title: Visualization of NO3⁻/NO2⁻ Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System.
abstract: Nitrate (NO3(-)) and nitrite (NO2(-)) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO3(-)/NO2(-) exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO3(-)/NO2(-) for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO3(-)/NO2(-) in physiology), by employing NO3(-)/NO2(-)-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO3(-) and NO2(-), and its FRET signal is changed in response to NO3(-)/NO2(-) in the micromolar range. Furthermore, both an increase and decrease in cellular NO3(-) concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO3(-)/NO2(-) dynamics in various organisms, including plants and animals.
authors.affiliation: From the Department of Molecular Cell Science, Tohoku University, Sendai 981-8555.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Hidaka
authors.affiliation: From the Department of Molecular Cell Science, Tohoku University, Sendai 981-8555.
authors.forename: Aina
authors.initials: A
authors.lastname: Gotoh
authors.affiliation: From the Department of Molecular Cell Science, Tohoku University, Sendai 981-8555.
authors.forename: Taiki
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: the Department of Environmental Life Sciences, Tohoku University, Sendai 980-8577, and.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Minamisawa
authors.affiliation: The Hakubi Center for Advanced Research, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
authors.affiliation: From the Department of Molecular Cell Science, Tohoku University, Sendai 981-8555, uchidat@biochem.tohoku.ac.jp.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Uchida
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D020369:Bradyrhizobium; D058440:Denitrification; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D009566:Nitrates / Q000737:chemistry*; D009569:Nitric Oxide; D009573:Nitrites / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D018517:Plant Roots / Q000382:microbiology; D025941:Protein Interaction Mapping; D012231:Rhizobium; D015398:Signal Transduction
chemical_list: D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; D009584:Nitrogen
keywords: NO3−/NO2− in physiology; bacterial signal transduction; biosensor; fluorescence resonance energy transfer (FRET); in vivo imaging; nitrogen metabolism; protein-protein interaction