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Updated: Jun 12, 2026
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level
Rodaria Roussou1,2, Dirk Metzler1, Francesco Padovani3
1Faculty of Biology, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany.
Abstract:
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.
Insights
Tracking mitochondrial DNA (mtDNA) variants in live yeast revealed asymmetric partitioning during cell division and limited mitochondrial fusion/fission. These factors drive mtDNA heteroplasmy dynamics and variant segregation.
Area of Science:
- Cell Biology
- Genetics
- Biophysics
Background:
- Mitochondrial DNA (mtDNA) exists in multiple copies per cell, crucial for ATP production.
- Cellular mtDNA exhibits sequence variation (heteroplasmy), but its dynamic changes are poorly understood due to real-time monitoring limitations.
Purpose of the Study:
- To develop and apply a novel method for real-time, single-cell tracking of mtDNA variants in live heteroplasmic yeast.
- To elucidate the key mechanisms driving the dynamic changes in mitochondrial DNA heteroplasmy.
Main Methods:
- Utilized mtDNA-based fluorescent markers for tracking genetic variants.
- Employed microfluidics and automated cell tracking for live population analysis.
- Integrated direct mtDNA tracking with data-driven mathematical modeling.
Main Results:
- Demonstrated asymmetric partitioning of mtDNA copies during yeast cell division.
- Quantified limited frequencies of mitochondrial fusion and fission events.
- Identified these processes as critical drivers of mtDNA variant segregation.
Conclusions:
- The developed approach enables real-time monitoring of mtDNA heteroplasmy dynamics at the single-cell level.
- Asymmetric partitioning and limited mitochondrial dynamics are key to mtDNA variant segregation.
- This methodology is valuable for studying mtDNA purifying selection mechanisms.
