Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Konovalova, Svetlana

doi:10.3791/59269

Cited by 10 publications

(4 citation statements)

References 10 publications

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“…4A). Our results coincided with Konovalova's report that nuclear-encoded components of CI (NDUFA9: NADH ubiquinone oxidoreductase subunit A9) and CIII (Core II) were also downregulated in chloramphenicol-treated human neuroblastoma SH-SY5Y cells (Konovalova, 2019). Therefore, depletion of mitochondrially encoded components could affect other protein expressions of nuclear-encoded components of respiratory chain complexes.…”

Section: Direct Comparison With Signals Of Iga and Western Blottingsupporting

confidence: 92%

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV

et al. 2019

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The in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.

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Section: Direct Comparison With Signals Of Iga and Western Blottingsupporting

confidence: 92%

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV

et al. 2019

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“…Next, mitochondrial and cytoplasmic fractions were isolated from Scrambled (scr.) and ARID1A KD ES-2 cells ( Figure 1 F) and the levels in the outer membrane of mitochondria (OMM) marker TOM20 and the mitochondrial complexes were analyzed via Western blot and native polyacrylamide gel electrophoresis (BN-PAGE) respectively [ 46 , 47 , 48 ]. As shown in Figure 1 G, the levels of TOM20 were higher in the mitochondrial fraction of ARID1A KD ES-2 cells as compared to scramble.…”

Section: Resultsmentioning

confidence: 99%

Targeting Mitochondrial Metabolism in Clear Cell Carcinoma of the Ovaries

Zhang

Shetty

Clemente

et al. 2021

IJMS

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Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.

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“…The assembly of mitochondrial respiratory chain complexes was evaluated with blue-native PAGE using isolated mitochondria from iPSC and fibroblasts as described elsewhere ( 62 ), followed by western blot analysis of mitochondrial Complex V ATP5 and Complex II SDHA. Antibodies used are indicated in Supplementary Material, Table S3 .…”

Section: Methodsmentioning

confidence: 99%

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism

Kenvin

Torregrosa‐Muñumer

Reidelbach

et al. 2021

Human Molecular Genetics

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Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

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Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Cited by 10 publications

References 10 publications

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV

Targeting Mitochondrial Metabolism in Clear Cell Carcinoma of the Ovaries

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism

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