Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase

Abstract

Edited by Wolfgang Peti Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22 phox) and three cytosolic subunits (p40 phox , p47 phox , and p67 phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy basedonFRET-fluorescencelifetimeimaging(FLIM)andfluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter-and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

“…The theoretical Förster distance, R 0 , at which the donor/acceptor FRET efficiency is 50 %, was calculated as 55 Å for the Aquamarine/dLanYFP monomer FRET pair (κ 2 =0,476 3 ). It is in the same order of magnitude as other CFP – YFP FRET pair (50-60 Å) and we decided to evaluate the possibility to use tdLanYFP as acceptor in FRET-based imaging applications.…”

“…We also evaluated the possibility to use tdLanYFP as a FRET donor in intermolecular FRET. As a model, we used two subunits of the NADPH oxidase, p67 phox and p40 phox that are known to interact in the resting state of the enzyme 3 . The subunits were transiently expressed in COS7 cells and FRET was monitored by TCSPC-FLIM (Fig.…”

“…For microscopy, cells were grown to 80 % confluence on glass coverslip (ibidi) and transiently transfected with the appropriate expression plasmids with XtremeGene HP (Roche Diagnostics) following the supplier’s instructions and used 24–48 h after the transfection. 3 AURKA FRET biosensor was expressed in U2OS cells. The cells were cultivated, transfected and synchronized at mitosis as previously described.…”

Consequences of the constitutive NOX2 activity in living cells: cytosol acidification, apoptosis, and localized lipid peroxidation

“…where I0 is the fluorescence intensity at t = 0, τD is the fluorescence lifetime of a donor alone, and C is the constant where long and short are the proportions of a long (τlong) and a short (τshort) lifetime components respectively (SI of [49]).…”

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

“…As described for FLIM-FRET experiments, cells were incubated for 24h, 48h and 72h and the medium was changed for phenol red-free Leibovitz's L-15 medium (Thermo Fisher Scientific), supplemented with 20% fetal bovine serum, 1% L-glutamine and 1% penicillin-streptomycin prior to analysis [90]. FCS measurements were performed using a confocal microscope with an FCS module as previously described [92,93]. Our setup was composed of a Leica SP8 (Leica) inverted confocal microscope with a 63X water objective with correcting ring (NA 1.2) and combined with a PicoQuant time-correlated single photon counting (TCSPC) module (PicoHarp, PicoQuant).…”