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

Analisi cAMP con cADDis: screening da singola cellula a alto rendimento

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.

Autore in primo piano: Avanzamento del rilevamento cAMP in tempo reale nelle cellule utilizzando il biosensore cADDis

Dinamica del cAMP in tempo reale in cellule vive utilizzando il rivelatore di differenza fluorescente cAMP in situ

We serve the organization of cycling KP micro domains with a focus on elucidating how these compartments are formed and sustained. We seek to uncover the key components that enable cells to discern spatial signals within these domains, exploring variation in their interpretation, and how these features are altered in diseases like asthma and COPD. Infecting primary cells using the Beckman vector is challenging.For this, the condition should be optimized for individual cells type in order to achieve sufficient expression of the biosensor. This protocol allows the real time detection and monitoring, ranging from minutes to hours, and measurement of cyclo campu dynamics within living cells. This biosensor has high specificity for abiding to cyclo camp P.Consequently, the signals generated by this essay directly correlates to cyclic P levels, significantly reducing the potential interference from the other cellular components, and providing reliable results.

Microscopia a fluorescenza invertita e analisi dei dati per valutare gli effetti dei farmaci sul cAMP nelle cellule HASM

Per iniziare, coltivare il vettore trasformato nelle vie aeree umane muscolari lisce, o cellule HASM. Per l'imaging, accendere l'hub centrale e il microscopio a fluorescenza invertito. Avvia il software designato e seleziona la funzione di acquisizione time lapse per la registrazione.Montare il piatto di coltura sul tavolino del microscopio. Utilizzando l'autofocus e la luminosità automatica, ottieni un'immagine delle singole celle. Regola il bilanciamento del nero e la lunghezza d'onda di eccitazione.Quindi fare clic su messa a fuoco automatica. Dopo aver trovato un'area di vista con diverse cellule citoplasmatiche fluorescenti, impostare l'intervallo di tempo di acquisizione su 30 secondi per un periodo totale di 20 minuti. Quindi fare clic su OK per iniziare l'acquisizione dei dati.Dopo tre minuti, aggiungere delicatamente 50 microlitri di farmaco nel pozzetto appropriato e lasciare che l'esperimento venga eseguito per altri 17 minuti. Aprire i file di immagine acquisiti durante l'esperimento. Utilizzando lo strumento poligono o cerchio, selezionare la regione di interesse di ciascuna cella ed eseguire l'analisi.Quindi apri i dati analizzati in un foglio Excel e calcola la luminosità media per ogni condizione in ogni foto. A partire dall'immagine poco prima di aggiungere l'agente farmacologico. Dopo aver calcolato la variazione di fluorescenza rispetto alla fluorescenza iniziale, per ogni valore medio in ogni condizione, caricare i valori in un software di analisi statistica e tracciare il delta F per F zero rispetto al tempo in secondi.Rispetto al controllo, è stata osservata una diminuzione osservabile dell'intensità della fluorescenza nel tempo dopo la stimolazione delle cellule HASM con 10 micromolari di forskolina e 10 nanomolari di isoproterenolo.

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 ...

Ricerca

Biochimica

Preparing HASM Cells for cAMP Detection Using Inverted Fluorescence Microscopy

Preparazione delle cellule HASM per la rilevazione di cAMP mediante microscopia a fluorescenza invertita