Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis.
Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-alpha-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-alpha-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.
Kawai
Hiroshi
H
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo.
Suzuki
Takuo
T
Kobayashi
Tetsu
T
Sakurai
Haruna
H
Ohata
Hisayuki
H
Honda
Kazuo
K
Momose
Kazutaka
K
Namekata
Iyuki
I
Tanaka
Hikaru
H
Shigenobu
Koki
K
Nakamura
Ryu
R
Hayakawa
Takao
T
Kawanishi
Toru
T
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D018613:Microscopy, Confocal; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D020169:Caspases