Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging.

Journal of Biophysics
R NiesnerM Gunzer

Abstract

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670 +/- 140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NAD...Continue Reading

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Citations

Sep 7, 2011·Journal of Fluorescence·Sebastian HerbrichKarl-Heinz Gericke
May 8, 2013·European Journal of Pharmacology·Rajkiran Mahalwar, Deepa Khanna
May 31, 2014·Nature Communications·Thomas S BlackerMichael R Duchen
Nov 9, 2010·Journal of Biomedical Optics·Yinghua SunLaura Marcu
Nov 2, 2015·Acta Neuropathologica·Agata A MossakowskiHelena Radbruch
Mar 26, 2016·Frontiers in Immunology·Helena RadbruchRaluca Niesner
Oct 22, 2013·British Journal of Haematology·Matthias Gunzer
Aug 16, 2016·Free Radical Biology & Medicine·Thomas S Blacker, Michael R Duchen
Sep 14, 2018·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Patrick M SchaeferBjoern von Einem
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Nov 11, 2019·International Journal of Molecular Sciences·Ruth LebenRaluca A Niesner
May 8, 2018·International Journal of Molecular Sciences·Randall L LindquistAnja E Hauser

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Methods Mentioned

BETA
FCS
scraping
PMA
fluorescence microscopy

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