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

In Situ Fluorescent cAMP Difference Detector를 사용한 살아있는 세포의 실시간 cAMP 역학

HASM 세포의 cAMP에 대한 약물 효과를 평가하기 위한 도립 형광 현미경 및 데이터 분석

먼저, 벡터로 변형된 인간 기도 평활근 또는 HASM 세포를 배양합니다. 이미징을 위해 중앙 허브와 도립 형광 현미경을 켭니다. 지정된 소프트웨어를 실행하고 녹화할 타임랩스 캡처 기능을 선택합니다.현미경 스테이지에 배양 접시를 장착합니다. 자동 초점 및 자동 밝기를 사용하여 개별 셀의 사진을 얻을 수 있습니다. 블랙 밸런스와 여기 파장을 조정합니다.그런 다음 자동 초점을 클릭합니다. 여러 세포질 형광 셀이 있는 시야 영역을 찾은 후 총 20분 동안 타임랩스 캡처를 30초로 설정합니다. 그런 다음 확인을 클릭하여 데이터 수집을 시작합니다.3분 후, 약물의 50 마이크로리터를 적절한 우물에 부드럽게 첨가하고 실험을 17분 더 실행합니다. 실험 중에 캡처된 이미지 파일을 엽니다. 폴리곤 또는 원 도구를 사용하여 각 셀의 관심 영역을 선택하고 분석을 수행합니다.그런 다음 분석된 데이터를 Excel 시트에서 열고 각 사진의 각 조건에 대한 평균 밝기를 계산합니다. 약리제를 첨가하기 직전의 사진부터 시작합니다. 초기 형광에서 형광의 변화를 계산한 후 각 조건의 각 평균값에 대해 값을 통계 분석 소프트웨어에 로드하고 시간에 대해 델타 F를 F 0으로 초 단위로 플로팅합니다.대조군과 비교하여, 10 마이크로몰 포스콜린 및 10 나노몰 이소프로테레놀로 HASM 세포를 자극할 때 시간 경과에 따른 형광 강도의 관찰 가능한 감소가 관찰되었습니다.

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 ...

연구

생화학

Preparing HASM Cells for cAMP Detection Using Inverted Fluorescence Microscopy

도립 형광 현미경을 사용한 cAMP 검출을 위한 HASM 세포 준비