Optimization of cell permeabilization in electron flow based mitochondrial function assays

Lei, Xiang-He; Bochner, Barry R.

doi:10.1016/j.freeradbiomed.2021.10.014

Cited by 9 publications

(7 citation statements)

References 57 publications

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“…The protein was aliquoted, snap frozen, and stored at −80°C until required. For each assay, a fresh aliquot was thawed, used immediately, and discarded afterwards, due to the instability of this protein [55].…”

Section: Methodsmentioning

confidence: 99%

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Needs

Pereira

Glover

et al. 2022

Preprint

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Mitochondrial biogenesis requires the import of most of their proteins from the cytosol. Therefore, efficient import apparatus is vital for eukaryotic cell function, particularly in highly energy demanding cells such as neurons and myocytes. Consequently, dysfunctional mitochondrial protein import is implicated in many diseases. This study explores the molecular basis and consequences of import failure in mammalian cells. We show that blocking import machinery has profound effects on mitochondrial ultra-structure and dynamics, but surprisingly little impact on import. The explanation is a remarkable mechanism in which healthy cells transfer mitochondria to cells with deficient mitochondrial import, and vice versa, through connecting tunnelling nanotubes. These observations suggest the existence of a widespread mechanism for rescue of mitochondrial import function in complex multicellular organisms.

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Section: Methodsmentioning

confidence: 99%

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Needs

Pereira

Glover

et al. 2022

Preprint

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“…A metabolic substrate that feeds complex II such as succinate will result in a strong flow of electrons via succinate dehydrogenase, which is expected to be inhibited by complex II and III (antimycin A and myxothiazol) inhibitors but not complex I blockers. A saponin concentration of 30 μg/mL has been shown to efficiently permeabilize cell membrane and abolish glucose metabolism without promoting any significant alteration of the mitochondrial metabolism of substrates such as malate and succinate [ 67 ]. Validation of the assay in the presence of succinate confirmed a lack of activity of the complex I inhibitors rotenone and pyridaben regardless of the presence or absence of the EMT phenotype.…”

Section: Resultsmentioning

confidence: 99%

“…The Biolog MitoPlate S-1™ assay directly measures electron flow into and through the ETC from various mitochondrial substrates that produce NADH and FADH 2 under conditions of saponin permeabilization. Notably, saponin permeabilizes only the plasma membrane, leaving the intracellular membranes of the mitochondria intact while equilibrating the intracellular spaces with incubation medium [ 67 , 91 ]. Our findings, therefore, support the notion that lactate could be imported and converted into pyruvate inside mitochondria to directly feed the TCA cycle in EMT cells.…”

Section: Discussionmentioning

confidence: 99%

Metabolomic and Mitochondrial Fingerprinting of the Epithelial-to-Mesenchymal Transition (EMT) in Non-Tumorigenic and Tumorigenic Human Breast Cells

Cuyàs

Fernández-Arroyo

Verdura

et al. 2022

Cancers

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Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial–cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. “Upper” and “lower” glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl–carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

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“…DNA synthesis was performed in lysolecithin-treated permeable cells [ 67 ] . This protocol was applied in permeable CHO cells synchronized in the S phase [ 68 ]. Smeared chromatin suggested abandoning the use of mild detergents for the isolation of interphase chromosomes.…”

Section: Methods Applied For Permeabilizationmentioning

confidence: 99%

Macromolecular Structure of Linearly Arranged Eukaryotic Chromosomes

Bánfalvi

2022

IJMS

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Eukaryotic chromosomes have not been visualized during the interphase. The fact that chromosomes cannot be seen during the interphase of the cell cycle does not mean that there are no means to make them visible. This work provides visual evidence that reversible permeabilization of the cell membrane followed by the regeneration of cell membranes allows getting a glimpse behind the nuclear curtain. Reversibly permeable eukaryotic cells have been used to synthesize nascent DNA, analyze the 5′-end of RNA primers, view individual replicons and visualize interphase chromosomes. Dextran T-150 in a slightly hypotonic permeabilizing buffer prevented cells from disruption. Upon reversal of permeabilization, the nucleus could be opened at any time during the interphase. A broad spectrum of a flexible chromatin folding pattern was revealed through a series of transient geometric forms of chromosomes. Linear attachment of chromosomes was visualized in several mammalian and lower eukaryotic cells. The linear connection of chromosomes is maintained throughout the cell cycle showing that rather than individual chromosomes, a linear array of chromosomes is the functional giant macromolecule. This study proves that not only the prokaryotic genome but also linearly attached eukaryotic chromosomes form a giant macromolecular unit.

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Optimization of cell permeabilization in electron flow based mitochondrial function assays

Cited by 9 publications

References 57 publications

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Metabolomic and Mitochondrial Fingerprinting of the Epithelial-to-Mesenchymal Transition (EMT) in Non-Tumorigenic and Tumorigenic Human Breast Cells

Macromolecular Structure of Linearly Arranged Eukaryotic Chromosomes

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