Dataset Information

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

ABSTRACT: Mitochondrial membrane potential (??m) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ??m were qualitative or semiquantitative. Here, we quantified intercellular differences in ??m in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ??m using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ??m. We showed that ??m is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ??m cells. Overall, our results showed that intercellular heterogeneity of ??m is mainly modulated by intramitochondrial factors, it is independent of the ??m indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

SUBMITTER: Rovini A

PROVIDER: S-EPMC7871195 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Rovini Amandine A Heslop Kareem K Hunt Elizabeth G EG Morris Morgan E ME Fang Diana D Gooz Monika M Gerencser Akos A AA Maldonado Eduardo N EN

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20201116 1

Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is ...[more]

PMID: 33196122

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Project description:Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

Dataset Information

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Publications

Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells.

Similar Datasets

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