Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

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 NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

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A novel technique for mapping viscosity in discrete subcellular locations with a BODIPY based fluorescent probe

10.1101/522532 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lior Pytowski ◽

Alex C. Foley ◽

Zayra E. Hernández ◽

Niall Moon ◽

Tim Donohoe ◽

...

Keyword(s):

Fluorescence Lifetime ◽

Fluorescence Lifetime Imaging ◽

Mitochondrial Matrix ◽

Molecular Rotor ◽

Cellular Processes ◽

Lifetime Imaging ◽

Novel Technique ◽

Absolute Viscosity ◽

Membrane Bound ◽

Fluorescent Molecules

AbstractNumerous cellular processes, including enzyme behaviour, signalling, and protein folding and transport are highly influenced by the local microviscosity environment within living cells. Molecular rotors are fluorescent molecules that respond to the viscosity of their environment through changes in both the intensity and lifetime of their fluorescence. We have synthesised a novel boron-dipyrrin (BODIPY) molecular rotor that is also a substrate for the SNAP-tag targeting system (named BG-BODIPY), allowing us to target the rotor to discrete locations within the living cell. We demonstrate that BG-BODIPY reports viscosity, and that this can be measured either through fluorescence lifetime or intensity ratiometric measurements. The relative microviscosities within the ER, Golgi, mitochondrial matrix, peroxisomes, lysosomes, cytoplasm, and nucleoplasm were significantly different. Additionally, this approach permitted fluorescence lifetime imaging microscopy (FLIM) to determine the absolute viscosity within both mitochondria and stress granules, showcasing BG-BODIPY’s usefulness in studying both membrane bound and membraneless organelles. These results highlight targeted BG-BODIPY’s broad usefulness for making measurements of cellular viscosity both with FLIM and conventional confocal microscopy, the latter option greatly extending the accessibility of the technique.

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