Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

Summary

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

Abstract

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Introduction

Adenosine 3′,5′-cyclic monophosphate, cAMP, plays a central role in cellular communication and the coordination of various physiological processes. cAMP acts as a second messenger, relaying external signals from hormones, neurotransmitters, or other extracellular molecules to initiate a cascade of intracellular events¹. Moreover, cAMP is intricately involved in various signaling pathways, including those associated with G-protein-coupled receptors (GPCRs) and adenylyl cyclases. Understanding the role of cAMP in cellular signaling is fundamental to unraveling the complex mechanisms that underlie normal cellular functions and the development of potential therapies for a wide range of medical conditions².

In the past, various methods have been employed to measure cAMP directly or indirectly. These included radiolabeling of cellular ATP pools followed by column purification, HPLC, radioimmunoassays, and enzyme-linked immunoassays^1,2. These legacy assays are limited by the fact that they are end-point measures, requiring a large number of samples to construct time-dependent responses. More recently, Fluorescence Resonance Energy Transfer (FRET) sensors were developed to create assays in living cells, producing real-time, dynamic data and allowing sensors to be targeted in different subcellular locations³. FRET leverages two fluorophores, one fluorescent donor, and one fluorescent acceptor that when in close proximity, the acceptor fluorophore will be excited by the donor fluorescent output. The two fluorophores most used are cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) since these have compatible excitation and emission properties. In addition to CFP and YFP, the utilization of the green fluorescent protein (GFP) and red fluorescent protein (RFP) is commonly used for FRET biosensors. cAMP FRET biosensors operate by having a donor and acceptor on opposite ends of the Epac2 cAMP binding protein. cAMP binding alters the confirmation of Epac and increases the distance between donor and acceptor fluorophores^3,4. This conformational change is detected by a loss of FRET, that is, the excitation of the acceptor fluorophore by energy transferred from the donor fluorophore drops³. While a seemingly simple process, there are an abundance of limitations and issues with the FRET biosensor for cAMP research⁵. One of which is the selection of fluorescent proteins, for example, GFP, which can dimerize naturally, thus reducing sensitivity⁶. FRET-based cAMP biosensors have been targeted to specific microdomains⁷, but there may be limitations owing to the large size of a construct with two fluorophores⁶. Another significant issue is the low signal-to-noise ratio of FRET signals resulting from the overlap between excitation and emission of the fluorophore, resulting in high sampling frequency and complicating analysis of the results^4,5.

Most recently, the novel biosensor (cAMP Difference Detector In Situ), cADDis has solved these and other limitations when it comes to studying the regulation of cAMP signaling⁸. One important improvement is the dependence on a single fluorophore. This allows for a rapid and efficient signal with a wider dynamic range and a high signal-to-noise ratio. As a result, there can be more accuracy as there is a less broad scope of wavelengths to comb through⁸. Like FRET probes, the biosensor has been targeted to subcellular locations, allowing research into the compartmentation theory and exploration in lipid rafts and non-rafts, and other subcellular domains⁹. Perhaps most important is the suitability of a single fluorophore biosensor for high-throughput screening, which has improved sensitivity and reproducibility over FRET-based biosensors. The biosensor is packaged into a BacMam vector for easy transduction of a wide array of cell types and precise control over protein expression.

Expression control via the BacMam vector can be particularly useful in assays using GPCR orthologs from different species to facilitate the interpretation of data from animal studies. Furthermore, control over receptor expression is critical for measuring the different degrees of drug efficacy (e.g., inverse agonists and partial agonists), and low levels of receptor expression are useful to mimic the low levels found in animal tissue. BacMam is a baculovirus vector that has been modified to transduce mammalian cells such as primary cell cultures and HEK-293 lines¹⁰. Dominant selectable markers allow for BacMam to provide more stability over traditional plasmid infections¹¹. Such selective promoters allow for more efficient gene delivery and expression. In addition, adding trichostatin A (a histone deacetylase inhibitor) enhances the reporter protein levels¹¹. Expression levels can be controlled via the titer of the BacMam virus used and should be optimized for each cell type. In the case of this biosensor, a red or green fluorescent protein is linked to the Epac at the N- and C-termini. When cAMP binds, a conformational change in the biosensor moves amino acids adjacent to the fluorescent protein. Such a shift moves the absorbance from the anionic state to the neutral state at 400 nm, thus decreasing the fluorescence.

There are 90-120 GPCRs expressed in a single cell that respond to a wide variety of neurohumoral signals¹². Therefore, it can be hypothesized that at least several dozen GPCRs per cell can stimulate or inhibit cAMP through Gs or Gi coupling, respectively. While there has been progress in monitoring this second messenger in real-time, such as FRET, more efficient methods are needed. The methodology for monitoring the synthesis and degradation of cAMP signals using cADDis in real-time is presented here. The change in fluorescence can be monitored in real-time using a fluorescence plate reader for high throughput assays or using a fluorescent microscope for single-cell assays. These methods are useful for a wide array of biological questions regarding GPCR signaling via cAMP.

Protocol

The details of all the reagents and equipment used for the study are listed in the Table of Materials.

1. Plate reading spectrophotometer high-throughput assay

1. Seeding HEK-293 cells with the cADDis BacMam vector in a 96-well plate (Day 1)
   1. Split and infect cells in a viral hood.
      1. Warm HEK media (Table 1) and 0.25% trypsin-EDTA in a 37 °C water bath.
      2. Disinfect the hood and the materials by wiping them with EtOH-soaked tissues.
      3. Remove and discard HEK media from the flask with a serological pipette.
      4. Add 5 mL of Mg²⁺ and Ca²⁺ free DPBS (37 °C) with a serological pipette. Tilt the flask back and forth to wash the bottom with DPBS. Remove and discard DPBS with a serological pipette.
      5. Trypsinize the cells by adding 3 mL of warmed 0.25% trypsin-EDTA with a serological pipette. Tilt the flask to ensure the reagent fully coats the bottom of the flask. Put the lid on the flask and let the reagent sit for 3-5 min to allow the cells to detach from the flask.
         NOTE: This experiment is optimized for a 75 cm² flask; volume adjustments for reagents may need to be made if a different-size culture apparatus is used.
      6. Draw 5 mL of fresh media containing antibiotics. Expel and draw the volume of the flask to wash the walls of the flask and physically detach cells.
      7. Collect the total volume of the flask (8 mL), and centrifuge (25 °C) in a 15 mL tube at 200 x g for 5 min.
      8. After centrifuging, remove the supernatant with a serological pipette, careful not to disturb the pellet.
      9. To wash the cells without disturbing the pellet, add 500 µL of DPBS without Mg²⁺ and Ca²⁺ DPBS to the side of the 15 mL tube. After gently adding the DPBS, remove and discard as much buffer as possible without disturbing the pellet. Repeat one more time.
      10. Count the cells and adjust to a cell density of 250,000 cells/mL cells.
      11. Make a master mix based on what reagent volumes are needed per well of a black, transparent bottom 96-well plate. This is referred to as the "tissue plate." See volumes below:
          NOTE: Example of 1 well (total volume 200 µL): 40 µL of the biosensor BacMam vector (stock of 2 x 10¹⁰ viral genes /mL), 2 µL trichostatin A (stock of 100 µM; final concentration 1 µM), 158 µL of 250,000 cells/mL cell density. Example of 10 wells (total volume 2 mL): 400 µL of the biosensor BacMam vector (stock of 2 x 10¹⁰ viral genes /mL), 20 µL of trichostatin A (stock of 100 µM), 1,580 µL media with 250,000 cells/mL cell density.
      12. Add 200 µL of master mix per well. Incubate the cells at 37 °C and 5% CO₂ in an incubator for 24 h before running on the plate reading spectrophotometer.
2. Running on a plate reader
   1. On a benchtop, carefully remove all media in each well and replace with 180 µL of DPBS with Mg²⁺ and Ca²⁺ at 37 °C.
   2. Inspect cells on a microscope to confirm cell health.
   3. Wrap the plate in aluminum foil and incubate at 37 °C for 30 min-1 h.
   4. While incubating, prepare the drugs for the reading at 1:10 dilution (also called 10-fold) for the desired final dose. Thus, prepare 100 µM forskolin and 100 nM isoproterenol.
   5. Add 60 µL of each drug in a columnar fashion to a clear 96-well plate (referred to as the "drug plate").
      NOTE: With the proper drug dilution and to optimize the reading, the drugs are added to a clear 96-well plate, referred to as the "drug plate", to be transferred at the time of the reading to the "tissue plate" using a 96-multi pipettor, allowing the drug to be added to all wells simultaneously and be rapidly read by the fluorescent spectrophotometer.
3. Fluorescence plate reader setup
   1. Adjust the settings of the fluorescence plate reader.
      1. Read Mode: fluorescence. Read Type: kinetic. Wavelengths reading: 494 nm and 522 nm.
      2. Reading time: Enter 3 min for the baseline fluorescent read and 7 min for the post-drug addition."Interval" is entered as 00:00:30 for 30 s between each read.
         NOTE: The reading time can be optimized and increased based on the required time.
4. Data acquisition
   1. Remove the tissue plate from the incubator, remove the aluminum foil and the plate lid, and place the tissue plate in the plate reader. Close the reader drawer to allow the tissue plate to rest and equilibrate to the reader's temperature (37 °C).
   2. Place the clear 96-well plate (drug plate) with the drug dilutions under the 96-multi pipette and practice drawing 20 µL from the wells. Ensure that there is an even volume of drug pulled in the pipettor tips.
   3. Initiate a "baseline" measurement run.
      NOTE: 40 RFU or above is considered viable data. If data is below this value, the experiment should be stopped and discarded.
   4. The tissue plate will eject from the reader at the end of the baseline run. Swiftly and carefully add 20 µL from the clear drug plate to the black tissue plate. Then, quickly begin the "treatment" run. The tissue plate should not be out of the reader for more than 30 s after drugs are added.
      NOTE: If the drug is added too quickly, the cells detach from the tissue plate, creating an unusable experiment. In addition, the cells are infected with a light-sensitive green fluorescent protein; thus, swiftly and promptly adding the drugs and beginning the second read is imperative to avoid missing the early part of the response.
   5. Once the second reading is complete, ensure that the tissue plate is ejected from the reader and data collection is concluded.
      NOTE: Before disposing of the tissue plate, it is best practice to verify that the cells are still attached to the plate. Observe the cells under a microscope. If the cells detach, the results are invalid and should be discarded. The experiment will need to be repeated.
   6. Export the resulting data as an Excel file.
   7. Open the exported Excel readable data from the plate reader and copy and paste relevant information into the Excel conversion template. The data will transform from the plate reader set up to single well, column readouts to the right of the copied data.
      NOTE: Columns of fluorescence for a well over time are listed at RFUs (titled column transformation) and as a decimal change relative to the RFU reading at initial reading (t₀ titled Delta F). In the Excel template, the Delta F table is set to the last baseline reading before drug addition. This is the 6^th data collection point.
   8. After data transformation, copy the data from Delta F and paste it into data analysis software as decay or relative decay in fluorescence over time.
   9. Change the X-title to "time (s)" and the Y-title to "ΔF/F₀" for relative decimal values.

2. Single-cell assay using an inverted fluorescent microscope

1. Seeding (HASM) cells with the cADDis BacMam vector in a 35 mm dish (Day 1)
   1. Split and infect cells in a viral hood.
      1. Warm HASM media (Table 1), 0.25% trypsin-EDTA in a 37 °C water bath.
      2. Disinfect the hood and the materials and wipe the hood with EtOH-soaked tissues.
      3. Remove and discard HASM media from the flask with a serological pipette.
      4. Add 5 mL of Mg²⁺ and Ca²⁺ free DPBS (37 °C) with a new serological pipette and tilt the flask to wash the bottom with the DPBS.
      5. Trypsinize cells by adding 3 mL of warmed 0.25% trypsin-EDTA with a serological pipette. Tilt the flask to ensure the reagent fully coats the bottom of the flask. Let the reagent sit for 3-5 min to allow the cells to detach from the flask.
      6. Draw 5 mL of fresh media containing antibiotics and expel the pipette to remove the cells from the bottom of the flask.
      7. Collect the total volume of the flask (8 mL) centrifuge in a centrifuge tube at 200 x g for 5 min.
      8. After centrifuging, remove the supernatant with a serological pipette, careful not to disturb the pellet.
      9. Without disturbing the pellet, add 500 µL of DPBS without Mg²⁺ and Ca²⁺ DPBS to the side of the centrifuge tube. Remove and discard as much liquid as possible and repeat one more time.
         NOTE: This experiment is optimized for a 75 cm² flask; volume adjustments for reagents may need to be made if a different-size culture apparatus is used.
      10. Count cells and adjust to 40,000 cells/mL cell density.
      11. Make a master mix based on the reagent volume needed per well of a 35 mm dish. See volumes below:
          NOTE: Example of 1 well (total volume 500 µL): 20 µL of the biosensor BacMam vector (stock of 2 x 10¹⁰ viral genes /mL), 5 µL trichostatin A (stock of 100 µM, final concentration 1 µM), 500 µL of 40,000 cells/mL cell density. Example for 4 wells (total volume 2 mL): 80 µL of the biosensor BacMam vector (stock of viral genes /mL), 20 µL of trichostatin A (stock of 100 µM, final concentration 1 µM), 2000 µL media 40,000 cells/mL cell density.
      12. Add 500 µL of master mix per well. Incubate the cells at 37 °C and 5% CO₂ for 24 h before continuing the experiment.
          NOTE: The cell number may vary according to the cell line. To avoid overlapping cells for the analysis, use low cell numbers.
2. Preparing pharmacological agents (Day 2)
   1. Carefully remove media in each well and replace with 450 µL of DPBS with Mg²⁺ and Ca²⁺ at 37 °C.
   2. Wrap the plate in aluminum foil and incubate the 35 mm disk at 37 °C for 30 min-1 h.
   3. Inspect cells on a microscope to confirm cell health.
   4. In the meantime, prepare the drugs for the reading 1 log unit above the desired final concentration (10-fold greater). Thus, prepare 100 µM forskolin and 100 nM isoproterenol.
   5. For log dose titers, prepare the drug at 10 mM and use serial dilutions to achieve 100 µM of forskolin and 100 nM of isoproterenol.
3. Data acquisition
   NOTE: The following settings are for a fluorescent inverted microscope. The software accompanying each microscope can differ; the general settings used in the current protocol are described here.
   1. Turn on the central hub, then the inverted fluorescent microscope.
   2. Open the software that will be used for the capture and choose the time-lapse capture option.
   3. Place a 35 mm dish sample holder in the microscope stage.
   4. Set objective lens to x20.
   5. Use autofocus and get as clear a picture as possible. If it is difficult to find individual cells, start on a 4x objective, then move to 20x. Focus the sample manually if the microscope lacks an autofocus feature.
      NOTE: The magnification of 40x can be used if more significant cell morphological detail is needed.
   6. Adjust the following settings for obtaining optimal data acquisition: Auto Brightness (exposure), Excitation Wavelength, Black Balance (to reduce background noise), Auto Focus.
   7. After finding an area of view with several cytoplasmic fluorescing cells, acquire data using time-lapse capture for 20 min every 30 s.
   8. Click on the go to begin the reading.
   9. Run for 3 min to collect baseline. As soon as the 3 min capture is taken, gently add 50 µL of the drug to the appropriate well. When the drugs are added, the final concentration on the dish will be 10 µM forskolin and 10 nM of isoproterenol.
      NOTE: The drug must be added carefully; do not touch the plate with the pipette tip, as this may move the field of view made and make the sample unable to be analyzed.
   10. Leave the experiment to run for 17 more minutes with data capture every 30 s.
   11. Once the test is completed, ensure that the data is ready for analysis.
       NOTE: While the current protocol does not incorporate a control for focal drift, it could be beneficial to include one in the experiment. For instance, the use of nuclear dyes can assist in managing focal drift during microscope analysis, especially when capturing images of live cells over prolonged durations. cADDis biosensors are typically distributed widely within the cell, making a broad focal plane the most effective capture and reducing the need for controlling focal drift⁹.
4. Analyzing the data
   NOTE: The software accompanying each microscope can differ, so the general settings that should be used to analyze the captured images are described here.
   1. Open the image files captured during the experiment.
   2. Use the polygon or circle tool to select the region of interest of each cell to perform the analysis. The analysis should give the brightness of each cell at each time point.
      NOTE: Best practice is to select individual cells and not cells touching the wall of the dish or other cells. This ensures the best data collection possible. Select a minimum of 5 cells for analysis within each condition, and replicate the experiment at least three times.
   3. After analyzing the data, open the data in an Excel sheet and calculate the average brightness for each condition at each photo, starting at the picture just before adding the pharmacological agents or vehicle. In this example, at the 3 min mark or the 6^th photo taken on the microscope, since each picture is taken every 30 s.
   4. Calculate ΔF/F₀ for each average value in each condition.
   5. Load into a statistical analysis software and plot ΔF/F₀ against time in s.
      NOTE: Figure 1 provides a schematic overview of the major steps of the protocols.

Results

The present study validated the cytosolic biosensor in both plate reader and microscope assays. Once cells expressed the biosensor, they were stimulated with either 10 µM forskolin (a direct activator of adenylyl cyclase), 10 nM isoproterenol (an agonist at ß₁AR and ß₂AR), or vehicle (Figure 1). The subsequent changes in fluorescence, indicative of cAMP production, were captured every 30 s.

The data was transformed as the chang...

Discussion

Accurate and sensitive measurement of cAMP is crucial for understanding its role in various cellular processes and for studying the activity of cAMP-dependent signaling pathways. There are several methods commonly employed to measure cAMP levels, including ELISA, radioimmunoassay, FRET biosensors, and the GloSensor cAMP assay^{14,15,16,17,18}. Each cAMP assay has...

Materials

Name	Company	Catalog Number	Comments
96-well plate (clear)	Fisherbrand	21-377-203
35 mm dish	Greiner Bio-One	627870	Cell culture dishes with glass bottom
96-well plate	Corning	3904	Black with clear flat bottom
Antibiotic-Antimycotic (100x)	Gibco	15240062	For HEK and HASM media
BZ-X fluorescence microscope	Keyence
Calcium chloride (IM)	Quality Biological Inc	E506	For HASM media
Centrifuge tube (15 mL)	Thermo Scientific	339651
DMEM (1x)	Gibco	11965092	HEK media
DPBS with Mg2+ and Ca2+	Gibco	14040-133
DPBS without Mg2+ and Ca2+	Corning	14040-133
Fetal Bovine Serum (FBS)	R&D systems	S11195	For HEK and HASM media
Forskolin	Millipore	344270	Drug
Green Down cADDis cAMP Assay Kit	Montana Molecular	#D0200G	Reagent
Ham's F-12K	Gibco	21127022	For HASM media
HEPES (1M)	Gibco	15630080	For HASM media
Isoproterenol	Sigma	I6504	Drug
L-glutamine 200 mM (100x)	Gibco	25030-081	For HASM media
Microcentrifuge tube (2 mL)	Eppendorf	22363352
Primocin	Invitrogen	ant-pm-1	Antibiotic for HASM media
RNAse away	Thermo Scientific	700511	Reagent
Sodium hydroxide solution	Sigma	S2770	For HASM media
Spectrmax M5 plate reader	Molecular Devices
Trichostatin A	TCI America	T2477	Reagent
Trypsin EDTA	Gibco	25200-056	Reagent