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A Single-Cell Resolution Imaging Protocol of Mitochondrial DNA Dynamics in Physiopathology, mTRIP, Which Also
Laurent Chatre1,2, Benjamin Montagne1,2, Miria Ricchetti3,4
1Team "Stability of Nuclear and Mitochondrial DNA" CNRS UMR 3525, Paris, France.
Abstract:
Mitochondria autonomously replicate and transcribe their own genome, which is present in multiple copies in the organelle. Transcription and replication of the mitochondrial DNA (mtDNA), which are defined here as mtDNA processing, are essential for mitochondrial function. The extent, efficiency, and coordination of mtDNA processing are key parameters of the mitochondrial state in living cells. Recently, single-cell analysis of mtDNA processing revealed a large and dynamic heterogeneity of mitochondrial populations in single cells, which is linked to mitochondrial function and is altered during disease. This was achieved using mitochondrial Transcription and Replication Imaging Protocol (mTRIP), a modified fluorescence in situ hybridization (FISH) approach that simultaneously reveals the mitochondrial RNA content and mtDNA engaged in initiation of replication at the single-cell level. mTRIP can also be coupled to immunofluorescence or MitoTracker, resulting in the additional labeling of proteins or active mitochondria, respectively. Therefore, mTRIP detects quantitative and qualitative alterations of the dynamics of mtDNA processing in human cells that respond to physiological changes or result from diseases. In addition, we show here that mTRIP is a rather sensitive tool for detecting mitochondrial alterations that may lead to loss of cell viability, and is thereby a useful tool for monitoring sublethal cytotoxicity for instance during chronic drug treatment.
Insights
Mitochondrial DNA (mtDNA) processing, including replication and transcription, shows dynamic heterogeneity in single cells. The new mTRIP method visualizes this, aiding disease and drug toxicity studies.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitochondria possess their own genome (mtDNA) essential for cellular function.
- mtDNA replication and transcription (mtDNA processing) are critical for mitochondrial health.
- Cellular states and diseases are linked to variations in mtDNA processing.
Purpose of the Study:
- To investigate the heterogeneity of mitochondrial DNA processing in single cells.
- To introduce and validate a novel method for analyzing mtDNA processing dynamics.
- To assess the utility of this method in disease and toxicology research.
Main Methods:
- Developed and utilized the mitochondrial Transcription and Replication Imaging Protocol (mTRIP).
- mTRIP is a modified fluorescence in situ hybridization (FISH) technique.
- mTRIP was combined with immunofluorescence and MitoTracker for multi-parametric analysis.
Main Results:
- mTRIP revealed significant heterogeneity in mtDNA processing within single cells.
- The method quantitatively and qualitatively detects alterations in mtDNA processing dynamics.
- mTRIP demonstrated sensitivity in detecting mitochondrial damage and cytotoxicity.
Conclusions:
- Single-cell analysis of mtDNA processing using mTRIP provides crucial insights into mitochondrial function.
- mTRIP is a valuable tool for studying mitochondrial alterations in disease contexts.
- The technique shows promise for monitoring sublethal cytotoxicity during drug treatment.