使用 4 维（x、y、z 和 +）高光谱 FRET 成像和分析测量活细胞中的三维 cAMP 分布
Summary
由于基于 Fürster 共振能量转移 （FRET） 传感器的固有的低信号与噪声比 （SNR），CAMP 信号的测量一直具有挑战性，尤其是在三个空间维度方面。在这里，我们描述了一个高光谱FRET成像和分析方法，允许测量三个空间尺寸的CAMP分布。
Abstract
循环AMP是第二个信使，参与广泛的细胞和生理活动。一些研究表明，cAMP 信号是分隔的，隔间化有助于 cAMP 信号通路中的信号特异性。基于Fürster共振能量转移（FRET）的生物传感器的发展，进一步增强了测量和可视化细胞中CAMP信号的能力。但是，这些测量通常局限于两个空间维度，这可能导致对数据的误解。迄今为止，由于使用本身信号与噪声比 （SNR） 较低的 FRET 传感器的技术限制，在三个空间维度（x、y 和 z）中对 CAMP 信号的测量非常有限。此外，由于光谱相声、信号强度有限和自发光等一系列因素，传统的基于滤波器的成像方法通常对精确测量局部子细胞区域的 CAMP 信号无效。为了克服这些限制，并允许基于FRET的生物传感器与多个荧光素一起使用，我们开发了高光谱FRET成像和分析方法，为计算FRET效率提供光谱特异性，并能够将FRET信号与混淆的自发光和/或来自其他荧光标签的信号隔离开来。在这里，我们介绍了实施高光谱FRET成像的方法，以及需要构建一个适当的光谱库，既不采样不足，也不采样过采，以执行光谱未混合。虽然我们提出了测量肺微血管内皮细胞（PMVECs）中三维CAMP分布的方法，但这种方法可用于研究CAMP在一系列细胞类型的空间分布。
Introduction
环腺苷单磷酸盐（cAMP）是参与关键细胞和生理过程的第二个信使，包括细胞分裂、钙的涌入、基因转录和信号转导。越来越多的证据表明，细胞中存在cAMP隔间，通过这些隔间可以达到1、2、3、4、5、6、7的信号特异性。直到最近，CAMP分隔断是根据不同的G耦合受体激动剂8，9，10，11诱导的独特的生理或细胞效应推断的。最近，基于FRET的荧光成像探测器为直接测量和观察12、13、14细胞中的CAMP信号提供了新的方法。
Fürster共振能量转移（FRET）是一种物理现象，当分子接近15、16时，能量转移以非辐射的方式发生在供体和接受分子之间。随着基于FRET的荧光指标的发展，这种物理现象已用于生物应用，研究蛋白质-蛋白质相互作用17，蛋白质共定位18，Ca+2信号19，基因表达20，细胞分裂21和环核苷酸信号。基于FRET的cAMP指标通常包括一个CAMP结合域，一个捐赠者氟和一个接受荧光22。例如，H188 cAMP 传感器12，22用于此方法包括从 Epac 获得的 cAMP 结合域，夹在绿松石（捐赠者）和金星（接受者）荧光之间。在基础条件下（未绑定），绿松石和金星处于一个方向，因此FRET发生在萤管之间。将 cAMP 与绑定域结合后，会发生构象变化，使绿松石和金星分开，导致 FRET 减少。
基于 FRET 的成像方法为研究细胞内的 cAMP 信号提供了一个很有前途的工具。然而，目前的基于FRET的微观成像技术往往只部分成功达到足够的信号强度，以测量具有亚细胞空间清晰度的FRET。这是由于几个因素，包括许多FRET记者的信号强度有限，准确量化FRET效率变化所需的高精度，以及存在混淆因素，如细胞自发光23，24。结果往往是受弱 SNR 困扰的 FRET 图像，使得 FRET 的子细胞变化的可视化变得非常困难。此外，空间定位 CAMP 信号的调查几乎只在两个空间维度中执行，轴向 CAMP 分布很少被视为25。这可能是因为低 SNR 妨碍了在三个空间维度中测量和可视化 cAMP 梯度的能力。为了克服使用低SNR的FRET传感器的限制，我们采用了高光谱成像和分析方法，在单个细胞25、26、27中测量FRET。
美国宇航局开发了超光谱成像方法，以区分卫星图像28、29中存在的地球物体。此后，这些技术被翻译成荧光显微镜场30，几个商业共聚焦显微镜系统提供光谱探测器。在传统的（非光谱）荧光成像中，样品使用带通滤镜或激光线激发，并且使用第二个带通滤波器收集发射，通常选择以匹配荧光的峰值发射波长。相比之下，高光谱成像方法寻求以特定波长间隔对荧光发射26、31、32或激发33、34的完整光谱剖面进行采样。在我们之前的研究中，我们发现，与传统的基于过滤器的FRET成像技术相比，高光谱成像和分析方法可以改善细胞中FRET信号的量化。在这里，我们提出了一种执行 4 维（x、y、z 和 +）高光谱 FRET 成像和分析的方法，以测量和可视化三个空间维度的 cAMP 分布。这些方法使单细胞25中激动剂诱导的CAMP空间梯度可视化。有趣的是，根据激动剂的不同，CAMP梯度在细胞中可能很明显。此处提供的方法利用不均匀背景和细胞自发光的光谱解密，以提高 FRET 测量的准确性。虽然这种方法在使用 cAMP FRET 生物传感器的肺微血管内皮细胞 （PMVECs） 中得到了证明，但该方法可以很容易地修改，用于替代 FRET 记者或替代细胞系。
Protocol
该协议遵循南阿拉巴马大学机构动物护理和使用委员会批准的程序。 1. 细胞、样品和试剂成像准备 孤立大鼠肺微血管内皮细胞（PMVECs），如前所述35。注：细胞被隔离和培养的细胞培养核心在南阿拉巴马大学，移动，AL在100毫米细胞培养菜。 种子分离PMVECs在25毫米圆形玻璃盖片上，并让他们在37°C的孵化器中生长，直到细胞达到至少80%的汇?…
Representative Results
本协议描述了使用高光谱 FRET 成像和分析方法测量活细胞中三个空间维度的 cAMP 梯度。生成这些结果涉及几个关键步骤，在分析和量化数据时需要仔细注意这些步骤。这些关键步骤包括构建适当的光谱库、背景光谱未混合、识别细胞边框的阈值以及 FRET 效率计算。 图1 说明了测量活细胞中FRET效率和cAMP水平所涉及的所有步骤的示意图流。如果执行得当，这些成像和分析步骤?…
Discussion
FRET生物传感器的研制使单细胞环核苷酸信号的测量和可视化，对13、22、37、38等亚细胞信号事件的可视化大有可为。然而，使用FRET生物传感器存在一些局限性，包括许多荧光蛋白基FRET记者的低信号到噪声特性，以及FRET记者的弱透射或表达效率（这在某些细胞系中可能特别具有挑战性，如PMVECs）23、24…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢Kees Jalink博士（荷兰癌症研究所和荷兰阿姆斯特丹范利乌文霍克高级显微镜中心）为我们提供了H188 cAMP FRET生物传感器和肯尼·特里恩（南阿拉巴马大学工程学院）的技术帮助，以减少运行我们自定义开发的编程脚本所花的时间。
作者要感谢资金来源：美国心脏协会（16PRE27130004），国家科学基金会：（1725937） NIH， S100D020149， S10RR027535， R01HL058506， P01HL066299）.）
Materials
| Attofluor Cell Chamber | Invitrogen | A7816 | Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage |
| Dimethyl Sulfoxide (DMSO) | Fisher Scientific | BP231-100 | Solvent used to prepare stock solution forskolin. |
| DRAQ5 Fluoroscent Probe Solution | Thermo Scientific | 62252 | Nuclear label |
| Dulbecco Modified Eagle Medium (DMEM) | Gibco | 11965-092 | Contains nutrients and growth factors for the cells to grow and divide in the culture dishes. |
| Fetal Bovine Serum (FBS) | Sigma | F6178 | Growth factor suppliment that is added to culture medium, DMEM |
| Forskolin | Sigma | F3917 | Adenyly cyclase activator. |
| H188 Cyclic AMP FRET biosensor | Netherland Cancer Institute, Dr. K. Jalink | Gift | Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac. |
| Image J | image.net | Free download | Another image processing platform used to extact spectral information and image processing. |
| Integrating Sphere | Ocean Optics | FOIS-1 | Used to measure illumination intensity of the laser line at different laser intensities (?). |
| Laminin Mouse Protein, Natural | Invitrogen | 23017-015 | Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell. |
| Lipofectamine 3000 Transfection Kit | Invitrogen | L3000-015 | Transfection reagent used to transfect cells with H188 FRET biosensor |
| MATLAB | Mathworks | R2019a | Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment |
| Nikon A1R confocal microscope | Nikon Instruments | Nikon A1R | Spectral image acquisition is performed using confocal microscope. |
| Nikon Elements Software | Nikon Instruments | Software dongle | used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R |
| NIST-Traceable Calibration Lamp | Ocean Optics | LS-1-CAL-INT | A lamp with a known spectrum for use as a standard |
| PBS pH 7.4 (1X) | Gibco | 10010-023 | coomonly used buffer suring cell culture |
| Pulmonary Microvascular Endothelial Cells (PMVECs) | In house – Cell culture core, Univeristy of South Alabama | Isolated from Rat pulmonary microvasculature | PMVECs form inner lining of a blood vessel. |
| Penicillin-Streptomycin (10,000 U/ml) | Gibco | 15140-122 | antibiotics are added to culture medum to prevent contamination of the cells. |
| Pre-Cleaned Gold Seal Micro Slides | Clay Adams | 3010 | Microscope slides used for cell fixation |
| ProLong Diamond Antifade Mounting Media | Invitrogen | P36961 | If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading. |
| Spectrometer | Ocean Optics | QE65000 | Used to measure spectral response of the light source (?) |
| Trypsin-EDTA (0.25%) | Gibco | 25200-056 | Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating. |
| Tyrodes Buffer | Made in-house | Made in-house | Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition. |
| 6 Well Cell Culture Plate | Corning | 3506 | Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well. |
| 25 mm Round Microscope Cover Slips | Fisher Scientific | 12545102 | Cells were grown on round glass coverslips |
| 60X Ojective | Nikon Instruments | Plan Apo VC 60X/1.2 WI ∞/0.15-0.18 WD 0.27 | water immersion and commonly used objective for cells |
Referencias
- Corbin, J. D., Sugden, P. H., Lincoln, T. M., Keely, S. L. Compartmentalization of adenosine 3′:5′-monophosphate and adenosine 3′:5′-monophosphate-dependent protein kinase in heart tissue. The Journal of Biological Chemistry. 252, 3854-3861 (1977).
- Terrin, A., et al. PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. The Journal of Cell Biology. 175, 441-451 (2006).
- Bacskai, B. J., et al. Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science. 260, 222-226 (1993).
- Iancu, R. V., Ramamurthy, G., Harvey, R. D. Spatial and temporal aspects of cAMP signalling in cardiac myocytes. Clinical and Experimental Pharmacology & Physiology. 35, 1343-1348 (2008).
- Brunton, L. L., Hayes, J. S., Mayer, S. E. Functional compartmentation of cyclic AMP and protein kinase in heart. Advances in Cyclic Nucleotide Research. 14, 391-397 (1981).
- Hohl, C. M., Li, Q. Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients. Circulation Research. 69, 1369-1379 (1991).
- Rich, T. C., et al. A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proceedings of the National Academy of Sciences of the United States of America. 98, 13049-13054 (2001).
- Sayner, S. L., Alexeyev, M., Dessauer, C. W., Stevens, T. Soluble adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelial cell barrier. Circulation Research. 98, 675-681 (2006).
- Rich, T. C., Tse, T. E., Rohan, J. G., Schaack, J., Karpen, J. W. In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. The Journal of General Physiology. 118, 63-78 (2001).
- Blackman, B. E., et al. PDE4D and PDE4B function in distinct subcellular compartments in mouse embryonic fibroblasts. Journal of Biological Chemistry. 286, 12590-12601 (2011).
- Sayner, S. L., et al. Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY. Circulation Research. 95, 196-203 (2004).
- Klarenbeek, J., Jalink, K. Detecting cAMP with an EPAC-based FRET sensor in single living cells. Methods in Molecular Biology. 1071, 49-58 (2014).
- Surdo, N. C., et al. FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility. Nature Communications. 8, 15031 (2017).
- Ponsioen, B., et al. Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Reports. 5, 1176-1180 (2004).
- Vogel, S. S., Thaler, C., Koushik, S. V. Fanciful FRET. Science’s STKE. 2006, (2006).
- Clegg, R. M. The History of FRET: From conception through the labors of birth. Reviews in Fluorescence. 3, (2006).
- Giepmans, B. N. G., Adams, S. R., Ellisman, M. H., Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science. 312, 217-224 (2006).
- Manzella-Lapeira, J., Brzostowski, J. A. Imaging protein-protein interactions by Förster resonance energy transfer (FRET) microscopy in live cells. Current Protocols in Protein Science. 93, 58 (2018).
- Cooper, D. M. F., Mons, N., Karpen, J. W. Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature. 374, 421-424 (1995).
- Sassone-Corsi, P. Coupling gene expression to cAMP signalling: role of CREB and CREM. The International Journal of Biochemistry & Cell Biology. 30, 27-38 (1998).
- Rebhun, L. I. Cyclic nucleotides, calcium, and cell division. International Review of Cytology. 49, 1-54 (1977).
- Klarenbeek, J., Goedhart, J., Van Batenburg, A., Groenewald, D., Jalink, K. Fourth-generation Epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PLOS ONE. 10, 0122513 (2015).
- Leavesley, S. J., Rich, T. C. FRET: signals hidden within the noise. Cytometry Part A. 85, 918-920 (2014).
- Rich, T. C., Webb, K. J., Leavesley, S. J. Can we decipher the information content contained within cyclic nucleotide signals. The Journal of General Physiology. 143, 17-27 (2014).
- Annamdevula, N. S., et al. Spectral imaging of FRET-based sensors reveals sustained cAMP gradients in three spatial dimensions. Cytometry Part A. 93 (10), 1029-1038 (2018).
- Leavesley, S. J., Britain, A. L., Cichon, L. K., Nikolaev, V. O., Rich, T. C. Assessing FRET using spectral techniques. Cytometry Part A. 83, 898-912 (2013).
- Leavesley, S. J., Rich, T. C. Overcoming limitations of FRET measurements. Cytometry Part A. 89, 325-327 (2016).
- Fink, D. J. Monitoring Earcths Resources from Space. Technology Review. 75, 32-41 (1973).
- Goetz, A. F. H., Vane, G., Solomon, J. E., Rock, B. N. Imaging Spectrometry for Earth Remote Sensing. Science. 228, 1147-1153 (1985).
- Bücherl, C. A., Bader, A., Westphal, A. H., Laptenok, S. P., Borst, J. W. FRET-FLIM applications in plant systems. Protoplasma. 251, 383-394 (2014).
- Chen, Y., Mauldin, J. P., Day, R. N., Periasamy, A. Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions. Journal of Microscopy. 228, 139-152 (2007).
- Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Letters. 531, 245-249 (2002).
- Griswold, J. R., Annamdevula, N., Deal, J., Rich, T., Leavesley, S. Estimating FRET Efficiency using Excitation-Scanning Hyperspectral Imaging. Biophysical Journal. 112, 586 (2017).
- Favreau, P. F., et al. Excitation-scanning hyperspectral imaging microscope. Journal of Biomedical Optics. 19, 046010 (2014).
- King, J., et al. Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes. Microvascular Research. 67, 139-151 (2004).
- Thaler, C., Koushik, S. V., Blank, P. S., Vogel, S. S. Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophysical Journal. 89, 2736-2749 (2005).
- Agarwal, S. R., et al. Compartmentalized cAMP signaling associated with lipid raft and non-raft membrane domains in adult ventricular myocytes. Frontiers in Pharmacology. 9, 332 (2018).
- Johnstone, T. B., Agarwal, S. R., Harvey, R. D., Ostrom, R. S. cAMP signaling compartmentation: Adenylyl cyclases as anchors of dynamic signaling complexes. Mol Pharmacol. , (2017).
- Zhang, J., Li, H., Chai, L., Zhang, L., Qu, J., Chen, T. Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction. Journal of Microscopy. 257, 104-116 (2015).
- Zhang, J., Lin, F., Chai, L., Wei, L., Chen, T. IIem-spFRET: improved Iem-spFRET method for robust FRET measurement. Journal of Biomedical Optics. 21, 105003 (2016).
- Levy, S., et al. SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency. Microscopy and Microanalysis. 17, 176-190 (2011).
- West, S. J., et al. Hyperspectral Measurements Allow Separation of FRET Signals from Non-Uniform Background Fluorescence. Biophysical Journal. 112, 453 (2017).
- Annamdevula, N. S., et al. An approach for characterizing and comparing hyperspectral microscopy systems. Sensors. 13, 9267-9293 (2013).
Citar este artículo
Annamdevula, N. S., Sweat, R., Gunn, H., Griswold, J. R., Britain, A. L., Rich, T. C., Leavesley, S. J. Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis. J. Vis. Exp. (164), e61720, doi:10.3791/61720 (2020).