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Updated: Nov 2, 2025
Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution
Published on: May 5, 2023
mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell
Laurent Chatre1,2, Miria Ricchetti3
1Institut Pasteur, Stem Cells and Development, Department of Developmental and Stem Cell Biology, CNRS UMR3738 Team "Stability of Nuclear and Mitochondrial DNA", Paris, France.
Abstract:
Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
Insights
We developed mTRIP, a novel imaging method to visualize mitochondrial DNA (mtDNA) replication and RNA (mtRNA) processing within single human cells. This technique aids in understanding mitochondrial dysfunction and disease diagnostics.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Molecular Biology
Background:
- Mitochondrial physiology and metabolism are intrinsically linked to mitochondrial DNA (mtDNA) replication and transcription.
- Current methods lack detailed single-cell resolution for characterizing mtDNA processing dynamics.
Purpose of the Study:
- To develop and validate a novel imaging technique for simultaneous, single-cell analysis of mtDNA replication and mtRNA content.
- To enable detailed investigation of mtDNA processing under various cellular conditions.
Main Methods:
- Developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), a modified fluorescence in situ hybridization (FISH) technique.
- mTRIP allows simultaneous visualization of mtDNA replication initiation sites and mtRNA content at the single-cell level.
- mTRIP can be combined with immunofluorescence or MitoTracker for co-detection of proteins or mitochondria.
Main Results:
- mTRIP successfully visualizes mtDNA replication and mtRNA in human cells.
- The method allows tracking of specific RNA regions, not just global RNA levels.
- Qualitative and quantitative alterations in mtDNA processing dynamics were detected in cells experiencing physiological changes, stress, or dysfunction.
- mTRIP elucidated mtDNA processing alterations in cancer cells.
Conclusions:
- mTRIP provides unprecedented single-cell resolution for studying mtDNA processing.
- The technique is valuable for investigating mitochondrial dysfunction in various cellular states, including disease.
- mTRIP holds potential for the diagnosis of mitochondrial diseases.
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