Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data

Divakaruni, Ajit S.; Paradyse, Alexander; Ferrick, David A.; Murphy, Anne N.; Jastroch, Martin

doi:10.1016/b978-0-12-801415-8.00016-3

Cited by 406 publications

(476 citation statements)

References 125 publications

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“…4A) or the extracellular acidification rate (ECAR) (Fig. 4B), a parameter used to assess glycolytic activity (Divakaruni et al 2014), even though FASTKD2 expression was efficiently silenced (Fig. 4C).…”

Section: Fastkd2 Is Required For Mitochondrial Translationmentioning

confidence: 99%

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FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation

Popow

Alleaume

Curk

et al. 2015

RNA

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Mitochondrial RNA processing is an essential step for the synthesis of the components of the electron transport chain in all eukaryotic organisms, yet several aspects of mitochondrial RNA biogenesis and regulation are not sufficiently understood. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNAbinding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. CRISPR-mediated deletion of FASTKD2 leads to aberrant processing and expression of RNR2 and ND6 mRNA that encodes a subunit of the respiratory complex I. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNAbinding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses.

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Section: Fastkd2 Is Required For Mitochondrial Translationmentioning

confidence: 99%

“…We first measured basal-, leak-, and maximal respiration by micro-respirometry (Divakaruni et al 2014) after adaptation of the protocol to HEK293 cells. Consistent with the less severe changes in mitochondrial RNA processing (Fig.…”

Section: Fastkd2 Is Required For Mitochondrial Translationmentioning

confidence: 99%

FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation

Popow

Alleaume

Curk

et al. 2015

RNA

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“…The glycolytic reserve capacity was calculated as the difference between ECAR Basal and ECAR Oligomycin . Adding 2-deoxyglucose (2DG) inhibited all glycolytic function and allowed a measure of the nonglycolytic acidification rate (18).…”

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confidence: 99%

Macrophages Promote Oxidative Metabolism To Drive Nitric Oxide Generation in Response to Trypanosoma cruzi

et al. 2016

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dTrypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (ms), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) ms were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent m stimulation and infection states, respectively. Ms incubated with lipopolysaccharide and gamma interferon (LPS/IFN-␥) and with interleukin-4 (IL-4) were used as controls. We monitored the cytokine profile (using enzyme-linked immunosorbent assay [ELISA]), reactive oxygen species (ROS; fluorescent probes), nitric oxide (·NO; Griess assay), and metabolic state using a customdesigned mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-␥ treatment of ms elicited a potent increase in production of tumor necrosis alpha (TNF-␣) at 3 h and of ROS and ·NO by 18 h. Upon SYL infection, murine ms elicited an inflammatory cytokine profile (TNF-␣ > > TGF-␤ ؉ IL-10) and low levels of ·NO and ROS production. LPS/IFN-␥ treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in ms, while IL-4-treated ms utilized oxidative metabolism to meet energy demands. SYL infection resulted in an intermediate functional metabolic state with increased mitoxosome gene expression and glycolysis, and IFN-␥ addition shut down the oxidative metabolism in SYL-infected ms. Further, TCC-and SYL-stimulated ms exhibited similar levels of cell proliferation and production of TNF-␣ and ROS, while TCC-stimulated ms exhibited up to 2-fold-higher levels of oxidative metabolism and ·NO production than SYL-infected ms. Inhibiting ATP-coupled O 2 consumption suppressed the ·NO generation in SYL-infected ms. Mitochondrial oxygen consumption constitutes a mechanism for stimulating ·NO production in ms during T. cruzi infection. Enhancing the oxidative metabolism provides an opportunity for increased ·NO production and pathogen clearance by ms to limit disease progression. Chagas cardiomyopathy is a neglected debilitating disease caused by the blood-borne parasite Trypanosoma cruzi. T. cruzi isolates are classified within six genetic groups (TcI to TcVI) which give insight into the evolution of the parasite (1). The virulent T. cruzi strain SylvioX10/4 (SYL) (2) and the nonpathogenic T. cruzi isolate (TCC) (3) are both of the TcI lineage which is considered to be the most common cause of disease in the Southern zone countries of South America, Central America, and Mexico. In mice and rats, SYL elicits acute parasitemia and persistent inflammatory infiltrate and injury in the myocardium and skeletal muscle during the chronic disease phase (4), while infection with the TCC isolate results in no detectable parasitemia or tissue injury (5).Macrophages (ms) serve as the first responders to T. cruzi infection, and their inflammatory activation exerts cytotoxic effects via NADPH oxidase (NOX)-mediated superoxide (O 2 · Ϫ ) ...

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“…4A) and cellular substrate preference (see below) were similar in WT and kin Ϫ cells. This renders PPR a reliable indicator of glycolytic turnover for this specific comparison (36,37).…”

Section: Bioenergetic Measurements In Wt and Kinmentioning

confidence: 99%

“…4A), implying mitochondrial dysfunction, a shift of ATP production to glycolysis, or an overall decrease in the rate of ATP utilization by the cells (37). To determine if CPT-cAMP induces mitochondrial dysfunction, activity of the electron transport chain was disengaged from cellular ATP turnover by the addition of oligomycin and the protonophore FCCP (Fig.…”

Section: Bioenergetic Measurements In Wt and Kinmentioning

confidence: 99%

Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A

Wilderman

Guo

Divakaruni

et al. 2015

Journal of Biological Chemistry

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Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data

Cited by 406 publications

References 125 publications

FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation

FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation

Macrophages Promote Oxidative Metabolism To Drive Nitric Oxide Generation in Response to Trypanosoma cruzi

Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A

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