title: A Single-Cell Resolution Imaging Protocol of Mitochondrial DNA Dynamics in Physiopathology, mTRIP, Which Also Evaluates Sublethal Cytotoxicity.
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.
authors.affiliation: Team "Stability of Nuclear and Mitochondrial DNA" CNRS UMR 3525, Paris, France.|Stem Cells and Development, Department of Developmental & Stem Cell Biology, Institut Pasteur, 25-28 Rue du Dr. Roux, Paris, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chatre
authors.affiliation: Team "Stability of Nuclear and Mitochondrial DNA" CNRS UMR 3525, Paris, France.|Stem Cells and Development, Department of Developmental & Stem Cell Biology, Institut Pasteur, 25-28 Rue du Dr. Roux, Paris, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Montagne
authors.affiliation: Team "Stability of Nuclear and Mitochondrial DNA" CNRS UMR 3525, Paris, France. mricch@pasteur.fr.|Stem Cells and Development, Department of Developmental & Stem Cell Biology, Institut Pasteur, 25-28 Rue du Dr. Roux, Paris, France. mricch@pasteur.fr.
authors.forename: Miria
authors.initials: M
authors.lastname: Ricchetti
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D054629:Genome, Mitochondrial / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000235:genetics; D063154:Mitochondrial Dynamics / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000447:Aldehydes; D015342:DNA Probes; D004272:DNA, Mitochondrial; D008565:Membrane Proteins; C507919:MitoTracker Red 580; D009930:Organic Chemicals; C111472:mitotracker green FM
keywords: Cytotoxicity; DNA replication; FISH; Imaging; Long-term drug treatment; Metabolism; Mitochondrial DNA; Single-cell; Transcription