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Real-Time Imaging of Nitric Oxide Signals in Individual Cells Using geNOps

  • Emrah Eroglu
  • Helmut Bischof
  • Suphachai Charoensin
  • Markus Waldeck-Weiermaier
  • Wolfgang F. Graier
  • Roland Malli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

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.

Key words

Fluorescence microscopy Genetically encoded probes Nitric oxide imaging Single cell analysis 

Notes

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.

References

  1. 1.
    Forstermann U, Closs EI, Pollock JS et al (1994) Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23(6 Pt 2):1121–1131CrossRefPubMedGoogle Scholar
  2. 2.
    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):267–277CrossRefPubMedGoogle Scholar
  3. 3.
    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 CrossRefPubMedGoogle Scholar
  4. 4.
    Han J, Burgess K (2010) Fluorescent indicators for intracellular pH. Chem Rev 110(5):2709–2728. http://sci-hub.tw/10.1021/cr900249z CrossRefPubMedGoogle Scholar
  5. 5.
    Kojima H, Urano Y, Kikuchi K et al (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed Engl 38(21):3209–3212CrossRefPubMedGoogle Scholar
  6. 6.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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 CrossRefPubMedGoogle Scholar
  9. 9.
    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 CrossRefPubMedGoogle Scholar
  10. 10.
    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 CrossRefPubMedGoogle Scholar
  11. 11.
    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 CrossRefPubMedGoogle Scholar
  12. 12.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. http://sci-hub.tw/10.1146/annurev.biochem.67.1.509 CrossRefPubMedGoogle Scholar
  14. 14.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    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 CrossRefGoogle Scholar
  16. 16.
    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 CrossRefPubMedGoogle Scholar
  17. 17.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    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 CrossRefPubMedGoogle Scholar
  20. 20.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    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 CrossRefPubMedGoogle Scholar
  22. 22.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    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 CrossRefPubMedGoogle Scholar
  25. 25.
    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 CrossRefPubMedGoogle Scholar
  26. 26.
    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 CrossRefPubMedGoogle Scholar
  27. 27.
    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):205–213CrossRefPubMedGoogle Scholar
  28. 28.
    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
  29. 29.
    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 CrossRefGoogle Scholar
  30. 30.
    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 CrossRefPubMedCentralGoogle Scholar
  31. 31.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Souslova EA, Chudakov DM (2007) Genetically encoded intracellular sensors based on fluorescent proteins. Biochemistry (Mosc) 72(7):683–697CrossRefGoogle Scholar
  33. 33.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    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 Google Scholar
  35. 35.
    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 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    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 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Emrah Eroglu
    • 1
  • Helmut Bischof
    • 1
  • Suphachai Charoensin
    • 1
  • Markus Waldeck-Weiermaier
    • 1
  • Wolfgang F. Graier
    • 1
  • Roland Malli
    • 1
  1. 1.Molecular Biology and Biochemistry, Gottfried Schatz Research CenterMedical University of GrazGrazAustria

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