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A Single-Cell Resolution Imaging Protocol of Mitochondrial DNA Dynamics in Physiopathology, mTRIP, Which Also Evaluates Sublethal Cytotoxicity

  • Laurent Chatre
  • Benjamin Montagne
  • Miria RicchettiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

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.

Key words

Mitochondrial DNA FISH Imaging Metabolism Transcription DNA replication Single-cell Cytotoxicity Long-term drug treatment 

Notes

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.”

References

  1. 1.
    Falkenberg M, Larsson NG, Gustafsson CM (2007) DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem 76:679–699CrossRefPubMedGoogle Scholar
  2. 2.
    Scarpulla RC (2008) Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88:611–638CrossRefPubMedGoogle Scholar
  3. 3.
    Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470–474CrossRefPubMedGoogle Scholar
  4. 4.
    Chang DD, Clayton DA (1985) Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc Natl Acad Sci U S A 82:351–355CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Holt IJ, Lorimer HE, Jacobs HT (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100:515–524CrossRefPubMedGoogle Scholar
  6. 6.
    Pohjoismaki JL, Goffart S (2011) Of circles, forks and humanity: topological organisation and replication of mammalian mitochondrial DNA. Bioessays 33:290–299CrossRefPubMedGoogle Scholar
  7. 7.
    Reyes A, Kazak L, Wood SR, Yasukawa T, Jacobs HT, Holt IJ (2013) Mitochondrial DNA replication proceeds via a ‘bootlace’ mechanism involving the incorporation of processed transcripts. Nucleic Acids Res 41:5837–5850CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nicholls TJ, Minczuk M (2014) In D-loop: 40 years of mitochondrial 7S DNA. Exp Gerontol 56:175–181CrossRefPubMedGoogle Scholar
  9. 9.
    Chatre L, Ricchetti M (2013) Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle. Nucleic Acids Res 41:3068–3078CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lee S, Kim S, Sun X, Lee JH, Cho H (2007) Cell cycle-dependent mitochondrial biogenesis and dynamics in mammalian cells. Biochem Biophys Res Commun 357:111–117CrossRefPubMedGoogle Scholar
  11. 11.
    Mitra K, Wunder C, Roysam B, Lin G, Lippincott-Schwartz J (2009) A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase. Proc Natl Acad Sci U S A 106:11960–11965CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, Clayton DA (2011) Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol Cell Biol 31:4994–5010CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kukat C, Wurm CA, Spahr H, Falkenberg M, Larsson NG, Jakobs S (2011) Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc Natl Acad Sci U S A 108:13534–13539CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Chatre L, Ricchetti M (2013) Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging. J Cell Sci 126:914–926CrossRefPubMedGoogle Scholar
  15. 15.
    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 W, Edeas M (eds) Mitochondrial medicine: volume I, probing mitochondrial function. Methods in Molecular Biology, vol 1264 Springer, New York. Vol. I. pp 133–147Google Scholar
  16. 16.
    Aras MA, Hartnett KA, Aizenman E (2008) Assessment of cell viability in primary neuronal cultures. Curr Protoc Neurosci Chapter 7, Unit 7 18Google Scholar
  17. 17.
    Lee CF, Liu CY, Hsieh RH, Wei YH (2005) Oxidative stress-induced depolymerization of microtubules and alteration of mitochondrial mass in human cells. Ann N Y Acad Sci 1042:246–254CrossRefPubMedGoogle Scholar
  18. 18.
    Yamaguchi T, Katoh I, Kurata S (2002) Azidothymidine causes functional and structural destruction of mitochondria, glutathione deficiency and HIV-1 promoter sensitization. Eur J Biochem 269:2782–2788CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Laurent Chatre
    • 1
    • 2
  • Benjamin Montagne
    • 1
    • 2
  • Miria Ricchetti
    • 1
    • 2
    Email author
  1. 1.Team “Stability of Nuclear and Mitochondrial DNA” CNRS UMR 3525ParisFrance
  2. 2.Stem Cells and Development, Department of Developmental & Stem Cell BiologyInstitut PasteurParisFrance

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