inverted fluorescence microscopy and data analysis to assess drug effects on camp in hasm cells

cAMP Analysis with cADDis: Single-Cell to High-Throughput Screening

Adenosine 3&#8242;,5&#8242;-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.

Author Spotlight: Advancing Real-Time cAMP Detection in Cells Using cADDis Biosensor

Echtzeit-cAMP-Dynamik in lebenden Zellen mit dem fluoreszierenden cAMP-Differenzdetektor in situ

Invertierte Fluoreszenzmikroskopie und Datenanalyse zur Bewertung der Auswirkungen von Medikamenten auf cAMP in HASM-Zellen

Kultivieren Sie zunächst den Vektor, transformierte menschliche Atemwegs-, glatte Muskel- oder HASM-Zellen. Schalten Sie für die Bildgebung den zentralen Hub und das inverse Fluoreszenzmikroskop ein. Starten Sie die entsprechende Software und wählen Sie die Zeitrafferaufnahmefunktion für die Aufnahme aus.Montieren Sie die Kulturschale auf dem Mikroskoptisch. Mit Autofokus und automatischer Helligkeit können Sie sich ein Bild von einzelnen Zellen machen. Passen Sie den Schwarzabgleich und die Anregungswellenlänge an.Klicken Sie dann auf Autofokus. Nachdem Sie einen Sichtbereich mit mehreren zytoplasmatischen fluoreszierenden Zellen gefunden haben, stellen Sie die Zeitrafferaufnahme für einen Gesamtzeitraum von 20 Minuten auf 30 Sekunden ein. Klicken Sie dann auf OK, um die Datenerfassung zu starten.Geben Sie nach drei Minuten vorsichtig 50 Mikroliter des Medikaments in die entsprechende Vertiefung und lassen Sie das Experiment weitere 17 Minuten laufen. Öffnen Sie die Bilddateien, die während des Experiments erfasst wurden. Wählen Sie mit dem Polygon- oder Kreiswerkzeug den Interessenbereich jeder Zelle aus, und führen Sie die Analyse durch.Öffnen Sie dann die analysierten Daten in einer Excel-Tabelle und berechnen Sie die durchschnittliche Helligkeit für jede Bedingung in jedem Foto. Beginnen Sie mit dem Bild kurz vor der Zugabe des pharmakologischen Wirkstoffs. Nachdem Sie die Änderung der Fluoreszenz gegenüber der anfänglichen Fluoreszenz berechnet haben, laden Sie die Werte für jeden Durchschnittswert in jeder Bedingung in eine statistische Analysesoftware und stellen Sie Delta F x F Null gegen die Zeit in Sekunden dar.Im Vergleich zur Kontrolle wurde eine beobachtbare Abnahme der Fluoreszenzintensität im Laufe der Zeit nach Stimulation von HASM-Zellen mit 10 mikromolaren Forskolin und 10 nanomolaren Isoproterenol beobachtet.

Research

JoVE Journal

Biochemistry

Inverted Fluorescence Microscopy and Data Analysis to Assess Drug Effects on cAMP in HASM Cells

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

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 ...

Forschung

Biochemie

Preparing HASM Cells for cAMP Detection Using Inverted Fluorescence Microscopy

Vorbereitung von HASM-Zellen für die cAMP-Detektion mittels invertierter Fluoreszenzmikroskopie