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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, Miria Ricchetti
1Team "Stability of Nuclear and Mitochondrial," Unité de Génétique Moléculaire des Levures, Institut Pasteur, CNRS UMR3525, 25-28 Rue du Dr. Roux, Paris, France, laurent.chatre@pasteur.fr.
Abstract:
Mitochondrial physiology and metabolism are closely linked to replication and transcription of the genome of the organelle, the mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. Here, we describe mTRIP (mitochondrial transcription and replication imaging protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures engaged in mtDNA initiation of replication and global mitochondrial RNA (mtRNA) content at the single-cell level in human cells. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing simultaneous labelling 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, with a potential for diagnostic of mitochondrial diseases.
Insights
We developed mTRIP, a novel imaging method to visualize mitochondrial DNA (mtDNA) replication and RNA content within single human cells. This technique aids in understanding mitochondrial dysfunction and disease diagnostics.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Genetics
Background:
- Mitochondrial DNA (mtDNA) replication and transcription are crucial for cellular energy production.
- Current methods lack single-cell resolution for characterizing mtDNA processing dynamics.
- Understanding mtDNA processing is key to diagnosing mitochondrial disorders.
Purpose of the Study:
- To develop a novel imaging protocol for simultaneous visualization of mtDNA replication and RNA content at the single-cell level.
- To characterize alterations in mtDNA processing during cellular stress and dysfunction.
- To explore the diagnostic potential of this method for mitochondrial diseases.
Main Methods:
- Developed mTRIP (mitochondrial transcription and replication imaging protocol), a modified fluorescence in situ hybridization (FISH) technique.
- mTRIP allows simultaneous detection of mtDNA replication initiation sites and mitochondrial RNA (mtRNA) content.
- Protocol can be combined with immunofluorescence or MitoTracker for protein or mitochondrial labeling.
Main Results:
- mTRIP successfully visualizes mtDNA replication and mtRNA content in single human cells.
- The method detects qualitative and quantitative changes in mtDNA processing dynamics.
- Demonstrated utility in human cells experiencing physiological changes, stress, and dysfunction.
Conclusions:
- mTRIP provides unprecedented single-cell resolution for studying mtDNA processing.
- This imaging approach offers insights into mitochondrial dynamics under various cellular conditions.
- mTRIP holds potential for the diagnosis and understanding of mitochondrial diseases.
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