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.
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Acknowledgment
This work was supported by the Association Nationale contre le Cancer (ARC 4022 and SFI20111204038), PTR-Institut Pasteur (PTR217), DARRI-Institut Pasteur (project P790319), Association Française contre les Myopathies (AFM, project 2012-0905 n° 16290), and Agence Nationale pour la Recherche (ANR 11BSV202502). L. C. was the recipient of a Bourse Roux Institut Pasteur and was supported by Mr. François R. Lacoste.
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Chatre, L., Ricchetti, M. (2015). mTRIP: An Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1264. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2257-4_13
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DOI: https://doi.org/10.1007/978-1-4939-2257-4_13
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