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Imaging Specific Cell Surface Protease Activity in Living Cells Using Reengineered Bacterial Cytotoxins

  • John P. Hobson
  • , Shihui Liu
  • Stephen H. Leppla
  • Thomas H. BuggeEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 539)

Summary

The scarcity of methods to visualize the activity of individual cell surface proteases in situ has hampered basic research and drug development efforts. In this chapter, we describe a simple, sensitive, and noninvasive assay that uses nontoxic reengineered bacterial cytotoxins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay takes advantage of the absolute requirement for site-specific endoproteolytic cleavage of cell surface-bound anthrax toxin protective antigen for its capacity to translocate an anthrax toxin lethal factor-β-lactamase fusion protein to the cytoplasm. A fluorogenic β-lactamase substrate is then used to visualize the cytoplasmically translocated anthrax toxin lethal factor-β-lactamase fusion protein. By using anthrax toxin protective antigen variants that are reengineered to be cleaved by furin, urokinase plasminogen activator, or metalloproteinases, the cell surface activities of each of these proteases can be specifically and quantitatively determined with single cell resolution. The imaging assay is excellently suited for fluorescence microscope, fluorescence plate reader, and flow cytometry formats, and it can be used for a variety of purposes.

Key words:

Anthrax toxin β-lactamase CCF2/AM Cell surface proteolysis Flow cytometry Fluorescence microscopy Fluorescence plate reader Furin Metalloproteinases Urokinase plasminogen activator. 

Notes

Acknowledgments

We thank Drs. Silvio Gutkind and Mary Jo Danton for critically reviewing this manuscript, and Drs. Kevin L. Holmes and David Stephany for expert assistance with flow cytometry. This work was supported by the NIH Intramural Research Program, by NIAID Support of Intramural Biodefense Research from ICs other than NIAID and by the Department of Defense (DAMD-17-02-1-0693) to Dr. Thomas H. Bugge. For imaging reagents, contact S. H. Leppla (Leppla@nih.gov) or T. H. Bugge (thomas.bugge@nih.gov).

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • John P. Hobson
  • , Shihui Liu
  • Stephen H. Leppla
  • Thomas H. Bugge
    • 1
    Email author
  1. 1.Proteases and Tissue Remodeling Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial ResearchNational Institutes of HealthBethesda

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