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. 2012;2(2):215-26.
doi: 10.7150/thno.3885. Epub 2012 Feb 15.

FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins

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Free PMC article

FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins

Alexander P Savitsky et al. Theranostics. .
Free PMC article

Abstract

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: FLIM; FRET; caspase; lentiviral vector.; molecular dynamics (MD); red fluorescent proteins (RFP).

Conflict of interest statement

Conflict of Interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1
TagRFP-23-KFP tetramer. The monomers are shown in red, orange, green and violet. The inset shows the distances (in angstroms) between bridge carbon atoms of the chromophore groups of TagRFP (shown in sticks) and KFP (shown in balls and sticks). Here, carbon, oxygen, nitrogen, hydrogen and sulfur atoms are shown in green, red, blue, white and yellow respectively.
Figure 2
Figure 2
Complex of caspase-3 dimer (shown in cyan) and TagRFP-23-KFP (colors are the same with those from the figure 1). Monomer of TagRFP-23-KFP that forms the complex with caspase-3 is shown in orange.
Figure 3
Figure 3
Lentiviral transduction of lung adenocarcinoma A549. (A) Genetically encoded caspase-3 sensor TagRFP-23aa-KFP (TR23K) based on red fluorescent proteins TagRFP and KFP linked by 23 a.a. with DEVD, site specific for cleavage by caspase-3. (B) Lentivector pLVT-GFP (www.evrogen.ru). The DNA fragment of TagRFP or TagRFP-23aa-KFP (TR23K) was inserted into pLVT-GFP between BamH1 и Sal1 restriction sites after GFP encoding fragment deleting. (C) Lung adenocarcinoma А549, transduced with lentivector pLVT–TagRFP, 7 passage. Nikon Eclipse TE2000-U, 20х. (D) Lung adenocarcinoma А549, transduced with lentivector pLVT –TR23K, 7 passage. Nikon Eclipse TE2000-U, 20х.
Figure 4
Figure 4
Detecting of caspase-3 activation. (A)Lung adenocarcinoma А549–TR23K, 14 passage. Nikon Eclipse TE2000-U, 20х. (B) Lung adenocarcinoma А549–TR23K, 14 passage, after induction of apoptosis with staurosporine 0,5 ng/ml for 2 h. Nikon Eclipse TE2000-U, 20х. (C) Every 5x105 of cells were tested in parallel for viability and activation of caspase-3 by fluorescence plate reader Labsystems Fluoroscan II. Caspase activity was analyzed by measuring change in intensity of TagRFP’s fluorescence with excitation filter 544 nm and emission filter 607nm. Caspase activity was analyzed with Caspase -3 Detection Kit (Calbiochem) by measuring FITС (fluorescein isothiocyanate) with excitation filter 485 nm and emission filter 538 nm. Cells viability was detected by FDA (fluorescein diacetate) test with excitation filter 485 nm and emission filter 538 nm.
Figure 5
Figure 5
Fluorescence lifetime images of A549 cells expressing TagRFP-23-KFP (A) before and (B) after incubation with H2O2. Lifetime distribution over the whole detecting area (C) before and (D) after incubation with H2O2.
Figure 6
Figure 6
Fluorescence lifetime images of A549 cells expressing TagRFP-23-KFP (A) nonapoptotic and (B) apoptotic cell. Lifetime distribution of (C) nonapoptotic and (D) apoptotic cell.

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References

    1. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science. 2006;312:217–24. - PubMed
    1. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–44. - PubMed
    1. Remington SJ. Structural basis for understanding spectral variations in green fluorescent protein. Biolumin Chemilumin C. 2000;305:196–211. - PubMed
    1. Zimmer M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev. 2002;102:759–81. - PubMed
    1. Du W, Wang Y, Luo Q, Liu BF. Optical molecular imaging for systems biology: from molecule to organism. Anal Bioanal Chem. 2006;386:444–57. - PMC - PubMed
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