Abstract
Nitric oxide (NO•) is a versatile signaling molecule which regulates fundamental cellular processes in all domains of life. However, due to the radical nature of NO• it has a very short half-life that makes it challenging to trace its formation, diffusion, and degradation on the level of individual cells. Very recently, we expanded the family of genetically encoded sensors by introducing a novel class of single fluorescent protein-based NO• probes—the geNOps. Once expressed in cells of interest, geNOps selectively respond to NO• by fluorescence quench, which enables real-time monitoring of cellular NO• signals. Here, we describe detailed methods suitable for imaging of NO• signals in mammalian cells. This novel approach may facilitate a broad range of studies to (re)investigate the complex NO• biochemistry in living cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Forstermann U, Closs EI, Pollock JS et al (1994) Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23(6 Pt 2):1121–1131
Hakim TS, Sugimori K, Camporesi EM et al (1996) Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiol Meas 17(4):267–277
Boens N, Leen V, Dehaen W (2012) Fluorescent indicators based on BODIPY. Chem Soc Rev 41(3):1130–1172. http://sci-hub.tw/10.1039/C1CS15132K
Han J, Burgess K (2010) Fluorescent indicators for intracellular pH. Chem Rev 110(5):2709–2728. http://sci-hub.tw/10.1021/cr900249z
Kojima H, Urano Y, Kikuchi K et al (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed Engl 38(21):3209–3212
Wang J, Zhao Y, Wang C et al (2015) Organelle-specific nitric oxide detection in living cells via HaloTag protein labeling. PLoS One 10(4):e0123986. http://sci-hub.tw/10.1371/journal.pone.0123986
Sato M, Hida N, Umezawa Y (2005) Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells. Proc Natl Acad Sci U S A 102(41):14515–14520. http://sci-hub.tw/10.1073/pnas.0505136102
Planchet E, Kaiser WM (2006) Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources. J Exp Bot 57(12):3043–3055. http://sci-hub.tw/10.1093/jxb/erl070
Namin SM, Nofallah S, Joshi MS et al (2013) Kinetic analysis of DAF-FM activation by NO: toward calibration of a NO-sensitive fluorescent dye. Nitric Oxide 28:39–46. http://sci-hub.tw/10.1016/j.niox.2012.10.001
Li H, Wan A (2015) Fluorescent probes for real-time measurement of nitric oxide in living cells. Analyst 140(21):7129–7141. http://sci-hub.tw/10.1039/C5AN01628B
Domaille DW, Que EL, Chang CJ (2008) Synthetic fluorescent sensors for studying the cell biology of metals. Nat Chem Biol 4(3):168–175. http://sci-hub.tw/10.1038/nchembio.69
Rogers JK, Church GM (2016) Genetically encoded sensors enable real-time observation of metabolite production. Proc Natl Acad Sci U S A 113(9):2388–2393. http://sci-hub.tw/10.1073/pnas.1600375113
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. http://sci-hub.tw/10.1146/annurev.biochem.67.1.509
Oldach L, Zhang J (2014) Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation. Chem Biol 21(2):186–197. http://sci-hub.tw/10.1016/j.chembiol.2013.12.012
Waldeck-Weiermair M, Bischof H, Blass S et al (2015) Generation of red-shifted Cameleons for imaging Ca2+ dynamics of the endoplasmic reticulum. Sensors (Basel) 15(6):13052–13068. http://sci-hub.tw/10.3390/s150613052
Hessels AM, Merkx M (2015) Genetically-encoded FRET-based sensors for monitoring Zn(2+) in living cells. Metallomics 7(2):258–266. http://sci-hub.tw/10.1039/c4mt00179f
Vishnu N, Jadoon Khan M, Karsten F et al (2014) ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release. Mol Biol Cell 25(3):368–379. http://sci-hub.tw/10.1091/mbc.E13-07-0433
Waldeck-Weiermair M, Alam MR, Khan MJ et al (2012) Spatiotemporal correlations between cytosolic and mitochondrial Ca2+ signals using a novel red-shifted mitochondrial targeted cameleon. PLoS One 7(9):e45917. http://sci-hub.tw/10.1371/journal.pone.0045917
Chiu WK, Towheed A, Palladino MJ (2014) Genetically encoded redox sensors. Methods Enzymol 542:263–287. http://sci-hub.tw/10.1016/B978-0-12-416618-9.00014-5
Eroglu E, Gottschalk B, Charoensin S et al (2016) Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nat Commun 7:10623. http://sci-hub.tw/10.1038/ncomms10623
Storace D, Rad MS, Han Z et al (2015) Genetically encoded protein sensors of membrane potential. Adv Exp Med Biol 859:493–509. http://sci-hub.tw/10.1007/978-3-319-17641-3_20
Deuschle K, Fehr M, Hilpert M et al (2005) Genetically encoded sensors for metabolites. Cytometry A 64(1):3–9. http://sci-hub.tw/10.1002/cyto.a.20119
Zhao Y, Araki S, Wu J et al (2011) An expanded palette of genetically encoded Ca2+ indicators. Science 333(6051):1888–1891. http://sci-hub.tw/10.1126/science.1208592
Lukyanov KA, Belousov VV (2014) Genetically encoded fluorescent redox sensors. Biochim Biophys Acta 1840(2):745–756. http://sci-hub.tw/10.1016/j.bbagen.2013.05.030
Taylor SC, Ferguson AD, Bergeron JJM et al (2004) The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nat Struct Mol Biol 11(2):128–134. http://sci-hub.tw/10.1038/nsmb715
Osibow K, Malli R, Kostner GM et al (2006) A new type of non-Ca2+-buffering Apo(a)-based fluorescent indicator for intraluminal Ca2+ in the endoplasmic reticulum. J Biol Chem 281(8):5017–5025. http://sci-hub.tw/10.1074/jbc.M508583200
Rosado CJ, Mijaljica D, Hatzinisiriou I et al (2008) Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. Autophagy 4(2):205–213
Tang HL, Tang HM, Fung MC et al (2016) In vivo biosensor tracks non-apoptotic caspase activity in drosophila. J Vis Exp 117. http://sci-hub.tw/10.3791/53992
Ivnitskii DM, Rishpon J (1993) Biosensor based on direct detection of membrane potential induced by immobilized hydrolytic enzymes. Anal Chim Acta 282(3):517–525. http://sci-hub.tw/10.1016/0003-2670(93)80115-2
Germond A, Fujita H, Ichimura T et al (2016) Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters. Biophys Rev 8:121–138. http://sci-hub.tw/10.1007/s12551-016-0195-9
Looger LL, Lalonde S, Frommer WB (2005) Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells. Plant Physiol 138(2):555–557. http://sci-hub.tw/10.1104/pp.104.900151
Souslova EA, Chudakov DM (2007) Genetically encoded intracellular sensors based on fluorescent proteins. Biochemistry (Mosc) 72(7):683–697
Raimondo JV, Joyce B, Kay L et al (2013) A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system. Front Cell Neurosci 7:202. http://sci-hub.tw/10.3389/fncel.2013.00202
Eroglu E, Rost R, Bischof H et al (2017) Application of genetically encoded fluorescent nitric oxide (NO•) probes, the geNOps, for real-time imaging of NO• signals in single cells. J Vis Exp 121:e55486. http://sci-hub.tw/10.3791/55486
Opelt M, Eroglu E, Waldeck-Weiermair M et al (2016) Formation of nitric oxide by aldehyde dehydrogenase-2 is necessary and sufficient for vascular bioactivation of nitroglycerin. J Biol Chem 291(46):24076–24084. http://sci-hub.tw/10.1074/jbc.M116.752071
Charoensin S, Eroglu E, Opelt M et al (2017) Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS). Free Radic Biol Med 102:248–259. http://sci-hub.tw/10.1016/j.freeradbiomed.2016.11.049
Acknowledgments
The authors thank the scientific advisory board of NGFI (Next Generation Fluorescence Imaging GmbH, Graz, Austria, http://www.ngfi.eu/) for their support.
Sources of Funding
This work is supported by Nikon Austria within the Nikon-Center of Excellence Graz. The researchers are also supported by the Ph.D. program Metabolic and Cardiovascular Disease (DK-W1226) of the Medical University of Graz, and also by the FWF project P 28529-B27. Microscopic equipment is part of the Nikon-Center of Excellence, Graz that is supported by the Austrian infrastructure program 2013/2014, Nikon Austria Inc., and BioTechMed, Graz.
Disclosure
E.E, M.W., R.M., and W.F.G., staff members of the Medical University of Graz, have filed a U.K. patent application (patent application number WO2015EP74877 20151027, priority number GB20140019073 20141027) that describe parts of the research in this manuscript. Licenses related to this patent are provided to Next Generation Fluorescence Imaging (NGFI) GmbH (http://www.ngfi.eu/), a spin-off company of the Medical University of Graz.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Eroglu, E., Bischof, H., Charoensin, S., Waldeck-Weiermaier, M., Graier, W.F., Malli, R. (2018). Real-Time Imaging of Nitric Oxide Signals in Individual Cells Using geNOps. In: Mengel, A., Lindermayr, C. (eds) Nitric Oxide. Methods in Molecular Biology, vol 1747. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7695-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7695-9_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7694-2
Online ISBN: 978-1-4939-7695-9
eBook Packages: Springer Protocols