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Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured cells harboring
C Sobreira1, M Davidson, M P King
1Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, USA.
Abstract:
Several human diseases have been found to be caused by mitochondrial DNA (mtDNA) mutations. Pathogenic mutated (mut) mtDNAs are usually "heteroplasmic," coexisting intracellularly with wild-type (wt) mtDNAs. For some mtDNA mutations, cells have normal levels of respiratory chain function unless the percentage of mut-mtDNA is very high. Although progress in understanding the molecular basis of mitochondrial diseases has been remarkable, the heterogeneity of mut-mtDNA distribution, even among cells of the same tissue, makes it difficult to clearly delineate the relationships between mtDNA mutations, gene dosage, and clinical phenotypes. In a search for screening methods for identifying cultured cells with deficient mitochondrial function, we incubated living cells harboring mut-mtDNAs with dihydrorhodamine 123 (DHR123), an uncharged, nonfluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining was observed in cells that respired normally. Fluorescence was significantly reduced in cells with mitochondrial respiratory chain dysfunction resulting from very high levels of mut-mtDNAs. The data show that DHR123 is useful for assessing mitochondrial function in single cells, and can be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures.
Insights
Researchers developed a new method using dihydrorhodamine 123 (DHR123) to detect mitochondrial DNA (mtDNA) mutations. This technique helps identify cells with impaired mitochondrial function and isolate them from mixed cell populations.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Mitochondrial DNA (mtDNA) mutations cause human diseases, often existing heteroplasmically with wild-type mtDNA.
- Cellular respiratory function can remain normal until mutated mtDNA levels are very high.
- Heterogeneous mtDNA mutation distribution complicates understanding disease mechanisms and phenotypes.
Purpose of the Study:
- To develop a screening method for identifying cultured cells with mitochondrial dysfunction.
- To assess the utility of dihydrorhodamine 123 (DHR123) for evaluating mitochondrial function at the single-cell level.
Main Methods:
- Incubation of living cells with mutated mtDNA using dihydrorhodamine 123 (DHR123).
- DHR123 is converted to fluorescent rhodamine 123 (R123) by mitochondrial oxidation.
- Assessment of fluorescence intensity to indicate mitochondrial respiratory function.
Main Results:
- Normal respiring cells showed bright mitochondrial staining with R123.
- Cells with high levels of mutated mtDNA and respiratory dysfunction exhibited significantly reduced fluorescence.
- DHR123 effectively distinguishes between cells with normal and deficient mitochondrial function.
Conclusions:
- Dihydrorhodamine 123 (DHR123) is a valuable tool for assessing mitochondrial function in single cells.
- This method allows for the isolation of viable, respiratory chain-deficient cells from heterogeneous cultures.
- The DHR123 assay aids in studying mitochondrial diseases caused by mtDNA mutations.