Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe.
BACKGROUND
A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells.


METHODS
This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry.


RESULTS
DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently preceded caspase-6 by approximately 30 min following induction of apoptosis.


CONCLUSIONS
We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway.
Wu
Xiaoli
X
Flow Cytometry Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland 20892, USA.
Simone
James
J
Hewgill
Derek
D
Siegel
Richard
R
Lipsky
Peter E
PE
He
Liusheng
L