Skip to main content
SpringerLink
Log in

Atomic Force Microscopy of Isolated Mitochondria

  • Protocol
  • First Online:
Atomic Force Microscopy in Biomedical Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 736))

Abstract

This chapter describes methods for isolating and imaging metabolically and toxicologically challenged mitochondria with atomic force microscopy. Mitochondria were isolated from rat dorsal root ganglia or brain and exposed to glucose or dinitrobenzene (DNB) to simulate the cellular environment of a diabetic animal that has been exposed to excess glucose or to DNB. It is one of only a few articles to present images of membrane structures, such as voltage-dependent, anion-selective channel pores, on intact organelles. The purpose of the chapter is not to report on the metabolic or toxic effects, but to communicate in more detail than a typical journal paper allows the methods used to image isolated organelles. We also provide a series images revealing the outer membrane and outer membrane pores. An image of an isolated nucleus as well as a set of notes written to avoid common pitfalls in isolation, labeling, and imaging is also included.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Layton, B.E., et al. (2004) In situ imaging of mito-chondrial outer membrane pores using atomic force microscopy. BioTechniques 37: 564–573.

    Google Scholar 

  2. Feldman, E.L. (2003) Oxidative stress and diabetic neuropathy: a new understanding of an old problem. Journal of Clinical Investigation 111(4): 431–3.

    Google Scholar 

  3. Verweij, B.H., et al. (1997) Mitochondrial dysfunction after experimental and human brain injury and its possible reversal with a selective N-type calcium channel antagonist (SNX-111). Neurol Res 19(3): 334–9.

    Google Scholar 

  4. Papadopoulos, V., et al. (1994) Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor. Mol Cell Endocrinol 104(1): R5–9.

    Google Scholar 

  5. Goncalves, R.P., N. Buzhysnskyy, and S. Scheuring (2008) Mini review on the structure and supramolecular assembly of VDAC. J Bioenerg Biomembr 40(3): 133–8.

    Google Scholar 

  6. Goncalves, R.P., et al. (2007) Supramolecular assembly of VDAC in native mitochondrial outer membranes. J Mol Biol 369(2): 413–8.

    Google Scholar 

  7. Kramer, A., et al. (2008) Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina. Proceedings of the National Academy of Sciences of the United States of America 105(32): 11236–11241.

    Google Scholar 

  8. Joshi, N.V., et al. (2000) Ultrastructural investigation of human sperm using atomic force microscopy. Archives of Andrology 44(1): 51–57.

    Google Scholar 

  9. Parpura, V., P.G. Haydon, and E. Henderson (1993) Three-dimensional imaging of living neurons and glia with the atomic force microscope. J Cell Sci 104 ( Pt 2): 427–32.

    Google Scholar 

  10. Silberberg, Y.R., et al. (2008) Mitochondrial displacements in response to nanomechanical forces. Journal of Molecular Recognition 21(1): 30–36.

    Google Scholar 

  11. Arechaga, I. and D. Fotiadis (2007) Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis. Journal of Structural Biology 160(3): 287–294.

    Google Scholar 

  12. Hoogenboom, B.W., et al. (2007) The supramolecular assemblies of voltage-dependent anion channels in the native membrane. Journal of Molecular Biology 370(2): 246–255.

    Google Scholar 

  13. Neff, D., et al. (1997) Chloroplast F0F1 ATP synthase imaged by atomic force microscopy. Journal of Structural Biology 119(2): 139–148.

    Google Scholar 

  14. Schillers, H., et al. (2000) Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy. Cellular Physiology and Biochemistry 10(1–2): 99–107.

    Google Scholar 

  15. De Stasio, G., et al. (2004) Imaging the cell surface: Argon sputtering to expose inner cell structures. Microscopy Research and Technique 63(2): 115–121.

    Google Scholar 

  16. Boujrad, N., B. Vidic, and V. Papadopoulos (1996) Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Endocrinology 137(12): 5727–30.

    Google Scholar 

  17. Schillers, H., et al. (2000) Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy. Cell Physiol Biochem 10(1–2): 99–107.

    Google Scholar 

  18. Mannella, C.A., et al. (1998) Electron microscopic tomography of rat-liver mitochondria and their interaction with the endoplasmic reticulum. Biofactors 8(3–4): 225–8.

    Google Scholar 

  19. Handran, S.D., et al. (1997) Mitochondrial morphology and intracellular calcium homeostasis in cytochrome oxidase-deficient human fibroblasts. Neurobiology of Disease 3(4): 287–298.

    Google Scholar 

  20. Russell, J.W., et al. (2002) High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. Faseb J 16(13): 1738–48.

    Google Scholar 

  21. Clippinger, A.J. and M.J. Bouchard (2008) Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential. J Virol 82(14): 6798–811.

    Google Scholar 

  22. Clippinger, A.J., T.L. Gearhart, and M.J. Bouchard (2009) Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore. J Virol 83(10): 4718–31.

    Google Scholar 

  23. McClain, S.L., et al. (2007) Hepatitis B virus replication is associated with an HBx-dependent mitochondrion-regulated increase in cytosolic calcium levels. J Virol 81(21): 12061–5.

    Google Scholar 

  24. Saccone, C., et al. (2003) Molecular clock and gene function. J Mol Evol 57 Suppl 1: S277–85.

    Google Scholar 

  25. Mannella, C.A. (1998) Conformational changes in the mitochondrial channel protein, VDAC, and their functional implications. J Struct Biol 121(2): 207–18.

    Google Scholar 

  26. Holmuhamedov, E. and J.J. Lemasters (2009) Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes. Arch Biochem Biophys 481(2): 226–33.

    Google Scholar 

  27. Nitzan, Y., K. Orlovsky, and I. Pechatnikov (1999) Characterization of porins isolated from the outer membrane of Serratia liquefaciens. Curr Microbiol 38(2): 71–9.

    Google Scholar 

  28. Kroemer, G., L. Galluzzi, and C. Brenner (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87(1): 99–163.

    Google Scholar 

  29. Malin, S.A., B.M. Davis, and D.C. Molliver (2007) Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2(1): 152–60.

    Google Scholar 

  30. Schillers, H., et al. (2001) Plasma membrane protein clusters appear in CFTB-expressing Xenopus laevis oocytes after cAMP stimulation. Journal of Membrane Biology 180(3): 205–212.

    Google Scholar 

  31. Wang, H., B.E. Layton, and A.M. Sastry (2003) Nerve collagens from diabetic and nondiabetic Sprague-Dawley and biobreeding rats: an atomic force microscopy study. Diabetes Metabolism Research and Reviews 19(4): 288–298.

    Google Scholar 

  32. Layton, B.E. (2008) Recent patents in bionanotechnologies: nanolithography, bionanocomposites, cell-based computing and entropy production. Recent Patents in Nanotechnology 2(1): 1–12.

    Google Scholar 

  33. Scheuring, S., T. Boudier, and J.N. Sturgis (2007) From high-resolution AFM topographs to atomic models of supramolecular assemblies. J Struct Biol 159(2): 268–76.

    Google Scholar 

  34. Domenech, O., et al. (2006) Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane. Biochim Biophys Acta 1758(2): 213–21.

    Google Scholar 

  35. Chen, Y., et al. (2005) Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures. Ultramicroscopy 103(3): 173–82.

    Google Scholar 

  36. Layton, B.E., et al. (2008) Collagen’s triglycine repeat length may help to explain an interdomain transfer event from a eukaryote into Trichodesmium erythraeum. Journal of Molecular Evolution 66(6): 539–554.

    Google Scholar 

Download references

Acknowledgments

The authors thank Michael Bouchard for helpful discussions on mitochondrial structure and function, Chia-Wei Wang and Hui Wang for AFM assistance, Carrie Backus and Terry Miller for mitochondrial isolation assistance, Eva Feldman for funding through the Michigan Diabetes Research and Training Center, Martin Filbert for donation of animal specimens, Ann Marie Sastry for atomic force microscope access, and the Keck Foundation for support.

Author information

Authors and Affiliations

  1. Applied Computing and Electronics, The University of Montana College of Technology, Missoula, MT, USA

    Bradley E. Layton

Authors
  1. Bradley E. Layton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Brent Boyd
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bradley E. Layton .

Editor information

Editors and Affiliations

  1. School of Medicine, Department of Pharmacology, University of Milan, Via Vanvitelli 32, Milano, 20129, Italy

    Pier Carlo Braga

  2. , Robotics, Brain & Cognitive Sciences, Italian Institute of Technology, Via Morego 30, Genova, 16163, Italy

    Davide Ricci

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Layton, B.E., Boyd, M.B. (2011). Atomic Force Microscopy of Isolated Mitochondria. In: Braga, P., Ricci, D. (eds) Atomic Force Microscopy in Biomedical Research. Methods in Molecular Biology, vol 736. Humana Press. https://doi.org/10.1007/978-1-61779-105-5_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-105-5_10

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-104-8

  • Online ISBN: 978-1-61779-105-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation