Visualization of NO3−/NO2− Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

摘要

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.