Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Area of Science:

• Cell Biology
• Genetics
• Biochemistry

Background:

• Heteroplasmy, the co-existence of wild-type and mutant mitochondrial DNA (mtDNA) within cells, dynamically changes during development, disease, and aging.
• The mechanisms driving these heteroplasmy shifts—selection versus drift, and at cellular versus intracellular levels—remain poorly understood.

Purpose of the Study:

• To investigate the dynamics of heteroplasmy in dividing cells.
• To determine whether selection or drift shapes heteroplasmy levels.
• To understand if selection acts at the cellular or intracellular level.

Main Methods:

• Utilized precise mtDNA base editing (DdCBE) for targeted mutations.
• Employed SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression) for ultra-high throughput single-cell heteroplasmy tracking.
• Engineered cells with synonymous or nonsynonymous complex I mtDNA mutations.

Main Results:

• Nonsynonymous mtDNA variants were purged, while synonymous variants were maintained in standard cell cultures, indicating selection dominates over drift.
• Single-cell heteroplasmy and ancestry tracking revealed stable lineage heteroplasmy despite population shifts, suggesting selection acts on cell fitness.
• Cells accumulated high levels of truncating complex I mtDNA heteroplasmy in specific environments where loss of complex I activity conferred a fitness benefit.

Conclusions:

• In dividing cells, selection, rather than simple drift, shapes population heteroplasmy.
• Selection acts at the level of cell fitness, influenced by the cellular environment.
• The impact of nonsynonymous mtDNA heteroplasmy on cell fitness is context-dependent, ranging from harmful to beneficial based on environmental conditions.