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.

  • Brief Communication
  • Published:

Imaging specific cell-surface proteolytic activity in single living cells

Nature Methods volume 3, pages 259–261 (2006)Cite this article

Abstract

We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin–β-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell-surface proteolytic activity in single living cells. The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator and metalloprotease activity. We have adapted the assay for fluorescence microscopy, flow cytometry and fluorescent plate reader formats, and it is amenable for automation and high-throughput analysis.

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: Schematic of cell-surface protease activity imaging assay.
Figure 2: Specific imaging of furin proteolytic activity in single living cells.
Figure 3: Specific imaging of cell surface uPA activity.

Similar content being viewed by others

References

  1. Sloane, B.F., Sameni, M., Podgorski, I., Cavallo-Medved, D. & Moin, K. Annu. Rev. Pharmacol. Toxicol. 46, 301–315 (2006).

    Article  CAS  Google Scholar 

  2. Coussens, L.M., Fingleton, B. & Matrisian, L.M. Science 295, 2387–2392 (2002).

    Article  CAS  Google Scholar 

  3. Klimpel, K.R., Molloy, S.S., Thomas, G. & Leppla, S.H. Proc. Natl. Acad. Sci. USA 89, 10277–10281 (1992).

    Article  CAS  Google Scholar 

  4. Liu, S., Bugge, T.H. & Leppla, S.H. J. Biol. Chem. 276, 17976–17984 (2001).

    Article  CAS  Google Scholar 

  5. Liu, S., Netzel-Arnett, S., Birkedal-Hansen, H. & Leppla, S.H. Cancer Res. 60, 6061–6067 (2000).

    CAS  PubMed  Google Scholar 

  6. Arora, N. & Leppla, S.H. J. Biol. Chem. 268, 3334–3341 (1993).

    CAS  PubMed  Google Scholar 

  7. Leppla, S.H., Arora, N. & Varughese, M. J. Appl. Microbiol. 87, 284 (1999).

    Article  CAS  Google Scholar 

  8. Liu, S., Aaronson, H., Mitola, D.J., Leppla, S.H. & Bugge, T.H. Proc. Natl. Acad. Sci. USA 100, 657–662 (2003).

    Article  CAS  Google Scholar 

  9. Mogridge, J., Cunningham, K. & Collier, R.J. Biochemistry 41, 1079–1082 (2002).

    Article  CAS  Google Scholar 

  10. Scobie, H.M., Rainey, G.J., Bradley, K.A. & Young, J.A. Proc. Natl. Acad. Sci. USA 100, 5170–5174 (2003).

    Article  CAS  Google Scholar 

  11. Zlokarnik, G. et al. Science 279, 84–88 (1998).

    Article  CAS  Google Scholar 

  12. Gordon, V.M., Klimpel, K.R., Arora, N., Henderson, M.A. & Leppla, S.H. Infect. Immun. 63, 82–87 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Dano, K. et al. APMIS 107, 120–127 (1999).

    Article  CAS  Google Scholar 

  14. Netzel-Arnett, S. et al. J. Biol. Chem. 277, 45154–45161 (2002).

    Article  CAS  Google Scholar 

  15. Sameni, M., Dosescu, J., Moin, K. & Sloane, B.F. Mol. Imaging 2, 159–175 (2003).

    Article  Google Scholar 

  16. Ke, S.H. et al. J. Biol. Chem. 272, 16603–16609 (1997).

    Article  CAS  Google Scholar 

  17. Gao, W.B.X., Tsien, R.Y. & Rao, J. J. Am. Chem. Soc. 125, 11146–11147 (2003).

    Article  CAS  Google Scholar 

  18. Xing, B., Khanamiryan, A. & Rao, J. J. Am. Chem. Soc. 127, 4158–4159 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Angerer, S. Gutkind and M.J. Danton for comments, and K. Holmes and D. Stephany for assistance with flow cytometry experiments. Supported by National Institute of Allergy and Infectious Diseases Support of Intramural Biodefense Research, and by Department of Defense (DAMD-17-02-1-0693) to T.H.B., by a doctorate fellowship from the State University Hospital, Copenhagen, Denmark to B.R., and National Institutes of Health Intramural support to T.H.B., and S.H.L.

Author information

Authors and Affiliations

  1. Proteases and Tissue Remodeling Unit, National Institute of Dental and Craniofacial Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 30 Convent Drive, Bethesda, 20892, Maryland, USA

    John P Hobson, Birgitte Rønø & Thomas H Bugge

  2. Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 30 Convent Drive, Bethesda, 20892, Maryland, USA

    Shihui Liu & Stephen H Leppla

  3. Finsen Laboratory, Strandboulevarden 49, Copenhagen Ø, DK-2100, Denmark

    Birgitte Rønø

Authors
  1. John P Hobson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shihui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Birgitte Rønø
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephen H Leppla
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas H Bugge
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Stephen H Leppla or Thomas H Bugge.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Imaging of metalloprotease activity in human tumor cells. (PDF 1021 kb)

Supplementary Fig. 2

Adaptation of the protease imaging assay to a fluorescent plate reader format. (PDF 357 kb)

Supplementary Fig. 3

Visualization of specific cell surface protease activity by flow cytometry. (PDF 440 kb)

Supplementary Data (DOC 43 kb)

Supplementary Methods (DOC 97 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hobson, J., Liu, S., Rønø, B. et al. Imaging specific cell-surface proteolytic activity in single living cells. Nat Methods 3, 259–261 (2006). https://doi.org/10.1038/nmeth862

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth862

This article is cited by

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