Skip to main content
SpringerLink
Log in

Monitoring the Mitochondrial Dynamics in Mammalian Cells

  • Protocol
  • First Online:
Mitochondrial Bioenergetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1782))

Abstract

Mitochondria exist in a dynamic state inside mammalian cells. They undergo processes of fusion and fission to adjust their shape according to the different cell needs. Different proteins tightly regulate these dynamics: Opa-1 and Mitofusin-1 and Mitofusin-2 are the main profusion proteins, while Drp1 and its different receptors (Mff, Fis1, MiD49, MiD51) regulate mitochondrial fission. The dynamic nature of the mitochondrial network has become evident and detectable, thanks to recent advances in live imaging video microscopy and to the availability of mitochondria-tagged fluorescent proteins. High-resolution confocal reconstruction of mitochondria over time allows researchers to visualize mitochondria shape changes in living cells, under different experimental conditions. Moreover, in recent years, different techniques in living cells have been developed to study the process of mitochondria fusion in more details. Among them are fluorescence recovery after photobleaching (FRAP) of mitochondria-tagged GFP (mtGFP), use of photoactivatable mtGFP, polyethylene glycol (PEG)-based fusion of mtGFP and mtRFP cells, and Renilla luciferase assay (for population studies). In addition, in combination with imaging, the analysis of the expression levels of the different mitochondria-shaping proteins, along with that of their activation status, represents a powerful tool to investigate potential modulations of the mitochondrial network. Here, we review this aspect and then mention a number of techniques, with particular attention to their relative protocols.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Detmer SA, Chan DC (2007) Functions and dysfunctions of mitochondrial dynamics. Nat Rev Mol Cell Biol 8(11):870–879

    Article  CAS  PubMed  Google Scholar 

  2. Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC, Perales-Clemente E, Salviati L, Fernandez-Silva P, Enriquez JA, Scorrano L (2013) Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell 155(1):160–171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Prieto J, León M, Ponsoda X, Sendra R, Bort R, Ferrer-Lorente R, Raya A, López-García C, Torres J (2016) Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming. Nat Commun 7:11124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS (2008) Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 27(2):433–446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Karbowski M, Lee YJ, Gaume B, Jeong SY, Frank S, Nechushtan A, Santel A, Fuller M, Smith CL, Youle RJ (2002) Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J Cell Biol 159(6):931–938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Campello S, Lacalle RA, Bettella M, Mañes S, Scorrano L, Viola A (2006) Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J Exp Med 203(13):2879–2886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhao J, Zhang J, Yu M, Xie Y, Huang Y, Wolff DW, Abel PW, Tu Y (2013) Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Oncogene 32(40):4814–4824

    Article  CAS  PubMed  Google Scholar 

  8. Qian W, Choi S, Gibson GA, Watkins SC, Bakkenist CJ, Van Houten B (2012) Mitochondrial hyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress. J Cell Sci 125(Pt 23):5745–5757

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen H, Chomyn A, Chan DC (2005) Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J Biol Chem 280(28):26185–26192

    Article  CAS  PubMed  Google Scholar 

  10. Eura Y, Ishihara N, Yokota S, Mihara K (2003) Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion. J Biochem 134(3):333–344

    Article  CAS  PubMed  Google Scholar 

  11. Gegg ME, Cooper JM, Chau KY, Rojo M, Schapira AH, Taanman JW (2010) Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy. Hum Mol Genet 19(24):4861–4870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yue W, Chen Z, Liu H, Yan C, Chen M, Feng D, Wu H, Du L, Wang Y, Liu J, Huang X, Xia L, Liu L, Wang X, Jin H, Wang J, Song Z, Hao X, Chen Q (2014) A small natural molecule promotes mitochondrial fusion through inhibition of the deubiquitinase USP30. Cell Res 24(4):482–496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc Natl Acad Sci U S A 101(45):15927–15932

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Anand R, Wai T, Baker MJ, Kladt N, Schauss AC, Rugarli E, Langer T (2014) The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission. J Cell Biol 204(6):919–929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, Scorrano L (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126(1):177–189

    Article  CAS  PubMed  Google Scholar 

  16. Gal A, Balicza P, Weaver D, Naghdi S, Joseph SK, Várnai P, Gyuris T, Horváth A, Nagy L, Seifert EL, Molnar MJ, Hajnóczky G (2017) MSTO1 is a cytoplasmic pro-mitochondrial fusion protein, whose mutation induces myopathy and ataxia in humans. EMBO Mol Med 9(7):967–984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Otera H, Ishihara N, Mihara K (2013) New insights into the function and regulation of mitochondrial fission. Biochim Biophys Acta 1833(5):1256–1268

    Article  CAS  PubMed  Google Scholar 

  18. Smirnova E, Griparic L, Shurland DL, van der Bliek AM (2001) Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell 12(8):2245–2256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. James DI, Parone PA, Mattenberger Y, Martinou JC (2003) hFis1, a novel component of the mammalian mitochondrial fission machinery. J Biol Chem 278(38):36373–36379

    Article  CAS  PubMed  Google Scholar 

  20. Otera H, Wang C, Cleland MM, Setoguchi K, Yokota S, Youle RJ, Mihara K (2010) Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells. J Cell Biol 191(6):1141–1158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Palmer CS, Osellame LD, Laine D, Koutsopoulos OS, Frazier AE, Ryan MT (2011) MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO Rep 12(6):565–573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chou CH, Lin CC, Yang MC, Wei CC, Liao HD, Lin RC, Tu WY, Kao TC, Hsu CM, Cheng JT, Chou AK, Lee CI, Loh JK, Howng SL, Hong YR (2012) GSK3beta-mediated Drp1 phosphorylation induced elongated mitochondrial morphology against oxidative stress. PLoS One 7(11):e49112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride HM (2007) The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J Cell Sci 120(Pt 7):1178–1188

    Article  CAS  PubMed  Google Scholar 

  24. Karbowski M, Neutzner A, Youle RJ (2007) The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. J Cell Biol 178(1):71–84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang X, Yan MH, Fujioka H, Liu J, Wilson-Delfosse A, Chen SG, Perry G, Casadesus G, Zhu X (2012) LRRK2 regulates mitochondrial dynamics and function through direct interaction with DLP1. Hum Mol Genet 21(9):1931–1944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lee JE, Westrate LM, Wu H, Page C, Voeltz GK (2016) Multiple dynamin family members collaborate to drive mitochondrial division. Nature 540(7631):139–143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rizzuto R, Brini M, De Giorgi F, Rossi R, Heim R, Tsien RY, Pozzan T (1996) Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol 6(2):183–188

    Article  CAS  PubMed  Google Scholar 

  28. Legros F, Lombès A, Frachon P, Rojo M (2002) Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol Biol Cell 13(12):4343–4354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mitra K, Lippincott-Schwartz J (2010) Analysis of mitochondrial dynamics and functions using imaging approaches. Curr Protoc Cell Biol Chapter 4 Unit 4.25.1–21

    Google Scholar 

  30. Sterky FH, Lee S, Wibom R, Olson L, Larsson NG (2011) Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo. Proc Natl Acad Sci U S A 108(31):12937–12942

    Article  PubMed  PubMed Central  Google Scholar 

  31. Scaduto RC, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76(1 Pt 1):469–477

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Poot M, Zhang YZ, Krämer JA, Wells KS, Jones LJ, Hanzel DK, Lugade AG, Singer VL, Haugland RP (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem 44(12):1363–1372

    Article  CAS  PubMed  Google Scholar 

  33. Dagda RK, Cherra SJ, Kulich SM, Tandon A, Park D, Chu CT (2009) Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem 284(20):13843–13855

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, Kurth MJ, Shaw JT, Hinshaw JE, Green DR, Nunnari J (2008) Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell 14(2):193–204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rosdah AA, Holien JK, Delbridge LM, Dusting GJ, Lim SY (2016) Mitochondrial fission - a drug target for cytoprotection or cytodestruction? Pharmacol Res Perspect 4(3):e00235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kushnareva Y, Andreyev AY, Kuwana T, Newmeyer DD (2012) Bax activation initiates the assembly of a multimeric catalyst that facilitates Bax pore formation in mitochondrial outer membranes. PLoS Biol 10(9):e1001394

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bordt EA, Clerc P, Roelofs BA, Saladino AJ, Tretter L, Adam-Vizi V, Cherok E, Khalil A, Yadava N, Ge SX, Francis TC, Kennedy NW, Picton LK, Kumar T, Uppuluri S, Miller AM, Itoh K, Karbowski M, Sesaki H, Hill RB, Polster BM (2017) The putative Drp1 inhibitor mdivi-1 is a reversible mitochondrial complex I inhibitor that modulates reactive oxygen species. Dev Cell 40(6):583–594.e6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Qi X, Qvit N, Su YC, Mochly-Rosen D (2013) A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity. J Cell Sci 126(Pt 3):789–802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wang D, Wang J, Bonamy GM, Meeusen S, Brusch RG, Turk C, Yang P, Schultz PG (2012) A small molecule promotes mitochondrial fusion in mammalian cells. Angew Chem Int Ed Engl 51(37):9302–9305

    Article  CAS  PubMed  Google Scholar 

  40. Woods LC, Berbusse GW, Naylor K (2016) Microtubules are essential for mitochondrial dynamics-fission, fusion, and motility-in Dictyostelium discoideum. Front Cell Dev Biol 4:19

    Article  PubMed  PubMed Central  Google Scholar 

  41. Tanaka Y, Kanai Y, Okada Y, Nonaka S, Takeda S, Harada A, Hirokawa N (1998) Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93(7):1147–1158

    Article  CAS  PubMed  Google Scholar 

  42. da Silva AF, Mariotti FR, Máximo V, Campello S (2014) Mitochondria dynamism: of shape, transport and cell migration. Cell Mol Life Sci 71(12):2313–2324

    PubMed  Google Scholar 

  43. Cereghetti GM, Costa V, Scorrano L (2010) Inhibition of Drp1-dependent mitochondrial fragmentation and apoptosis by a polypeptide antagonist of calcineurin. Cell Death Differ 17(11):1785–1794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Goodwin JS, Kenworthy AK (2005) Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. Methods 37(2):154–164

    Article  CAS  PubMed  Google Scholar 

  45. Karbowski M, Cleland MM, Roelofs BA (2014) Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells. Methods Enzymol 547:57–73

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Huang H, Choi SY, Frohman MA (2010) A quantitative assay for mitochondrial fusion using Renilla luciferase complementation. Mitochondrion 10(5):559–566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4(9):429–434

    Article  PubMed  PubMed Central  Google Scholar 

  48. Patten DA, Wong J, Khacho M, Soubannier V, Mailloux RJ, Pilon-Larose K, MacLaurin JG, Park DS, McBride HM, Trinkle-Mulcahy L, Harper ME, Germain M, Slack RS (2014) OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand. EMBO J 33(22):2676–2691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB, Korsmeyer SJ (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14(16):2060–2071

    PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the Italian Ministry of Health (GR-2011-02351643) and IG-19826 by AIRC to SC and by grants from Fondazione Roma and FISM.

Author information

Authors and Affiliations

  1. Department of Biology, University of Rome Tor Vergata, Rome, Italy

    Luca Simula & Silvia Campello

  2. Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children’s Hospital, Rome, Italy

    Luca Simula

  3. IRCCS, Fondazione Santa Lucia, Rome, Italy

    Silvia Campello

Authors
  1. Luca Simula
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silvia Campello
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Silvia Campello .

Editor information

Editors and Affiliations

  1. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    Carlos M. Palmeira

  2. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    António J. Moreno

1 Movie Caption

 Time-lapse confocal video microscopy of a mtYFP-expressing Jurkat cell. Confocal 3D reconstruction of the mitochondrial network in a mtYFP-expressing Jurkat cell over time. Acquisitions have been performed with a 488 nm laser, by 0.4 μm z-stack steps, each 10 s (MP4 269 kb)

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Simula, L., Campello, S. (2018). Monitoring the Mitochondrial Dynamics in Mammalian Cells. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 1782. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7831-1_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7831-1_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7830-4

  • Online ISBN: 978-1-4939-7831-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation