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 immunoassays1,2. 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 fluorophores3,4. 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 results4,5.
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
Representative Results
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 assay14,15,16,17,18. Each cAMP assay has…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This study was supported by the National Heart, Lung, and Blood Institute (NHLBI) (HL169522).
Materials
| 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 |
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