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Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

A Publisher Correction to this article was published on 15 February 2018

This article has been updated

Abstract

Although mitochondria are ubiquitous organelles, they exhibit tissue-specific morphology, dynamics and function. Here, we describe a robust approach to isolate mitochondria from specific cells of diverse tissue systems in Caenorhabditis elegans. Cell-specific mitochondrial affinity purification (CS-MAP) yields intact and functional mitochondria with exceptional purity and sensitivity (>96% enrichment, >96% purity, and single-cell and single-animal resolution), enabling comparative analyses of protein and nucleic acid composition between organelles isolated from distinct cellular lineages. In animals harbouring a mixture of mutant and wild-type mitochondrial genomes, we use CS-MAP to reveal subtle mosaic patterns of cell-type-specific heteroplasmy across large populations of animals (>10,000 individuals). We demonstrate that the germline is more prone to propagating deleterious mitochondrial genomes than somatic lineages, which we propose is caused by enhanced mtDNA replication in this tissue.

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Fig. 1: Cell-specific mitochondrial affinity purification (CS-MAP).
Fig. 2: Validation of CS-MAP purity.
Fig. 3: Purified mitochondria are intact and functional.
Fig. 4: CS-MAP in a variety of major tissues.
Fig. 5: Single-animal and single-cell CS-MAP.
Fig. 6: CS-MAP reveals mosaicism of mtDNA heteroplasmy.

Change history

  • 15 February 2018

    In the version of this Technical Report originally published, chromosome representations (indicated by black lines) were missing from Fig. 2a due to a technical error. The corrected version of Fig. 2a is shown below. This has now been amended in all online versions of the Technical Report.

References

  1. 1.

    West, A. P. & Shadel, G. S. Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat. Rev. Immunol. 17, 363–375 (2017).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Friedman, J. R. & Nunnari, J. Mitochondrial form and function. Nature 505, 335–343 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Prim. 2, 16080 (2016).

    Article  PubMed  Google Scholar 

  4. 4.

    Tuppen, H. A., Blakely, E. L., Turnbull, D. M. & Taylor, R. W. Mitochondrial DNA mutations and human disease. Biochim. Biophys. Acta 1797, 113–128 (2010).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Wallace, D. C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu. Rev. Genet. 39, 359–407 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wallace, D. C. & Chalkia, D. Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring Harb. Perspect. Biol. 5, a021220 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Frokjaer-Jensen, C. et al. Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat. Methods 11, 529–534 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Billing, O., Kao, G. & Naredi, P. Mitochondrial function is required for secretion of DAF-28/insulin in C. elegans. PLoS. ONE 6, e14507 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Vrablik, T. L., Wang, W. Q., Upadhyay, A. & Hanna-Rose, W. Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans. Dev. Biol. 349, 387–394 (2011).

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Tsang, W. Y. & Lemire, B. D. Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes. Biochem. Cell. Biol. 80, 645–654 (2002).

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Fukushige, T., Hawkins, M. G. & McGhee, J. D. The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev. Biol. 198, 286–302 (1998).

    CAS  PubMed  Google Scholar 

  12. 12.

    Dayama, G., Emery, S. B., Kidd, J. M. & Mills, R. E. The genomic landscape of polymorphic human nuclear mitochondrial insertions. Nucleic Acids Res. 42, 12640–12649 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Hazkani-Covo, E., Zeller, R. M. & Martin, W. Molecular poltergeists: mitochondrial DNA copies (numts) in sequenced nuclear genomes. PLoS. Genet. 6, e1000834 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Elson, J. L., Samuels, D. C., Turnbull, D. M. & Chinnery, P. F. Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age. Am. J. Human. Genet. 68, 802–806 (2001).

    CAS  Article  Google Scholar 

  15. 15.

    Menzies, R. A. & Gold, P. H. The turnover of mitochondria in a variety of tissues of young adult and aged rats. J. Biol. Chem. 246, 2425–2429 (1971).

    CAS  PubMed  Google Scholar 

  16. 16.

    Jenuth, J. P., Peterson, A. C. & Shoubridge, E. A. Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice. Nat. Genet. 16, 93–95 (1997).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Gitschlag, B. L. et al. Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans. Cell. Metab. 24, 91–103 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Lemire, B. in WormBook (eds. The C. elegans Research Community) https://doi.org/10.1895/wormbook.1.25.1 (2005).

  19. 19.

    Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate parkin. PLoS. Biol. 8, e1000298 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Lin, Y. F. et al. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response. Nature 533, 416–419 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Diaz, F. et al. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 30, 4626–4633 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Bergstrom, C. T. & Pritchard, J. Germline bottlenecks and the evolutionary maintenance of mitochondrial genomes. Genetics 149, 2135–2146 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Stewart, J. B., Freyer, C., Elson, J. L. & Larsson, N. G. Purifying selection of mtDNA and its implications for understanding evolution and mitochondrial disease. Nat. Rev. Genet. 9, 657–662 (2008).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Soong, N. W., Hinton, D. R., Cortopassi, G. & Arnheim, N. Mosaicism for a specific somatic mitochondrial-DNA mutation in adult human brain. Nat. Genet. 2, 318–323 (1992).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Chen, Z. et al. Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function. Mol. Biol. Cell. 26, 674–684 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Wachsmuth, M., Hubner, A., Li, M., Madea, B. & Stoneking, M. Age-related and heteroplasmy-related variation in human mtDNA copy number. PLoS. Genet. 12, e1005939 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Chen, W. W., Freinkman, E., Wang, T., Birsoy, K. & Sabatini, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324–1337 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Hornig-Do, H. T. et al. Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal. Biochem. 389, 1–5 (2009).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Kayo, S., Bahnemann, J., Klauser, M., Portner, R. & Zeng, A. P. A microfluidic device for immuno-affinity-based separation of mitochondria from cell culture. Lab. Chip 13, 4467–4475 (2013).

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Cao, L. Q. et al. The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat. Genet. 39, 386–390 (2007).

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Cree, L. M. et al. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nat. Genet. 40, 249–254 (2008).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Daniele, J. R., Heydari, K., Arriaga, E. A. & Dillin, A. Identification and characterization of mitochondrial subtypes in Caenorhabditis elegans via analysis of individual mitochondria by flow cytometry. Anal. Chem. 88, 6309–6316 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Mattiasson, G. Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death. Cytom. A 60, 145–154 (2004).

    Article  Google Scholar 

  34. 34.

    Saunders, J. E., Beeson, C. C. & Schnellmann, R. G. Characterization of functionally distinct mitochondrial subpopulations. J. Bioenerg. Biomembr. 45, 87–99 (2013).

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Wang, H. et al. cGAL, a temperature-robust GAL4-UAS system for Caenorhabditis elegans. Nat. Methods 14, 145–148 (2017).

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Hasegawa, E., Karashima, T., Sumiyoshi, E. & Yamamoto, M. C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development. Dev. Biol. 295, 689–699 (2006).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).

    CAS  Article  Google Scholar 

  38. 38.

    Varkey, J. P., Muhlrad, P. J., Minniti, A. N., Do, B. & Ward, S. The Caenorhabditis elegans Spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins Kelch and Scruin. Genes. Dev. 9, 1074–1086 (1995).

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zeiser, E., Frokjaer-Jensen, C., Jorgensen, E. & Ahringer, J. MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline. PLoS. ONE 6, e20082 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Ahier, A. & Jarriault, S. Simultaneous expression of multiple proteins under a single promoter in Caenorhabditis elegans via a versatile 2A-based toolkit. Genetics 196, 605–613 (2014).

    Article  Google Scholar 

  42. 42.

    Nichols, A. L. et al. The apoptotic engulfment machinery regulates axonal degeneration in C. elegans neurons. Cell. Rep. 14, 1673–1683 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Ahier, A. & Zuryn, S. Cell-specific mitochondrial affinity purification (CS-MAP) protocol. Protoc. Exch. https://doi.org/10.1038/protex.2017.152 (2017).

  44. 44.

    Kagias, K., Ahier, A., Fischer, N. & Jarriault, S. Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo. Proc. Natl. Acad. Sci. USA 109, 6596–6601 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Restif, C. et al. CeleST: computer vision software for quantitative analysis of C. elegans swim behavior reveals novel features of locomotion. PLoS. Comput. Biol. 10, e1003702 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Miller, M. A. Sperm and oocyte isolation methods for biochemical and proteomic analysis. Methods Mol. Biol. 351, 193–201 (2006).

    PubMed  Google Scholar 

  47. 47.

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–258 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–350 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank R. Tweedale and L. Richards for comments on the manuscript, M. Hilliard and members of the Zuryn laboratory for discussions and comments, M. Hilliard and T. Bredy for sharing reagents and equipment, R. Amor and L. Hammond for support with microscopy, and A. Ho for support with statistics. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by NHMRC Project Grant 1128381, a University of Queensland Early Career Researcher Grant (UQECR1608181) and a Stafford Fox Senior Research Fellowship to S.Z., and a University of Queensland International Scholarship to C.Y.D.

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A.A. carried out most experiments. C.-Y.D., A.T., A.B.-G., I.K. and S.Z. contributed some experiments. A.A. and S.Z. designed and interpreted experiments and wrote the paper.

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Correspondence to Steven Zuryn.

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A correction to this article is available online at https://doi.org/10.1038/s41556-018-0055-x.

Supplementary information

Supplementary Information

Supplementary Figures 1–7 and Supplementary References.

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Supplementary Table 1

Statistical source data for Figure 6.

Supplementary Table 2

List of primers used in this study.

Supplementary Table 3

List of generated transgenic strains used in this study.

Supplementary Table 4

Antibodies and the working dilutions used in this study.

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Ahier, A., Dai, CY., Tweedie, A. et al. Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism. Nat Cell Biol 20, 352–360 (2018). https://doi.org/10.1038/s41556-017-0023-x

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