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Mitochondria in cultured human muscle cells depleted of mitochondrial DNA
N H Herzberg1, E Middelkoop, M Adorf
1Department of Neurology, University of Amsterdam, The Netherlands.
Abstract:
Cultured human muscle cells were depleted of mitochondrial DNA (mtDNA) by prolonged treatment with ethidium bromide (EB). In these respiration-deficient muscle cells neither cytochrome c oxidase activity nor mtDNA were detectable. However, mitochondrial matrix enzymes remained present and were localized in mitochondria-like organelles, as shown by subcellular fractionation. Metabolic labeling showed synthesis of cytochrome c oxidase subunits coded by nuclear DNA (nDNA). These results indicate that depletion of mtDNA in cultured human myoblasts does not inhibit expression of nDNA-coded mitochondrial proteins. The characteristic thread-like pattern of mitochondria was lost in mtDNA-depleted myoblasts, as shown by immunofluorescence with antibodies against cytochrome c oxidase and the F1 part of the mitochondrial ATP synthase (F1-ATPase) and by fluorescence of the carbocyanine dye, 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)). The organelles visualized by these methods were round and swollen and had a localization different from lysosomes as shown by double-labeling with mitochondrial and lysosomal antibodies. These results indicate that not only synthesis, but also import of mitochondrial proteins into mitochondria-like organelles remains possible in respiration-deficient cells.
Insights
Human muscle cells lacking mitochondrial DNA (mtDNA) can still synthesize and import nuclear-coded mitochondrial proteins. This demonstrates that mitochondrial function is not entirely dependent on mtDNA for protein expression and localization.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Molecular Genetics
Background:
- Mitochondrial DNA (mtDNA) encodes essential components for cellular respiration.
- The interplay between nuclear DNA (nDNA) and mtDNA in mitochondrial protein synthesis is complex.
- Understanding mitochondrial biogenesis in the absence of mtDNA is crucial for studying mitochondrial diseases.
Purpose of the Study:
- To investigate the expression and localization of nuclear-coded mitochondrial proteins in human muscle cells depleted of mtDNA.
- To determine if mitochondrial protein import and assembly are affected by the absence of mtDNA.
- To elucidate the structural and functional consequences of mtDNA depletion on mitochondria-like organelles.
Main Methods:
- Prolonged treatment of cultured human muscle cells with ethidium bromide (EB) to deplete mtDNA.
- Assessment of cytochrome c oxidase activity and mtDNA presence.
- Subcellular fractionation to localize mitochondrial matrix enzymes.
- Metabolic labeling to detect synthesis of nDNA-coded proteins.
- Immunofluorescence and vital staining (DiOC5(3)) to visualize mitochondria-like organelles.
- Double-labeling with lysosomal antibodies to differentiate organelles.
Main Results:
- mtDNA-depleted muscle cells lacked detectable cytochrome c oxidase activity and mtDNA.
- Mitochondrial matrix enzymes were present and localized in mitochondria-like organelles.
- Synthesis of cytochrome c oxidase subunits coded by nDNA was observed.
- The characteristic thread-like mitochondrial structure was lost; organelles appeared round and swollen.
- Import of proteins into these mitochondria-like organelles was confirmed.
Conclusions:
- Depletion of mtDNA in human myoblasts does not prevent the expression of nDNA-coded mitochondrial proteins.
- Protein import into mitochondria-like organelles remains functional even in respiration-deficient cells.
- These findings highlight the adaptability of cellular machinery for mitochondrial protein handling independent of mtDNA presence.