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Updated: May 7, 2025
Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria
Published on: September 18, 2014
Flow-cytometry reveals mitochondrial DNA accumulation in Saccharomyces cerevisiae cells during cell cycle arrest
Elena Yu Potapenko1, Nataliia D Kashko2, Dmitry A Knorre1
1A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
Abstract:
Mitochondria are semi-autonomous organelles containing their own DNA (mtDNA), which is replicated independently of nuclear DNA (nDNA). While cell cycle arrest halts nDNA replication, mtDNA replication continues. In Saccharomyces cerevisiae, flow cytometry enables semi-quantitative estimation of mtDNA levels by measuring the difference in signals between cells lacking mtDNA and those containing mtDNA. In this study, we used flow cytometry to investigate mtDNA accumulation in yeast cells under G1 and G2 phase cell cycle arrest conditions utilising thermosensitive mutants cdc4-3 and cdc15-2. In line with the previous studies, cell cycle arrest induced a several-fold accumulation of mtDNA in both mutants. The total DNA levels in arrested cells correlated with cell forward scattering, suggesting a relationship between individual cell mtDNA quantity and size. In cell cycle-arrested cells, we observed no correlation between cell size and intercellular mtDNA copy number variability. This implies that as cell size increases during arrest, the mtDNA content remains within a specific limited range for each size class. This observation suggests that mtDNA quantity control mechanisms can function in cell cycle-arrested cells.
Insights
Cell cycle arrest in yeast causes mitochondrial DNA (mtDNA) to accumulate significantly. Despite increasing cell size, the mtDNA copy number remains stable within size classes, indicating functional quantity control mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitochondria are organelles with their own DNA (mtDNA), replicating independently of nuclear DNA (nDNA).
- Unlike nDNA replication, mtDNA replication persists during cell cycle arrest.
Purpose of the Study:
- To investigate mitochondrial DNA (mtDNA) accumulation in yeast cells during G1 and G2 phase cell cycle arrest.
- To explore the relationship between cell size and mtDNA copy number variability under cell cycle arrest.
Main Methods:
- Utilized thermosensitive yeast mutants (cdc4-3 and cdc15-2) to induce cell cycle arrest.
- Employed flow cytometry to quantify mtDNA levels by measuring signal differences in mtDNA-containing versus mtDNA-lacking cells.
- Correlated total DNA levels with cell forward scattering (cell size).
Main Results:
- Cell cycle arrest led to a several-fold accumulation of mtDNA in both yeast mutants.
- Total DNA levels in arrested cells correlated positively with cell forward scattering, indicating increased mtDNA per cell.
- No significant correlation was found between cell size and intercellular mtDNA copy number variability in arrested cells.
Conclusions:
- Mitochondrial DNA quantity control mechanisms remain functional even during cell cycle arrest.
- As yeast cells increase in size during arrest, mtDNA content is maintained within a specific range for each size class, suggesting regulated mtDNA replication or degradation.
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