Spontaneous Mutations in mtDNA Increase Cell-to-Cell Variation in mtDNA Amount.

In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated strains with large deletions in the mtDNA and strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the and strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the mtDNAs partially escape this regulation.