introduction
The coexistence of wild-type and mutant mitochondrial DNA (mtDNA) during cell development, disease, and aging is called heteroplasmy, and its levels vary dynamically in different biological states. The study, "Single-cell analysis reveals context-dependent, cell-level selection of mtDNA," published April 24 in Nature, looked at cell-level selection and heterogeneity of mitochondrial DNA (mtDNA). By combining precise mtDNA base editing technology (DdCBE) with a new method, SCI-LITE (Single Cell Combinatorial Index Technology), it was revealed that cell populations would clear non-synonymous mtDNA variants while retaining synonymous variants under standard culture conditions, suggesting that selection rather than simple drift plays a dominant role in shaping population heterogeneity. At the same time, the study also tracked single-cell mtDNA heterogeneity and its ancestry, and found that although population heterogeneity changed, the heterogeneity of individual cell lines remained stable, suggesting that the selection effect was at the level of cellular health.
In addition, by placing cells in a specific environment, studies have shown that in environments where complex I activity needs to be reduced, cells can accumulate high levels of truncated complex I mtDNA heterogeneity, which may be beneficial for cell health. These findings are not only important for understanding the dynamics of mtDNA in cells, but also provide new insights into the mechanisms of age-related degenerative diseases and their possible genetic basis.
Taken together, this study highlights the influence of the cellular environment on mtDNA heterogeneity dynamics and proposes the selective removal of non-synonymous heterogeneity during cell division, supporting a new view that mtDNA selection is not just a random event, but a complex process influenced by multiple factors, in which cell health levels and external environmental conditions play a decisive role. This study not only provides us with an insight into mtDNA heterogeneity, but also provides a theoretical basis for possible future therapeutic strategies.
Highlights
Precise mtDNA Base EditingThis study successfully applied Precise Mitochondrial DNA Base Editing (DdCBE), which enables precise modification of mtDNA in single cells to study the effects of different mtDNA variants on cell behavior. With this approach, the researchers were able to observe dynamic changes in mtDNA heteroplasmy at the cellular level and reveal how these changes are driven by selection rather than random drift.
高通量单细胞异质性分析工具SCI-LITE（High-Throughput Single-Cell Heteroplasmy Analysis with SCI-LITE）研究中引入了一种新的单细胞组合索引技术（SCI-LITE），该技术能够在超高通量情况下追踪单细胞的mtDNA异质性。 SCI-LITE的使用大幅提升了研究的灵敏度和扩展性，使得科学家可以更加详细地分析和理解mtDNA在单个细胞中的行为及其变化。
Environment-Dependent SelectionThe study found that cells exhibited different mtDNA heterogeneity dynamics in different culture environments. In some settings, specific nonsynonymous mtDNA variants are actively selected for their benefits for cell fitness. This finding suggests that changes in mtDNA heterogeneity are closely related to external environmental conditions, and that environmental factors can significantly affect the selection process of mtDNA.
The Impact on Cell Fitness study shows how mtDNA heterogeneity affects the growth and proliferation of cells. Through a detailed analysis of cells with different levels of heterogeneity, the study noted that non-synonymous heterogeneity often has a negative impact on cell health, but this effect can be turned positive under certain conditions. This phenomenon provides new insights in the field of mtDNA research, which is particularly important for understanding the role of complex I dysfunction in disease.
Strategies
The research team started with the dynamics of mitochondrial DNA heteroplasmy and explored the underlying mechanisms of these changes in cell development, disease and aging. Of particular interest is whether these changes are driven by random drift or selection, and whether these processes occur at the cellular or intracellular level.
Precision mtDNA Base Editing Technology (DdCBE) studies use a mitochondrial base editing technique called DdCBE, which allows researchers to precisely modify mtDNA without the use of CRISPR. With this technique, researchers can introduce synonymous and nonsynonymous mutations and then see how these mutations affect the heterogeneity level of mtDNA in cells.
单细胞组合索引技术SCI-LITE为了在单细胞水平上分析mtDNA异质性，研究团队开发了一种名为SCI-LITE的新方法。 这种方法结合了单细胞组合索引（single-cell combinatorial indexing, SCI）和灵敏的单细胞RNA测序技术，允许研究人员在极高的通量下测定特定转录物。 SCI-LITE技术通过多轮的池化（pooling）和分裂（splitting）过程，利用条形码（barcodes）和独特分子标识符（unique molecular identifiers, UMIs）对单个细胞进行标记。
Investigation studies of environmental factors further explore how environmental factors affect the selection of mtDNA heterogeneity. By placing cells in different energy sources that are dependent or independent of mitochondrial oxidative phosphorylation (OXPHOS), the research team observed how mtDNA with non-synonymous mutations was selectively retained or cleared under specific conditions. This finding suggests that the selection for mtDNA heterogeneity is strongly influenced not only by genetic variation, but also by the external environment.
Tracking Heterogeneity DynamicsWith SCI-LITE technology, researchers are able to track the dynamics of heterogeneity within cell populations as well as the heterogeneity stability of individual cell lines. The application of this technique provides a novel perspective on how mtDNA heterogeneity changes in response to different cellular states and environmental stresses.
Behind the Scenes
Mitochondrial DNA Base Editing Technology (DdCBE)Mitochondrial DNA Base Editing Technology (DdCBE) is a specially designed technique for precise editing of mitochondrial DNA (mtDNA) without relying on CRISPR systems. This technique utilizes an enzyme called "deaminase" that specifically modifies DNA bases to introduce mutations. In DdCBE, deaminase is fused to a specific DNA-binding protein, which allows the editing complex to precisely target mtDNA and introduce desired mutations.
The base switching mechanism, DdCBE technology, primarily utilizes a enzyme called cytidine deaminase, which converts cytosine (C) to uracil (U). During DNA replication, this conversion can cause the base pair to change from C-G to T-A, introducing point mutations in the DNA sequence. This editing technique does not require DNA cleavage or double-strand breaks, reducing the additional mutations that may be introduced during cell repair.
ProcedureBefore starting an experiment, the researcher needs to design a specific fusion protein, including selecting the appropriate DNA-binding domain to ensure that the protein can precisely target a specific region of mtDNA. This typically involves a detailed analysis of the sequence of mtDNA to identify sequences that can serve as binding sites. Constructing the desired fusion protein is the first step in the experiment. This involves the fusion of the deaminase gene with a specific DNA-binding protein gene (e.g., TALE or other DNA-binding module) through molecular cloning techniques. The constructed plasmid is then transfected into the cells by conventional cell biology methods. After transfection, the fusion protein enters the cell and ultimately targets the mitochondria. Protein localization tags, such as mitochondrial localization sequences, ensure that proteins are properly transported inside the mitochondria. Once the fusion protein reaches the mitochondria, the deaminase enzyme activates and begins editing bases near the target sequence. This process usually occurs during cell division, and when DNA is replicated, the introduced U is treated as T, resulting in a permanent change in the base pair. The final step of the experiment is to verify the effect of the edits. This typically involves amplifying the mtDNA of the edited region using PCR technology and then sequencing to determine whether the expected mutation was successfully introduced. In addition, the specificity and efficiency of the editing need to be assessed to ensure that no mutations have been introduced in non-target regions.
The high-throughput single-cell heterogeneity analysis tool SCI-LITESCI-LITE (Single-Cell Combinatorial Indexing Leveraged to Interrogate Targeted Expression) is a revolutionary high-throughput single-cell analysis tool for studying the heterogeneity of mitochondrial DNA (mtDNA). It combines Single-Cell Combinatorial Indexing (SCI) and targeted expression query techniques to enable the simultaneous analysis of multiple transcripts at the single-cell level. The SCI-LITE technology begins by utilizing a sample of fixed and permeabilized cells, which are dispensed into separate wells in a multi-well plate. In each well, gene-specific Reverse Transcription (RT) primers are used, which are specifically barcoded to allow subsequent sequence identification of transcripts from different cells. Cells are mixed and reassigned to new wells for paired ligation, with a second Unique Molecular Identifier (UMI) and a second barcode. This step provides further combinatorial diversity, greatly increasing the number of individual cells that can be labeled. Finally, PCR amplification is performed to introduce additional barcodes and prepare for sequencing.
Advantages of SCI-LITE High Throughput & Sensitivity The design of SCI-LITE technology allows it to have extremely high throughput and sensitivity, allowing it to process tens of thousands of single-cell samples while maintaining the unique identification of each cell. This high-throughput capability is particularly well-suited for large-scale biomedical research, such as exploring the role of heterogeneity in multiple diseases.
Cost-effectiveCompared to traditional single-cell RNA sequencing (scRNA-seq) technology, SCI-LITE reduces costs by reducing the required sequencing depth through the use of combinatorial indexes. This approach makes it a better choice for resource-constrained situations by reducing the number of required sequencing cycles while providing similar or higher data quality.
Flexibility & ScalabilitySCI-LITE allows researchers to perform custom analyses for specific gene sets, which increases the flexibility of the method and allows it to be optimized for different research purposes. In addition, the technology is designed to support scale-out and can be used for larger-scale sample processing.
Reduced Sample LossBy processing in multi-well plates and the addition of barcodes, SCI-LITE maximizes sample utilization, reducing cell loss that is common in traditional single-cell isolation and processing. This is especially important for handling precious or hard-to-obtain biological samples.
Environment-Dependent SelectionIn research, Environment-dependent selection refers to how mitochondrial DNA (mtDNA) heterogeneity is affected by natural selection under different environmental conditions, resulting in the retention or elimination of specific mtDNA variants in certain environments. This study investigated the selective response of cells to mtDNA variants under different energy-demanding environments. The researchers used SCI-LITE, a high-throughput single-cell heterogeneity analysis tool, to analyze cell populations that had undergone specific mtDNA gene editing. By changing the sources of sugars in the medium, such as glucose and galactose, different cellular metabolic demands are mimicked. Glucose acts as a highly efficient energy substance, while galactose forces cells to rely on mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Heterogeneity levels of mtDNA containing synonymous and non-synonymous mutations were observed and recorded under different culture conditions. Using SCI-LITE technology, researchers were able to accurately measure the frequency of specific mtDNA mutations in individual cells and track changes in these frequencies under different culture conditions. In the glucose environment, the level of mtDNA heterogeneity of non-synonymous mutations decreases, suggesting that these mutations may have a negative impact on cellular metabolism under high-energy conditions. This decline was more pronounced in the galactose environment, suggesting that non-synonymous mutations have a greater negative impact on cell survival when cells are dependent on OXPHOS. The level of heterogeneity of synonymous mutations remained stable under both conditions, suggesting that these mutations did not affect cellular energy metabolism.
Potential Limitations: High Dependency on Environmental Factors: This study highlights the importance of environmental factors on mtDNA heterogeneity and the impact of mtDNA heterogeneity on cell fitness. While environmental sensitivity is an innovative point of the study, it also means that its conclusions may not be applicable to different physiological or pathological settings. The high dependence of the environment limits the generalizability of the results, as cellular responses can be very different in different environments.
Limitations in Data Representativeness and ApplicabilityAlthough studies have used advanced single-cell sequencing technology (SCI-LITE) to analyze heterogeneity dynamics, the cell types and conditions studied may not be fully representative of the broader or more complex biological systems in the human body. In addition, the artificial setting of experimental conditions may affect the biomedical applicability of the study results, as in vitro experimental conditions often do not fully mimic the complex interactions and microenvironments in living organisms.
Challenges in Interpreting Heteroplasmy Dynamics The study has made detailed observations and analyses of changes in the level of heteroplasmy dynamics, but it remains challenging to explain these dynamics. The measurement and interpretation of heterogeneity relies on highly specialized techniques and analytical methods, which may limit the reproducibility of studies and the validation work of other scientists. In addition, the mechanistic understanding of the effects of heterogeneity is incomplete, and further studies may be needed to elucidate these complex molecular processes.
Potential research directions: Understanding mtDNA Heterogeneity Dynamics, which uses a combination of mtDNA base editing and a novel approach, SCI-LITE, to study how levels of heterogeneity change over time in individual cells. This reveals that heterogeneity can be influenced by genetic selection and environmental factors, depending on the cellular environment. Future studies could explore more deeply how these dynamics are affected by different cellular environments or stress conditions.
The single-cell mtDNA analysis technology SCI-LITE provides a highly scalable and cost-effective tool for detailed single-cell analysis. Further research could focus on improving the resolution or efficiency of this technique for wider application in biomedical research, such as in more complex or diverse tissue types.
Impact on mitochondrial diseases and aging studies suggest that heterogeneity levels can be critical in many diseases where mitochondrial dysfunction is a key factor, such as neurodegenerative diseases or metabolic disorders. Future studies could link specific patterns of heterogeneous change to disease progression or outcomes, potentially leading to targeted treatments.
The interaction between environment and genetic selection, the study also highlights how the cellular environment significantly influences the selective pressure on mtDNA variants. This interaction between genetic traits and environmental conditions provides a research pathway to study how the environment or therapeutic means beneficially modulate these dynamics.
The use of mtDNA editing in research and treatmentWith the use of tools such as CRISPR-free mtDNA base editing, there is potential for direct manipulation of mitochondrial genetics. Future directions may include the development of more precise editing tools or methods that can be safely applied in clinical settings to correct pathological mtDNA mutations.
Link to original article
Kotrys AV, Durham TJ, Guo XA, Vantaku VR, Parangi S, Mootha VK. Single-cell analysis reveals context-dependent, cell-level selection of mtDNA. Nature. 2024 Apr 24. doi: 10.1038/s41586-024-07332-0. Epub ahead of print. PMID: 38658765.
https://www.nature.com/articles/s41586-024-07332-0
Editor-in-charge|Explore Jun
Typography |
Please indicate the source of reprinting【Biological Exploration】
End