Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells.
A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.
Kawai
Hiroshi
H
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. kawai.nihs.go.jp
Suzuki
Takuo
T
Kobayashi
Tetsu
T
Mizuguchi
Hiroyuki
H
Hayakawa
Takao
T
Kawanishi
Toru
T
D000595:Amino Acid Sequence; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004594:Electrophysiology; D004789:Enzyme Activation; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology* / Q000502:physiology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases; D019311:Staurosporine