Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.

Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA. Synopsis: The basis for dynamic changes in mitochondria DNA variations in cell populations, known as heteroplasmy, remains unclear. Here, live-cell tracking of mtDNA variants in yeast populations reveals mechanistic principles underlying mtDNA variant segregation. mtDNA variants encoding fluorescent reporters facilitate microscopy approaches for monitoring mtDNA variant segregation in real-time in a dividing yeast population. Asymmetric partitioning of mtDNA copies between mother and daughter cells as well as mitochondrial fission rates determine the rate of mtDNA variant segregation. mtDNA variant segregation is delayed in ∆mrx6 cells with increased mtDNA copy number. The imaging approach allows monitoring of purifying selection against mutant mtDNA. Tracking mtDNA variants in live yeast populations reveals asymmetric partitioning of mtDNA copies during cell division as well as limited mitochondrial fusion/fission frequencies as critical drivers of mtDNA variant segregation. [ABSTRACT FROM AUTHOR]