title: Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells.
abstract: Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable. Recently, we expanded the pallet of NO• indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO•) probes (geNOps) that directly respond to cellular NO• fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO• signals in response to two different chemical NO•-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO• levels as compared with the inorganic NO• donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO• formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO• signals in diverse experimental setups.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Emrah
authors.initials: E
authors.lastname: Eroglu
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Rene
authors.initials: R
authors.lastname: Rost
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Helmut
authors.initials: H
authors.lastname: Bischof
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Sandra
authors.initials: S
authors.lastname: Blass
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Anna
authors.initials: A
authors.lastname: Schreilechner
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gottschalk
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Depaoli
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Christiane
authors.initials: C
authors.lastname: Klec
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Suphachai
authors.initials: S
authors.lastname: Charoensin
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Corina T
authors.initials: CT
authors.lastname: Madreiter-Sokolowski
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Jeta
authors.initials: J
authors.lastname: Ramadani
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Markus
authors.initials: M
authors.lastname: Waldeck-Weiermair
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz.
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.affiliation: Institute of Molecular Biology and Biochemistry, Medical University of Graz; roland.malli@medunigraz.at.
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus; D042783:Endothelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000737:chemistry; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D019219:Parvovirinae / Q000235:genetics
chemical_list: C115239:3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine; D005456:Fluorescent Dyes; D006834:Hydrazines; D020030:Nitric Oxide Donors; D009599:Nitroprusside; D009569:Nitric Oxide; D016257:Fura-2