Confirmation by FRET in Individual Living Cells of the Absence of Significant Amyloid β-Mediated Caspase 8 Activation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 99; no. 23; pp. 14716 - 14721
• Main Authors: Onuki, Reiko, Nagasaki, Akira, Kawasaki, Hiroaki, Baba, Tadashi, Taro Q. P. Uyeda, Taira, Kazunari
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 12.11.2002; National Acad Sciences
• Subjects: 3T3 Cells; Amyloid beta-Peptides - physiology; Animals; Apoptosis; Apoptosis - physiology; Bacterial Proteins - genetics; BH3 Interacting Domain Death Agonist Protein; Biochemistry; Biological Sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Caspase 8; Caspase 9; Caspases - metabolism; Cell lines; Cells; Cercopithecus aethiops; Cloning, Molecular; COS Cells; DNA, Complementary - genetics; Enzyme Activation; Fluorescence; Gene Library; Genes, Reporter; Geometric lines; Green Fluorescent Proteins; Humans; Ligands; Liver - metabolism; Luminescent Proteins - genetics; Mice; Microscopy; Mitochondria; NIH 3T3 cells; Proteins; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; Sequence Deletion; Spectrometry, Fluorescence; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.232177599

When cells are exposed to death-inducing molecules such as tumor necrosis factor-α or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Aβ or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Aβ- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.