Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels

Sevimli, Gulsah; Smith, Matthew J.; Caglar, Tuba Akgul; Bilir, Şükriye; Secilmis, Melike; Altun, Hamza Yusuf; Yiğit, Esra Nur; Yang, Fan; Keeley, Thomas P.; Malli, Roland; Öztürk, Gürkan; Mann, Giovanni E.; Eroğlu, Emrah

doi:10.1016/j.redox.2022.102319

Cited by 10 publications

(9 citation statements)

References 54 publications

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“…However, it has not been feasible to control exact concentrations of iron (II) to calibrate geNOps in cells. Additionally, the calibration of recombinant geNOps failed [ 25 ]. Thus, the assay only assesses subcellular iron availability, but does not provide an absolute quantification of subcellular iron (II) concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were maintained in a humidified CO 2 incubator (5% CO 2 , 37 °C). Stable EA.hy926 ] and HEK293T cell lines expressing O-geNOps-NES [ 24 ] used in this study were developed in previous studies [ 25 ]. The same protocols were applied to generate stable HeLa cells, as described previously [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

“…Ferrous but not ferric iron can activate the probe [ 20 , 21 , 22 , 23 , 24 ]. In a recent study, we exploited the geNOps technology to study the role of pericellular oxygen levels and cellular ferrous iron uptake [ 25 ]. The study unveiled that cells cultured under physiological levels of oxygen (5 kPa O 2 ) require lower iron (II) supplementation compared to cells adapted to hyperoxia (18 kPa O 2 ).…”

Section: Introductionmentioning

confidence: 99%

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Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Sevimli

Alston²,

Funk

et al. 2022

Biosensors

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Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Sevimli

Alston²,

Funk

et al. 2022

Biosensors

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“…The cells were pre-treated with Iron (II) supplementation (300 M FeSO4 and 500 M ascorbate) for 15 min for full activation of the geNOp biosensor prior to experiments as described previously [44].…”

Section: Iron Loading Proceduresmentioning

confidence: 99%

Visualizing H₂O₂and NO in endothelial cells: strategies and pitfalls

Altun

Secilmis

Caglar

et al. 2023

Preprint

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The relationship between hydrogen peroxide (H2O2) and nitric oxide (NO) in the vasculature is multifaceted and remains controversial because the dynamic detection of these reactive molecules is challenging. Genetically encoded biosensors (GEBs) allow visualizing real-time dynamics in living cells and permit multiparametric detection of several analytes. Although robust, GEBs' utility depends on several parameters that need fine-tuning for proper imaging and correct data analysis: i.e., camera binning, temperature, and the resolution power of the imaging instruments are some critical parameters that require optimization. We have generated a new double-stable transgenic endothelial cell line stably expressing the biosensors HyPer7 and O-geNOp and systematically tested different imaging modes and their impact on the performance of each biosensor. Ambient temperature and the type of imaging mode did not influence the results, while camera resolution settings significantly affected readouts of HyPer probes but not O-geNOp. Changing a single parameter in a co-imaging mode significantly altered the biosensor's dynamic measurements, potentially causing misinterpretation. This study provides a general guide and the pitfalls of employing GEBs in a multispectral imaging mode.

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“…However, it has been observed that data on reversibility of this sensor are not conclusive. Nevertheless, geNOps have been used recently for insightful physiological studies [ 23 ]. Chimeric proteins comprising myoglobin as a sensing unit and either mCherry, or a tandem fusion between Enhanced Yellow Fluorescent Protein (EYFP) (or citrine) and mCherry, as fluorescent reporters were recently proposed as NO sensors.…”

Section: Introductionmentioning

confidence: 99%

Nitric Oxide Sensing by a Blue Fluorescent Protein

et al. 2022

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S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

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Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels

Cited by 10 publications

References 54 publications

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Visualizing H₂O₂and NO in endothelial cells: strategies and pitfalls

Nitric Oxide Sensing by a Blue Fluorescent Protein

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Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels

Cited by 10 publications

References 54 publications

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Visualizing H2O2and NO in endothelial cells: strategies and pitfalls

Nitric Oxide Sensing by a Blue Fluorescent Protein

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Product

Resources

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Visualizing H₂O₂and NO in endothelial cells: strategies and pitfalls