Abstract
Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling 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. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
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Acknowledgments
This work was supported by Association Nationale contre le Cancer (ARC 4022 and SFI20111204038), PTR-Institut Pasteur (PTR217), DARRI-Institut Pasteur (projects P790319 and PasteurInnov 14/152), and Agence Nationale pour la Recherche (ANR 11BSV202502).
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Chatre, L., Ricchetti, M. (2021). 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 2275. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1262-0_15
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DOI: https://doi.org/10.1007/978-1-0716-1262-0_15
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