Theranostics 2012; 2(2):215-226. doi:10.7150/thno.3885
FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins
1. A.N.Bach Institute of Biochemistry of the Russian Academy of Science, Moscow 119071, Russia;
2. Evrogen JSC, Miklukho-Maklaya 16/10, Moscow, 117420, Russia;
3. Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia;
4. N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow 119334, Russia.
Savitsky AP, Rusanov AL, Zherdeva VV, Gorodnicheva TV, Khrenova MG, Nemukhin AV. FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins. Theranostics 2012; 2(2):215-226. doi:10.7150/thno.3885. Available from http://www.thno.org/v02p0215.htm
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.
Keywords: FRET, FLIM, red fluorescent proteins (RFP), caspase, molecular dynamics (MD), lentiviral vector.