pathways. cAMP is integral to basic cell function as it regulates proliferation, differentiation, migration, attachment, and many other processes. Hence, irregular cAMP signaling has been linked to several diseases. For example, activating mutations that lead to constitutive cAMP accumulation have been reported in thyroid hyperplasia and adenoma [1,2]. Diminished cAMP signaling has been found in certain mood disorders, Alzheimer’s disease, and multiple sclerosis [3,4]. Furthermore, a growing body of literature suggests dysregulation of localized cAMP signaling can elicit physiological consequences [5].

Steady-state intracellular cAMP levels are primarily determined by the rate of synthesis (i.e. adenylyl cyclases) and degradation (i.e. phosphodiesterases). Several reports have shown that the basal concentration of cellular cAMP is between 0.1—1 μM depending on the cell type [6,7]. Upon hormonal stimulation, this can increase to more than 10-fold [8,9]. Super-physiologic levels of cAMP (i.e. up to 90-fold) can be reached by stimulating adenylyl cyclases with forskolin and inhibiting phosphodiesterases with IBMX (3-isobutyl-1-methylxanthine) [10]. Cellular cAMP concentration fluctuates following three temporal patterns: transient, sustained, and oscillating (see Fig. 1a). Transient spikes of cAMP production after GPCR stimulation only last on the order of minutes due to phosphodiesterase activity [11-14] and cAMP-dependent protein kinase A (PKA)-driven negative feedback loops. Sustained cAMP production has been reported during GPCR endocytosis [15-20] and activating mutations along the pathway can lead to constitutive cAMP production [21-24]. Additionally, cAMP oscillations have been reported in pancreatic β cells [25,26], neurons [27,28], pituitary cells [29], and myocardiac cells [30].

Fig. 1.

Fig. 1

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(a) Diagram of cAMP concentration over time (t) following three temporal patterns. (b) Depiction of cellular cAMP compartmentalization or localized cAMP signaling from transmembrane adenylyl cyclases (tmAC) and soluble adenylyl cyclases (sAC). cAMP diffuses unevenly through the cell and gradients are largely shaped by phosphodiesterase activity.

The concentration and temporal patterns of cAMP accumulation alone do not fully account for how cAMP activates distinct pathways [7,31]. Over the last few decades, mounting evidence suggests cAMP is spatially regulated, forming regions of high and low concentrations (see Fig. 1b). This uneven distribution, or compartmentalization, allows preferential activation of specific signalosomes [32]. Under this model, studies suggest local cAMP signaling is regulated by adenylyl cyclase proximity to specific cAMP effectors [33], cAMP efflux [34], buffering by effector proteins [35,36], phosphodiesterase barriers or sinks [37], restricted diffusion due to steric hindrance from intracellular structures [38,39], and overall cell geometry [40,41]. Hence to further validate this model and mimic cAMP signaling from transmembrane and soluble adenylyl cyclases, tools with high spatial and temporal control are needed.

While there are several pharmacological agents available to modulate cAMP levels, chemical activation lacks cellular specificity and has limited spatio-temporal control. Genetically encoded tools can help overcome these issues. For example, the optogenetic photo-activated adenylyl cyclase from beggiatoa (bPAC) has been successfully used for tissue specific expression [42,43] and sub-cellular localization [44,45] of cAMP synthesis. bPAC is a ~40 kDa monomer containing a BLUF (sensors of blue-light using FAD) domain and Type III adenylyl cyclase domain [46,47]. As a dimer, it is activated by 435—455 nm light exposure within milliseconds and can produce up to a 300-fold increase in cAMP [47,48]. bPAC returns to its dark-state quickly (τoff=12 sec) although residual cAMP accumulation is reported (τ=23 ± 2 sec) [47]. bPAC offers unique optogenetic strategies to study cAMP; however, light delivery is not always feasible in some lab techniques, in vivo operations, and can be phototoxic if not regulated. To circumvent issues with light delivery and expand the methods of activation for optogenetic proteins, Hochgeschwender and colleagues created a fusion protein of the light-sensitive channelrhodopsin ion channel and Gaussia luciferase. This ‘luminopsin’ represented a new class of proteins with both optogenetic and chemical regulation of activation [49-51].

Following this concept, our group created a luminescence-activated adenylyl cyclase by fusing bPAC to nanoluciferase (nLuc). nLuc was selected for its small size (19 kDa) and blue-shifted luminescence in response to the luciferins furimazine (Fz) and h-coelenterazine (h-CTZ) [52]. A myc-tag was included between the two moieties to aid identification and visualization. The resulting bPAC-nLuc fusion protein (~62 kDa) can be activated by blue light or chemical stimulation of luminescence [53] (see Fig. 2a). This protocol describes the general working conditions and optimization process for using bPAC-nLuc in mammalian cell lines. bPAC-nLuc expressing cells must be kept away from light <500 nm. Upon stimulation with luciferins, such as furimazine, luminescence can be measured and used to confirm expression. For effective photo-activation using a blue LED light source, it is important to have even illumination across a sample. cAMP accumulation resulting from either chemical- or photo-activation can be measured using an enzyme linked immunoabsorbant assay (ELISA) or live-cell imaging with a cAMP biosensor. Here, a rat hepatoma cell line (HC1) [54,55] was selected for most assays because they exhibit low basal cAMP levels and high transfection efficiency. For live-cell imaging, rat follicular thyroid cells (PCCL3) [56] were used since they are more physiologically relevant to our interests. These techniques can be adopted to other cells lines, including human embryonic kidney cells (HEK293) [53]; however, the expression level of bPAC-nLuc and light/chemical dosage may need to be tuned to match endogenous cAMP levels.

Fig. 2.

Fig. 2

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(a) Schematic of luminescence activated cyclase, bPAC-nLuc, which can be activated by blue light and luciferins. (b) An image of an Arduino controlled LED mounted on a stage where cell culture dishes can be placed above. (c) The measured irradiance at 440 nm of a royal blue LED at varying intensities (mean ± SD of 54 measurements across the area of a 6-well dish). This research was originally published in the Journal of Biological Chemistry. Naim et al. Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis. J. Biol. Chem. 2018; 294:1095-1103. © the American Society for Biochemistry and Molecular Biology.

Dual regulation of bPAC-nLuc using light and chemical activation provides flexibility in experimental design. Transient and sustained cAMP synthesis can be tested using the same construct, creating a broad range of temporal signatures. Interestingly, new analogs of furimazine, such as Endurazine™ and Vivazine™ (Promega), have been created for extended luminescent lifetimes (>48 hr) and lower cytotoxicity. Furthermore, we found bPAC-nLuc was easily targeted to various sub-cellular locations by adding targeting motifs to the N- and C-termini [53]. Hence, bPAC-nLuc can be used to study location-biased cAMP signaling or compartmentalization with a high degree of temporal control, thus expanding the available toolkit to artificially regulate local cAMP synthesis.

2. Materials

2.1. Cell Culture Materials

  1. HC1 cells (kindly provided by Dr. Elliot Ross, Department of Pharmacology, University of Texas Southwestern Medical Center)

  2. Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning #10-013-CV)

  3. PCCL3 cells (kindly provided by Dr. James Fagin, Department of Medicine, Memorial Sloan Kettering Cancer Center)

  4. Complete Coon’s Media: Nutrient Mixture F-12 Ham (Coon’s modification, Sigma-Aldrich, F6636) supplemented with 2.68 g/L sodium bicarbonate, 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 2 mM L-glutamine, and 4 Hormone solution: insulin (1 μg/mL), apo-transferrin (5 μg/mL), hydrocortisone (1 nM), and thyroid stimulating hormone (1 IU/L).

  5. Starvation Coon’s media: Complete Coon’s Media (still containing 5% FBS) supplemented with 0.2 % bovine serum albumin but lacking insulin, hydrocortisone, and thyroid stimulating hormone

  6. Phosphate Buffered Solution without Ca+2 or Mg+2 (PBS)

  7. 0.25 % Trypsin – 22.1 mM EDTA (Corning #25-053-CI)

  8. Cell dissociation solution, non-ezymatic 1x (Sigma-Aldrich #C5914)

  9. 0.1 % gelatin (prepared in PBS)

  10. 10 cm and 6-well dishes (Corning)

  11. 15 mL conical tubes (Corning)

  12. 96-well dishes, white/opaque (Corning)

  13. 25-mm glass coverslips

  14. Opti-MEM, phenol-red free (Invitrogen)

  15. X-tremeGENE HP (Roche)

  16. Lipofectamine 3000 (Thermo Fisher Scientific)

  17. pcDNA3.1+ bPAC-myc-nLuc DNA [53]

  18. Red Dimerization Dependent Sensor for cAMP (Montana Molecular)

  19. Laminar Flow Hood for sterile cell culture

  20. 37 °C / 5 % CO2 incubator

  21. 37 °C hot water bath

  22. Hemocytometer

2.2. bPAC-nLuc Activation and Lighting Conditions

  1. Thor Labs Laser Power Meter (ThorLabs PM1100D, detector S130C)

  2. Blackout curtains and/or heavy-weight, opaque paper (optional for blocking light from doors/windows)

  3. Aluminum foil

  4. A heavy-duty cutting blade

  5. 13 W amber compact fluorescence bulb (Low Blue Lights, Photonic Developments LLC)

  6. Red safelight lamp (Kodak GBX-2 Safelight Filter)

  7. NanoGlo® Luciferase Substrate (Furimazine, Promega)

  8. Arduino-controlled LED system (full description and parts list in Naim et. al. 2018) [53]

  9. High-power LED (royal blue CREE XTE Tri-Star LED, LED Supply)

2.3. cAMP ELISA

  1. Monoclonal Anti-cAMP Antibody Based Direct cAMP ELISA Kit (non-acetylated, NewEast Biosciences)

  2. 0.1 M HCl (prepared in ultra-pure water)

  3. Pierce™ BCA Protein Assay Kit

  4. TeccanSpark 20M plate reader (SparkControl V1.2 software)

  5. 3-isobutyl-1-methylxanthine (IBMX, stock prepared in DMSO, Sigma-Aldrich)

2.4. Live Cell Imaging

  1. Imaging chamber

  2. Olympus IX70 microscope

  3. Till Polychrome V monochromator

  4. 60x/1.4 NA oil objective

  5. Hamamatsu CCD camera (Photonics Model C4742-80-12AG)

  6. Slidebook software (Intelligent Imaging Innovations Inc.)

  7. Emission filter FF01-620/52 (Semrock)

  8. Emission filter S470/30m (Chroma Technology Corp)

  9. 560 nm long pass dichroic (HQ570 LP, Chroma Technology Corp)

  10. Forskolin (stock prepared in DMSO, Sigma-Aldrich)

3. Methods

3.1. LED Calibration

  1. To approximate the amount of light through polystyrene cell culture dishes and ensure even light distribution, set up the LED light source mounted on a stage that fits a standard 6-well dish (see Note 1) (see Fig. 2b).

  2. Prepare the lid of a 6-well dish by carefully remove the edges from one or two sides of the lid using a heavy-duty cutting blade so that the laser power meter can lay flat against the plastic.

  3. Place the lid upside-down on the stage above the LED light source.

  4. Turn on the LED (see Note 2)

  5. Place the laser power meter wand flat against the lid and measure the 440 nm light intensity at multiple points across the lid, ensuring areas where wells would reside are well represented (i.e. 9 measurements per well of a 6-well dish)

  6. Calculate the irradiance (power per area, μW/mm2) using the measured intensity values and area of the probe sensor.

  7. Repeat process for each LED intensity used (see Fig. 2c).

3.2. Cell Culture Environment

All cells and samples containing functional bPAC-nLuc (i.e. during cell culture but not necessarily after cell lysis or fixation) should be kept from light below 500 nm to prevent protein activation. This can be accomplished by covering windows and doorways using blackout curtains, aluminum foil, or heavy-weight opaque paper. Use a laser power meter to detect diffuse light at 430—460 nm wavelengths. Light sources should be above 500 nm. Combining an amber light bulb in a red safelight lamp provides sufficient light for performing cell culture while maintaining low basal activity of bPAC-nLuc. All cell culture should be performed in a sterile environment using pre-warmed reagents. All buffers should be prepared in ultra-pure water unless otherwise noted.

3.3. HC1 Cell Culture & Transfection

  1. Culture HC1 cells in DMEM containing 10% FBS, penicillin, and streptomycin in a 10 cm dish at 37 °C, 5 % CO2. Passage every 2—3 days once cells are 80—90 % confluent using 1:5 dilutions.

  2. Aspirate media and wash in 5 mL of PBS.

  3. Aspirate PBS and incubate with 1 mL of trypsin for 3—5 min in an incubator.

  4. Once cells have detached, resuspend the cells in 4 mL of media and transfer to a 15 mL conical tube.

  5. If seeding cells for an experiment, count the cell density using a hemocytometer.

  6. Dilute the cell suspension in media to the desired final cell concentration.

  7. Pipette 1:5 cell suspension into a new 10 cm dish or the desired cell number for experimental dishes.

  8. For transient transfection, wait 24 hr after cell seeding.

  9. Provide cells with fresh media.

  10. Transfect each well with bPAC-nLuc using X-tremeGENE HP, following the manufacturer’s orders using a ratio of 1 μg DNA / 2 μL X-tremeGENE reagent / 100 μL Opti-MEM. Use 10 μL of transfection mixture per well of a 96-well dish, and 100 μL per well of a 6-well dish (see Note 3).

  11. Incubate 24 hr protected from light (i.e. using aluminum foil) (see Note 4).

3.4. Chemical Luminescence Activation Assay

  1. Seed 8,000 HC1 cells per well in a white, opaque 96-well dish using a 100 μL volume.

  2. Incubate 24 hr, transfect bPAC-nLuc as described above, and incubate 24 hr.

  3. Prepare furimazine solution in phenol red-free Opti-MEM for a two-fold serial dilution ranging from 1:20 – 1:2560. Prepare enough solution to run samples in triplicate.

  4. Aspirate media and wash wells once in PBS.

  5. For each dose, aspirate media from three wells and add 100 μL of furimazine to each.

  6. Measure luminescence on a plate reader immediately as the intensity decays within seconds.

  7. Repeat steps 12 and 13 for each dose (see Fig. 3a) (see Note 5).

Fig. 3.

Fig. 3

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(a) Measured relative luminescence (RLU) of HC1 cells expressing bPAC-nLuc or bPAC-mCherry stimulated with varying dilutions of Furimazine (mean ± SD of n=4). (b-d) Live cell, real-time monitoring of cAMP accumulation in PCCL3 cells co-expressing the Red Dimerization Dependent Sensor for cAMP with bPAC-nLuc or myc-empty vector (myc-eV) (representative cell traces shown). (b) Cells were stimulated with light pulses of varying duration at ~4 μW/mm2 (250 μM IBMX indicates sensor saturation point) or (c,d) with 25 μM h-coelenterazine (h-CTZ), a luciferin capable of activating nLuc (100 μM IBMX and 10 μM forskolin (Fsk) indicate sensor saturation). Panel B was originally published in the Journal of Biological Chemistry. Naim et al. Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis. J. Biol. Chem. 2018; 294:1095-1103. © the American Society for Biochemistry and Molecular Biology.

3.5. cAMP ELISA of bPAC-nLuc Activity

  1. Seed 1.5 * 105 HC1 cells per well in two 6-well dishes and incubate 24 hr.

  2. Transfect cells with bPAC-nLuc following the protocol described above and incubate 24 hr (see Note 6).

  3. Prepare stimulating conditions in 1 mL media containing either DMSO (vehicle), 100 μM IBMX (basal cAMP activity, see Note 7), 1:100 furimazine (activation), or 100 μM IBMX + 1:100 furimazine (max activation). Other dilutions of furimazine can be used after optimizing the protocol (see Note 8).

  4. Aspirate media from one dish and add the stimulating reagents.

  5. Incubate 10 min in incubator.

  6. Aspirate and wash with PBS.

  7. Aspirate and lyse in 100 μL of 0.1 M HCl for 10 min at room temperature (see Note 9).

  8. Collect samples by scraping cells with a rubber policeman and storing at −80 °C.

  9. Using second dish of cells, treat with DMSO (vehicle) or IBMX (max activation) for 10 min at 37 °C.

  10. During the last minute, stimulate with LED using a 1 min pulse of 440 nm light (see Note 10).

  11. Immediately wash with PBS and lyse as in steps 6—8.

  12. Measure cAMP using an ELISA kit following the manufacturer’s directions.

  13. Quantify cAMP accumulation per amount of protein loaded (pmol cAMP / μg protein lysate) which can be measured using a bicinchoninic acid assay (BCA).

3.6. Live-cell Monitoring of bPAC-nLuc Activation

  1. Culture PCCL3 cells in ‘Complete Coon’s Media’ (see materials) in a 10 cm dish at 37 °C, 5 % CO2. Passage every 2—3 days once cells are 80—90 % confluent using 1:3 dilutions.

  2. Place sterilized 25 mm glass coverslips into a 6-well dish.

  3. Coat coverslips in 0.1 % gelatin solution for 30 min.

  4. Aspirate solution and wash with PBS.

  5. Remove a ~90 % confluent 10 cm dish of PCCL3 cells from the incubator and aspirate media.

  6. Wash with PBS.

  7. Aspirate and add a mixture of 2 mL of cell dissociation solution and 50 μL trypsin.

  8. Incubate approximately 5 min until cells begin to round.

  9. Aspirate the solution and gently dislodge cells by pipetting 10 mL of Coon’s complete medium across the surface of the dish.

  10. Count the cell density using a hemocytometer.

  11. Dilute the cell suspension as needed to seed 1.5 * 106 cells per well of the 6-well dish.

  12. Incubate 24 hr.

  13. Provide 1.5 mL of fresh media and co-transfect bPAC-nLuc and Red Dimerization Dependent Sensor for cAMP using Lipofectamine 3000 following the manufacturer’s instructions, using a ratio of 0.25 μg bPAC-nLuc DNA / 0.75 μg Red Dimerization Dependent Sensor for cAMP / 2 μL P3000 reagent / 3 μL Lipofectamine 3000 reagent for each well (see Note 11).

  14. Incubate approximately 24 hr.

  15. Aspirate media and add ‘Coon’s Starvation Media’ (see Materials) to each well 3 hr before imaging.

  16. Start the monochromator, microscope components, Arduino-controlled LED, and imaging software.

  17. Invert the LED-mounted stage and position the LED light source above the microscopy stage, using the same distance used for LED Calibration (3.2).

  18. Wash a coverslip in PBS and transfer to an imaging chamber.

  19. Add 0.9 mL of Opti-MEM (phenol red-free) to the chamber.

  20. Transport the chamber, protected from light, to the microscope stage.

  21. Setup imaging conditions to monitor red fluorescence (e.g. 60x oil objective, 20 % monochromator intensity, 15 nm bandpass, 570 nm excitation, 560 nm long pass dichroic, 620 nm emission filter.

  22. Identify red fluorescent cells (see Note 12) to find a field with cells that show diffuse localization of the sensor (see Note 13).

  23. Take a test image to select an exposure time between 200-500 msec and adjust the gain as needed.

  24. Allow cells to equilibrate without any excitation or light exposure for approximately 5 min.

  25. Begin imaging the cells using a 10 sec capture rate.

  26. Select each cell as a region of interest and monitor the intensity in real-time.

  27. Once a stable base line has been established (5—10 min), deliver varying pulses of light between 100 msec to 100 sec and monitor decreases in fluorescent intensity. Allow the intensity to return to basal levels between each pulse (see Fig. 3b).

  28. At the end of each experiment, saturate the sensor by adding 10 μM forskolin and 100 μM IBMX directly to the chamber. This can be done by pipetting 100 μL of a 10x working solution prepared in Opti-MEM.

  29. Finally, demonstrate bPAC-nLuc expression via luminescence. Take an image of the cell field luminescence using a long (10—15 sec) exposure using a CFP emission filter (470/30 nm) but with no excitation.

  30. Add a 1:1000 dose of furimazine and take another image of the luminescence.

  31. To test the effect of furimazine in real-time, repeat steps 18-24.

  32. Take a ‘before’ picture of cell luminescence as in step 29.

  33. Begin the imaging time course to monitor red fluorescence.

  34. After establishing a baseline, stimulate with 1:1000 furimazine pre-diluted in Opti-MEM (see Fig. 3c, d).

  35. Monitor for >30 min or until signal returns to baseline.

  36. Saturate sensor as in step 28.

  37. Take an ‘after’ picture of luminescence as in step 29.

  38. Save all files and export raw data to Excel.

  39. Subtract the background fluorescence and graph the change in red fluorescent intensity over time. This can be normalized as a percentage of the maximum and minimum signal.

  40. Quantify the change in luminescence between before and after images with background corrections.

Acknowledgements

This research was supported by National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH), and the Molecular Pharmacology Training Program of the University of Pittsburgh under grant Awards Number R01-GM09975, R01-GM130612, T32-GM00842419/20/21, and the Winstar Morris’ Cotswold Foundation Fellowship.

Footnotes

1

To excite bPAC-nLuc with 440 nm light, we used a custom-built Arduino-compatible LED system (see Fig. 2b). A complete parts list, description of how it was built, and the code used to alter light pulse frequency, duration, and intensity was previously described [53]. Other systems can be used as long as the LED emits blue wavelengths in the 440 nm range with an intensity of at least 4 μW/mm2, which is the half maximal activation reported [47]. For example, step-by-step guides to build LED arrays have been described by other groups [57,58].

2

Do not look directly into the LED light source as it can cause damage to the eyes. Protective eyewear is recommended.

3

bPAC-nLuc constructs with sub-cellular targeting domains can be used instead. Additionally, if transient transfections are not ideal, lentiviral version of bPAC-nLuc have been created and stable cell lines can be prepared [53].

4

In addition to the luminescence assay, expression can be tested in transfected cells using the myc-tag located between the bPAC and nLuc moiety. The anti-myc antibody, clone 9E10, can be used for both immunofluorescence and western blot analysis.

5

Other luciferins can be used to activate bPAC-nLuc as long as they emit strong blue light (~440 nm). For example, we have used h-coelenterazine (NanoLight Technology) and a prototype of the NanoGlo Endurazine Live Cell Substrate (Furimazine-4377, Promega). When testing new luciferins, ensure the light emitted covers blue wavelengths be measuring the luminescent spectrum. Furimazine emitted maximum luminescence at 455 nm [53]. Luminescence can also be monitored over time to provide kinetic data on the lifetime of luminescence.

6

Controls for transfection must be included in luminescence assays, ELISAs, and live-cell imaging. For example, an myc-empty vector can be used to control for transfection while bPAC (not fused with nLuc but preferably myc-tagged, i.e. bPAC-myc-mCherry) can be used demonstrate specificity of chemical activation.

7

bPAC has a reported dark activity of 33 ± 5 pmol/min/mg of protein [47]. Treating cells with IBMX helps estimate the basal activity of bPAC-nLuc and can help determine if lighting conditions are suitably dark.

8

To optimize activation conditions, compare cAMP accumulation to other stimulating agents with physiologically relevant, such as stimulating GPCR signaling.

9

Lysis conditions may be different depending on the ELISA kit protocol used. Follow manufacturer’s instruction.

10

1 min of light activation at 4 μW/mm2 is a useful starting point since it should stimulate very high levels of cAMP. The intensity and duration can be reduced after establishing the assay works as expected.

11

Other cAMP biosensors can be used as long as wavelengths below 500 nm are not required. Using a lower amount of bPAC-nLuc DNA helps prevent overexpression of the construct and saturation of the biosensor.

12

Do not use bright field imaging to identify cells as it can activate bPAC-nLuc activity.

13

Red Dimerization Dependent Sensor for cAMP should not form punctate dots, rather it should be expressed evenly throughout the cytoplasm.

5. References

  • 1.Ledent C, Dumont JE, Vassart G, Parmentier M (1992) Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism. EMBO Journal 11 (2):537–542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kosugi S, Shenker A, Mori T (1994) Constitutive activation of cyclic AMP but not phosphatidylinositol signaling caused by four mutations in the 6th transmembrane helix of the human thyrotropin receptor. FEBS letters 356 (2-3):291–294. doi: 10.1016/0014-5793(94)01286-5 [DOI] [PubMed] [Google Scholar]
  • 3.Gould TD, Manji HK (2002) Signaling networks in the pathophysiology and treatment of mood disorders. Journal of Psychosomatic Research 53 (2):687–697. doi: 10.1016/S0022-3999(02)00426-9 [DOI] [PubMed] [Google Scholar]
  • 4.Poppinga WJ, Muñoz-Llancao P, González-Billault C, Schmidt M (2014) A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases. British Journal of Pharmacology 171 (24):5603–5623. doi: 10.1111/bph.12882 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Gold MG, Gonen T, Scott JD (2013) Local cAMP signaling in disease at a glance. Journal of cell science 126 (20):4537–4543. doi: 10.1242/jcs.133751 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Borner S, Schwede F, Schlipp A, Berisha F, Calebiro D, Lohse MJ, Nikolaev VO (2011) FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6 (4):427–438. doi: 10.1038/nprot.2010.198 [DOI] [PubMed] [Google Scholar]
  • 7.Koschinski A, Zaccolo M (2017) Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling. Scientific reports 7 (1):14090–14090. doi: 10.1038/s41598-017-13021-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Terasaki WL, Brooker G (1977) Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium. The Journal of biological chemistry 252 (3):1041–1050 [PubMed] [Google Scholar]
  • 9.Iancu RV, Ramamurthy G, Warrier S, Nikolaev VO, Lohse MJ, Jones SW, Harvey RD (2008) Cytoplasmic cAMP concentrations in intact cardiac myocytes. American journal of physiology Cell physiology 295 (2):C414–422. doi: 10.1152/ajpcell.00038.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Smith FD, Esseltine JL, Nygren PJ, Veesler D, Byrne DP, Vonderach M, Strashnov I, Eyers CE, Eyers PA, Langeberg LK, Scott JD (2017) Local protein kinase A action proceeds through intact holoenzymes. Science 356 (6344): 1288–1293. doi: 10.1126/science.aaj1669 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. The Journal of biological chemistry 279 (36):37215–37218. doi: 10.1074/jbc.C400302200 [DOI] [PubMed] [Google Scholar]
  • 12.Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2 (1):25–29. doi: 10.1038/71345 [DOI] [PubMed] [Google Scholar]
  • 13.Cui W, Smith A, Darby-King A, Harley CW, McLean JH (2007) A temporal-specific and transient cAMP increase characterizes odorant classical conditioning. Learning & Memory 14 (3):126–133. doi: 10.1101/lm.496007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Vedel L, Brauner-Osborne H, Mathiesen JM (2015) A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors. J Biomol Screen 20 (7):849–857. doi: 10.1177/1087057115580019 [DOI] [PubMed] [Google Scholar]
  • 15.Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, Persani L, Lohse MJ (2009) Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 7(8):e1000172. doi: 10.1371/journal.pbio.1000172 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ, Vilardaga J-P (2009) Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nature chemical biology 5 (10):734–742. doi: 10.1038/nchembio.206 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kuna RS, Girada SB, Asalla S, Vallentyne J, Maddika S, Patterson JT, Smiley DL, DiMarchi RD, Mitra P (2013) Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells. American Journal of Physiology-Endocrinology and Metabolism 305 (2):E161–E170. doi: 10.1152/ajpendo.00551.2012 [DOI] [PubMed] [Google Scholar]
  • 18.Merriam LA, Baran CN, Girard BM, Hardwick JC, May V, Parsons RL (2013) Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide-Induced Increase in Guinea Pig Cardiac Neuron Excitability. The Journal of Neuroscience 33 (10):4614–4622. doi: 10.1523/jneurosci.4999-12.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Inda C, Dos Santos Claro PA, Bonfiglio JJ, Senin SA, Maccarrone G, Turck CW, Silberstein S (2016) Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling. J Cell Biol 214 (2):181–195. doi: 10.1083/jcb.201512075 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Pavlos NJ, Friedman PA (2017) GPCR Signaling and Trafficking: The Long and Short of It. Trends in endocrinology and metabolism: TEM 28 (3):213–226. doi: 10.1016/j.tem.2016.10.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Persani L, Lania A, Alberti L, Romoli R, Mantovani G, Filetti S, Spada A, Conti M (2000) Induction of Specific Phosphodiesterase Isoforms by Constitutive Activation of the cAMP Pathway in Autonomous Thyroid Adenomas1. The Journal of Clinical Endocrinology & Metabolism 85 (8):2872–2878. doi: 10.1210/jcem.85.8.6712 [DOI] [PubMed] [Google Scholar]
  • 22.Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM (1991) Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 325 (24):1688–1695. doi: 10.1056/nejm199112123252403 [DOI] [PubMed] [Google Scholar]
  • 23.Boot AM, Lumbroso S, Verhoef-Post M, Richter-Unruh A, Looijenga LHJ, Funaro A, Beishuizen A, van Marle A, Drop SLS, Themmen APN (2011) Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene. The Journal of Clinical Endocrinology and Metabolism 96 (7):E1197–E1205. doi: 10.1210/jc.2010-3031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Min KS, Liu X, Fabritz J, Jaquette J, Abell AN, Ascoli M (1998) Mutations that induce constitutive activation and mutations that impair signal transduction modulate the basal and/or agonist-stimulated internalization of the Lutropin/Choriogonadotropin receptor. J Biol Chem 273 (52):34911–34919 [DOI] [PubMed] [Google Scholar]
  • 25.Dyachok O, Isakov Y, Sagetorp J, Tengholm A (2006) Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells. Nature 439 (7074):349–352. doi: 10.1038/nature04410 [DOI] [PubMed] [Google Scholar]
  • 26.Tian G, Sagetorp J, Xu Y, Shuai H, Degerman E, Tengholm A (2012) Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion. J Cell Sci 125 (Pt 21):5084–5095. doi: 10.1242/jcs.107201 [DOI] [PubMed] [Google Scholar]
  • 27.Vitalis EA, Costantin JL, Tsai P-S, Sakakibara H, Paruthiyil S, Iiri T, Martini J-F, Taga M, Choi ALH, Charles AC, Weiner RI (2000) Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells. Proceedings of the National Academy of Sciences 97 (4):1861–1866. doi: 10.1073/pnas.040545197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Nicol X, Voyatzis S, Muzerelle A, Narboux-Neme N, Sudhof TC, Miles R, Gaspar P (2007) cAMP oscillations and retinal activity are permissive for ephrin signaling during the establishment of the retinotopic map. Nat Neurosci 10 (3):340–347. doi: 10.1038/nn1842 [DOI] [PubMed] [Google Scholar]
  • 29.Haisenleder DJ, Yasin M, Marshall JC (1992) Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression. Endocrinology 131 (6):3027–3033. doi: 10.1210/endo.131.6.1280210 [DOI] [PubMed] [Google Scholar]
  • 30.Brooker G (1973) Oscillation of Cyclic Adenosine Monophosphate Concentration during the Myocardial Contraction Cycle. Science 182 (4115):933–934 [DOI] [PubMed] [Google Scholar]
  • 31.Rich TC, Fagan KA, Nakata H, Schaack J, Cooper DM, Karpen JW (2000) Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 116 (2):147–161 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ghigo A, Mika D (2019) cAMP/PKA signaling compartmentalization in cardiomyocytes: Lessons from FRET-based biosensors. Journal of molecular and cellular cardiology 131:112–121. doi: 10.1016/j.yjmcc.2019.04.020 [DOI] [PubMed] [Google Scholar]
  • 33.Agarwal SR, Gratwohl J, Cozad M, Yang PC, Clancy CE, Harvey RD (2018) Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes. Frontiers in pharmacology 9:332. doi: 10.3389/fphar.2018.00332 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sassi Y, Abi-Gerges A, Fauconnier J, Mougenot N, Reiken S, Haghighi K, Kranias EG, Marks AR, Lacampagne A, Engelhardt S, Hatem SN, Lompre A-M, Hulot J-S (2012) Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes. The FASEB Journal 26 (3):1009–1017. doi: 10.1096/fj.11-194027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chen C, Nakamura T, Koutalos Y (1999) Cyclic AMP diffusion coefficient in frog olfactory cilia. Biophys J 76 (5):2861–2867 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Agarwal SR, Clancy CE, Harvey RD (2016) Mechanisms Restricting Diffusion of Intracellular cAMP. Sci Rep 6:19577. doi: 10.1038/srep19577 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lohse C, Bock A, Maiellaro I, Hannawacker A, Schad LR, Lohse MJ, Bauer WR (2017) Experimental and mathematical analysis of cAMP nanodomains. PLoS One 12 (4):e0174856. doi: 10.1371/journal.pone.0174856 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Richards M, Lomas O, Jalink K, Ford KL, Vaughan-Jones RD, Lefkimmiatis K, Swietach P (2016) Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes. Cardiovasc Res 110 (3):395–407. doi: 10.1093/cvr/cvw080 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Monterisi S, Favia M, Guerra L, Cardone RA, Marzulli D, Reshkin SJ, Casavola V, Zaccolo M (2012) CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity. Journal of cell science 125 (Pt 5):1106–1117. doi: 10.1242/jcs.089086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Maiellaro I, Lohse MJ, Kittel RJ, Calebiro D (2016) cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons. Cell reports 17 (5):1238–1246. doi: 10.1016/j.celrep.2016.09.090 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Bacskai BJ, Hochner B, Mahaut-Smith M, Adams SR, Kaang BK, Kandel ER, Tsien RY (1993) Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science 260 (5105):222–226 [DOI] [PubMed] [Google Scholar]
  • 42.Efetova M, Petereit L, Rosiewicz K, Overend G, Haussig F, Hovemann BT, Cabrero P, Dow JA, Schwarzel M (2013) Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo. Journal of cell science 126 (Pt 3):778–788. doi: 10.1242/jcs.114140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Jansen V, Alvarez L, Balbach M, Strunker T, Hegemann P, Kaupp UB, Wachten D (2015) Controlling fertilization and cAMP signaling in sperm by optogenetics. Elife 4. doi: 10.7554/eLife.05161 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Tsvetanova NG, von Zastrow M (2014) Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis. Nat Chem Biol 10 (12):1061–1065. doi: 10.1038/nchembio.1665 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Averaimo S, Assali A, Ros O, Couvet S, Zagar Y, Genescu I, Rebsam A, Nicol X (2016) A plasma membrane microdomain compartmentalizes ephrin-generated cAMP signals to prune developing retinal axon arbors. Nat Commun 7:12896. doi: 10.1038/ncomms12896 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ryu MH, Moskvin OV, Siltberg-Liberles J, Gomelsky M (2010) Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications. The Journal of biological chemistry 285 (53):41501–41508. doi: 10.1074/jbc.M110.177600 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gartner W, Petereit L, Efetova M, Schwarzel M, Oertner TG, Nagel G, Hegemann P (2011) Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. The Journal of biological chemistry 286 (2):1181–1188. doi: 10.1074/jbc.M110.185496 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lindner R, Hartmann E, Tarnawski M, Winkler A, Frey D, Reinstein J, Meinhart A, Schlichting I (2017) Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase. J Mol Biol 429 (9):1336–1351. doi: 10.1016/j.jmb.2017.03.020 [DOI] [PubMed] [Google Scholar]
  • 49.Berglund K, Birkner E, Augustine GJ, Hochgeschwender U (2013) Light-emitting channelrhodopsins for combined optogenetic and chemical-genetic control of neurons. PloS one 8 (3):e59759. doi: 10.1371/journal.pone.0059759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Berglund K, Clissold K, Li HE, Wen L, Park SY, Gleixner J, Klein ME, Lu D, Barter JW, Rossi MA, Augustine GJ, Yin HH, Hochgeschwender U (2016) Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation. Proceedings of the National Academy of Sciences of the United States of America 113 (3):E358–E367. doi: 10.1073/pnas.1510899113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Park SY, Song SH, Palmateer B, Pal A, Petersen ED, Shall GP, Welchko RM, Ibata K, Miyawaki A, Augustine GJ, Hochgeschwender U (2017) Novel luciferase-opsin combinations for improved luminopsins. Journal of neuroscience research. doi: 10.1002/jnr.24152 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7 (11):1848–1857. doi: 10.1021/cb3002478 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Naim N, White AD, Reece JM, Wankhede M, Zhang X, Vilardaga JP, Altschuler DL (2018) Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis. The Journal of biological chemistry 294 (4):1095–1103. doi: 10.1074/jbc.AC118.004905 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Insel PA, Maguire ME, Gilman AG, Bourne HR, Coffino P, Melmon KL (1976) Beta adrenergic receptors and adenylate cyclase: products of separate genes? Molecular pharmacology 12 (6):1062–1069 [PubMed] [Google Scholar]
  • 55.Ross EM, Howlett AC, Ferguson KM, Gilman AG (1978) Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. The Journal of biological chemistry 253 (18):6401–6412 [PubMed] [Google Scholar]
  • 56.Kimura T, Van Keymeulen A, Golstein J, Fusco A, Dumont JE, Roger PP (2001) Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr Rev 22 (5):631–656. [DOI] [PubMed] [Google Scholar]
  • 57.Polstein LR, Gersbach CA (2014) Light-inducible gene regulation with engineered zinc finger proteins. Methods in molecular biology (Clifton, NJ) 1148:89–107. doi: 10.1007/978-1-4939-0470-9_7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Dietler J, Stabel R, Möglich A (2019) Pulsatile illumination for photobiology and optogenetics. In: Deiters A (ed) Methods in Enzymology, vol 624. Academic Press, pp 227–248. doi: 10.1016/bs.mie.2019.04.005 [DOI] [PubMed] [Google Scholar]

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