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

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.

journal_name

Theranostics

journal_title

Theranostics

authors

Savitsky AP,Rusanov AL,Zherdeva VV,Gorodnicheva TV,Khrenova MG,Nemukhin AV

doi

10.7150/thno.3885

subject

Has Abstract

pub_date

2012-01-01 00:00:00

pages

215-26

issue

2

issn

1838-7640

pii

thnov02p0215

journal_volume

2

pub_type

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