Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis

Nature Protocols volume 5, pages 1396–1405 (2010)Cite this article

Subjects

Abstract

Applications for noninvasive real-time imaging of apoptosis and neuronal degeneration are hindered by technical limitations in imaging strategies and by existing probes. Monitoring the progression of a cell through apoptosis could provide valuable insight into the temporal events that initiate cell death as well as the potential for rescue of apoptotic cells. We engineered an annexin-based biosensor to function as a polarity-sensitive indicator for viability and apoptosis (known as pSIVA) by binding to externalized phosphatidylserine (PS) exposed on apoptotic cell membranes. Constructed from a structure-based design strategy, pSIVA fluoresces only when bound to PS and remains effectively undetectable in solution. In this paper, we describe protocols for the design, expression, purification and labeling of pSIVA as well as for its application in time-lapse imaging of degenerating neurons in culture; the entire protocol can be completed in 2 weeks. The primary advantage of this method is the flexibility to use pSIVA, in combination with other probes and without perturbing experimental conditions, to explore the cellular mechanisms involved in apoptosis and degeneration in real time.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure-based design of pSIVA.
Figure 2: Application of pSIVA for monitoring the degeneration of DRG neurons.

Similar content being viewed by others

References

  1. Fadok, V.A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216 (1992).

    CAS  PubMed  Google Scholar 

  2. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Green, D.R. & Reed, J.C. Mitochondria and apoptosis. Science 281, 1309–1312 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Koopman, G. et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415–1420 (1994).

    CAS  PubMed  Google Scholar 

  5. Li, J. & Yuan, J. Caspases in apoptosis and beyond. Oncogene 27, 6194–6206 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Kim, Y.E., Chen, J., Chan, J.R. & Langen, R. Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration. Nat. Methods 7, 67–73 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Gerke, V. & Moss, S.E. Annexins: from structure to function. Physiol. Rev. 82, 331–371 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Seaton, B.A. ed. Annexins: Molecular Structure to Cellular Function (R.G. Landes Company, Austin, Texas, USA, 1996).

  9. Martin, S.J. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–1556 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Bretscher, M.S. Asymmetrical lipid bilayer structure for biological membranes. Nat. New. Biol. 236, 11–12 (1972).

    Article  CAS  PubMed  Google Scholar 

  11. Logue, S.E., Elgendy, M. & Martin, S.J. Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nat. Protoc. 4, 1383–1395 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. van Genderen, H. et al. In vitro measurement of cell death with the annexin A5 affinity assay. Nat. Protoc. 1, 363–367 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Isas, J.M. et al. Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in Annexin B12. Biochemistry 44, 16435–16444 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Isas, J.M., Langen, R., Haigler, H.T. & Hubbell, W.L. Structure and dynamics of a helical hairpin and loop region in annexin 12: a site-directed spin labeling study. Biochemistry 41, 1464–1473 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Isas, J.M., Langen, R., Hubbell, W.L. & Haigler, H.T. Structure and dynamics of a helical hairpin that mediates calcium-dependent membrane binding of Annexin B12. J. Biol. Chem. 279, 32492–32498 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Langen, R., Isas, J.M., Luecke, H., Haigler, H.T. & Hubbell, W.L. Membrane-mediated assembly of annexins studied by site-directed spin labeling. J. Biol. Chem. 273, 22453–22457 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Luecke, H., Chang, B.T., Mailliard, W.S., Schlaepfer, D.D. & Haigler, H.T. Crystal structure of the annexin XII hexamer and implications for bilayer insertion. Nature 378, 512–515 (1995).

    Article  CAS  PubMed  Google Scholar 

  18. Kerr, J.F., Wyllie, A.H. & Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bedner, E., Smolewski, P., Amstad, P. & Darzynkiewicz, Z. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 259, 308–313 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Nikolaev, A., McLaughlin, T., O′Leary, D.D. & Tessier-Lavigne, M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981–989 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Finn, J.T. et al. Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J. Neurosci. 20, 1333–1341 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cordeiro, M.F. et al. Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration. Proc. Natl. Acad. Sci. USA 101, 13352–13356 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schmitz-Valckenberg, S. et al. Real-time in vivo imaging of retinal cell apoptosis after laser exposure. Invest Ophthalmol. Vis. Sci. 49, 2773–2780 (2008).

    Article  PubMed  Google Scholar 

  24. Dumont, E.A. et al. Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart. Nat. Med. 7, 1352–1355 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Blankenberg, F.G., Vanderheyden, J.L., Strauss, H.W. & Tait, J.F. Radiolabeling of HYNIC-annexin V with technetium-99m for in vivo imaging of apoptosis. Nat. Protoc. 1, 108–110 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Kenis, H. et al. Cell surface-expressed phosphatidylserine and annexin A5 open a novel portal of cell entry. J. Biol. Chem. 279, 52623–52629 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Fadok, V.A., de Cathelineau, A., Daleke, D.L., Henson, P.M. & Bratton, D.L. Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J. Biol. Chem. 276, 1071–1077 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Hammill, A.K., Uhr, J.W. & Scheuermann, R.H. Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death. Exp. Cell. Res. 251, 16–21 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Rimon, G., Bazenet, C.E., Philpott, K.L. & Rubin, L.L. Increased surface phosphatidylserine is an early marker of neuronal apoptosis. J. Neurosci. Res. 48, 563–570 (1997).

    Article  CAS  PubMed  Google Scholar 

  30. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Mailliard, W.S., Luecke, H. & Haigler, H.T. Annexin XII forms calcium-dependent multimers in solution and on phospholipid bilayers: a chemical cross-linking study. Biochemistry 36, 9045–9050 (1997).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the members of the Chan and Langen labs for technical assistance. The development of this protocol was partially supported by the US National Multiple Sclerosis Society Career Transition Award (J.R.C) and EY12155 (J.C.). J.R.C. is a Harry Weaver Neuroscience Scholar (TA 3008A/T, JF 2142-A-2).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute at the Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Yujin E Kim, Ralf Langen & Jonah R Chan

  2. Department of Cell and Neurobiology, Zilkha Neurogenetic Institute at the Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Jeannie Chen

Authors
  1. Yujin E Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeannie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ralf Langen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jonah R Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.L. conceived the study and, together with J.C. and J.R.C., supervised the project. Y.E.K. designed and performed the experiments and, together with J.R.C., designed and performed the experiments involving neurons. R.L., J.C., J.R.C. and Y.E.K. analyzed the data. Y.E.K. and J.R.C. wrote the paper.

Corresponding authors

Correspondence to Ralf Langen or Jonah R Chan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, Y., Chen, J., Langen, R. et al. Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis. Nat Protoc 5, 1396–1405 (2010). https://doi.org/10.1038/nprot.2010.101

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.101

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing