Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells.
Activation of caspase-3 is a central event in apoptosis. We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3. To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.
Luo
K Q
KQ
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
Yu
V C
VC
Pu
Y
Y
Chang
D C
DC
D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D001426:Bacterial Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D004735:Energy Transfer; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases