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.
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Acknowledgement
The authors thank Dr. François R. Lacoste for the suggestion of using mTRIP to monitor mitochondrial activity in the context of long-term drug treatments. This work was supported by Association Nationale contre le Cancer (ARC 4022 and SFI20111204038), PTR-Institut Pasteur (PTR217), DARRI-Institut Pasteur (project P790319), and Agence Nationale pour la Recherche (ANR 11BSV202502). mTRIP tool is covered by patent applications: EP2500436 and WO2012123588 “Method, probe and kit for DNA in situ hybridization and use thereof.”
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Chatre, L., Montagne, B., Ricchetti, M. (2016). A Single-Cell Resolution Imaging Protocol of Mitochondrial DNA Dynamics in Physiopathology, mTRIP, Which Also Evaluates Sublethal Cytotoxicity. In: McKenzie, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 1351. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3040-1_5
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DOI: https://doi.org/10.1007/978-1-4939-3040-1_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3039-5
Online ISBN: 978-1-4939-3040-1
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