Imaging specific cell-surface proteolytic activity in single living cells.
We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin-beta-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.
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, Maryland 20892, USA.
John P
JP
Hobson
Shihui
S
Liu
Birgitte
B
Rønø
Stephen H
SH
Leppla
Thomas H
TH
Bugge
D000942:Antigens, Bacterial / Q000378:metabolism*; D001331:Automation; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D045683:Furin / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; D045726:Metalloproteases; D045683:Furin; D001618:beta-Lactamases