MitoNeoD: A Mitochondria-Targeted Superoxide Probe

Shchepinova, Maria M.; Cairns, Andrew G.; Prime, Tracy A.; Logan, Angela; James, Andrew M.; Hall, Andrew; Vidoni, Sara; Arndt, Sabine; Caldwell, Stuart T.; Prag, Hiran A.; Pell, Victoria R.; Krieg, Thomas; Mulvey, John; Yadav, Pooja; Cobley, James N.; Bright, Thomas P.; Senn, Hans Martin; Anderson, Robert F.; Murphy, Michael P.; Hartley, Richard C.

doi:10.1016/j.chembiol.2017.08.003

Cited by 83 publications

(64 citation statements)

References 55 publications

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“…Second, MitoSox can intercalate mtDNA resulting in an ~20-fold higher fluorescence that is independent of redox status [134]. Fortunately, newer mitochondrial superoxide probes, including MitoB [135,136] and MitoNeoD [137], have been developed to overcome these shortcomings. Another mitochondrial dye, which monitors temperature gradients (mito thermo yellow) has been used to suggest the provocative idea that mitochondria may be physiologically maintained around 50°C [138,139].…”

Section: 3 Resultsmentioning

confidence: 99%

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

Harwig

Viana

Egner

et al. 2018

Analytical Biochemistry

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Mitochondria are found in a variety of shapes, from small round punctate structures to a highly interconnected web. This morphological diversity is important for function, but complicates quantification. Consequently, early quantification efforts relied on various qualitative descriptors that understandably reduce the complexity of the network leading to challenges in consistency across the field. Recent application of state-of-the-art computational tools have resulted in more quantitative approaches. This prospective highlights the implementation of MitoGraph, an open-source image analysis platform for measuring mitochondrial morphology initially optimized for use with Saccharomyces cerevisiae. Here Mitograph was assessed on five different mammalian cells types, all of which were accurately segmented by MitoGraph analysis. MitoGraph also successfully differentiated between distinct mitochondrial morphologies that ranged from entirely fragmented to hyper-elongated. General recommendations are also provided for confocal imaging of labeled mitochondria (using mito-YFP, MitoTracker dyes and immunostaining parameters). Widespread adoption of MitoGraph will help achieve a long-sought goal of consistent and reproducible quantification of mitochondrial morphology.

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Section: 3 Resultsmentioning

confidence: 99%

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

Harwig

Viana

Egner

et al. 2018

Analytical Biochemistry

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“…Derivatives of HE for site-specific detection of O2 •-have been reported (64)(65)(66). Those probes share the same oxidative chemistry with HE (65).…”

Section: Superoxidementioning

confidence: 99%

“…In those reports, 2-OH-E + , 2-OH-Mito-E + , and 2-OH-Pr ++ were used as specific marker products for O2 •-. Hydroxylation of ethidium-based probes remains a method of choice for the detection of O2 •in cell-free and cellular systems (17,22,(64)(65)(66)79).…”

Section: -Hydroxyethidium As a Specific Marker For O2 •-mentioning

confidence: 99%

Tracking isotopically labeled oxidants using boronate-based redox probes

Ríos

Radí

Kalyanaraman

et al. 2020

Journal of Biological Chemistry

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Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18–labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

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“…First, Click PEGylation could be used to develop site-specific sentinels of mitochondrial superoxide production. Unravelling the providence of the superoxide detected by fluorescent reporters (e.g., MitoNeoD [165]) is challenging [166][167][168]. It is imperative to overcome existing challenges to rationally manipulate mitochondrial superoxide production to treat disease.…”

Section: Opportunities: How To Use Click Pegylation To Advance Knowlementioning

confidence: 99%

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions

Cobley

Husi

2020

Antioxidants

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To understand oxidative stress, antioxidant defense, and redox signaling in health and disease it is essential to assess protein thiol redox state. Protein thiol redox state is seldom assessed immunologically because of the inability to distinguish reduced and reversibly oxidized thiols by Western blotting. An underappreciated opportunity exists to use Click PEGylation to realize the transformative power of simple, time and cost-efficient immunological techniques. Click PEGylation harnesses selective, bio-orthogonal Click chemistry to separate reduced and reversibly oxidized thiols by selectively ligating a low molecular weight polyethylene glycol moiety to the redox state of interest. The resultant ability to disambiguate reduced and reversibly oxidized species by Western blotting enables Click PEGylation to assess protein thiol redox state. In the present review, to enable investigators to effectively harness immunological techniques to assess protein thiol redox state we critique the chemistry, promise and challenges of Click PEGylation.

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MitoNeoD: A Mitochondria-Targeted Superoxide Probe

Cited by 83 publications

References 55 publications

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

Methods for imaging mammalian mitochondrial morphology: A prospective on MitoGraph

Tracking isotopically labeled oxidants using boronate-based redox probes

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions

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