Confirmation by FRET in individual living cells of the absence of significant amyloid β-mediated caspase 8 activation

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.

[1]  S. Korsmeyer,et al.  Caspase Cleaved BID Targets Mitochondria and Is Required for Cytochrome c Release, while BCL-XL Prevents This Release but Not Tumor Necrosis Factor-R1/Fas Death* , 1999, The Journal of Biological Chemistry.

[2]  J. Safrit,et al.  Visualization and quantification of T cell–mediated cytotoxicity using cell-permeable fluorogenic caspase substrates , 2002, Nature Medicine.

[3]  Junying Yuan,et al.  Cross-Talk between Two Cysteine Protease Families , 2000, The Journal of cell biology.

[4]  Junying Yuan,et al.  Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in the Fas Pathway of Apoptosis , 1998, Cell.

[5]  Junying Yuan,et al.  Apoptosis in the nervous system , 2000, Nature.

[6]  V. Mootha,et al.  tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. , 2000, Genes & development.

[7]  T. Kudo,et al.  The unfolded protein response and Alzheimer's disease. , 2001, Biochimica et biophysica acta.

[8]  Fred S. Wouters,et al.  Imaging FRET between spectrally similar GFP molecules in single cells , 2001, Nature Biotechnology.

[9]  D. Payan,et al.  Detection of programmed cell death using fluorescence energy transfer. , 1998, Nucleic acids research.

[10]  A. Sorkin,et al.  Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy , 2000, Current Biology.

[11]  K. Taira,et al.  Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids. , 1998, Nucleic acids research.

[12]  T. Furuyama,et al.  The Cell Death-promoting Gene DP5, Which Interacts with the BCL2 Family, Is Induced during Neuronal Apoptosis Following Exposure to Amyloid β Protein* , 1999, The Journal of Biological Chemistry.

[13]  Y. Pu,et al.  Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. , 2001, Biochemical and biophysical research communications.

[14]  K. Taira,et al.  Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries , 2002, Nature Biotechnology.

[15]  R. Heim,et al.  Using GFP in FRET-based applications. , 1999, Trends in cell biology.

[16]  S. Estus,et al.  Aggregated Amyloid-β Protein Induces Cortical Neuronal Apoptosis and Concomitant “Apoptotic” Pattern of Gene Induction , 1997, The Journal of Neuroscience.

[17]  A. Khwaja,et al.  Resistance to the Cytotoxic Effects of Tumor Necrosis Factor α Can Be Overcome by Inhibition of a FADD/Caspase-dependent Signaling Pathway* , 1999, The Journal of Biological Chemistry.

[18]  K. Taira,et al.  A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries. , 2002, Nucleic acids research.

[19]  S. Korsmeyer,et al.  Solution Structure of the Proapoptotic Molecule BID A Structural Basis for Apoptotic Agonists and Antagonists , 1999, Cell.

[20]  M. Greenberg,et al.  β-Amyloid Induces Neuronal Apoptosis Via a Mechanism that Involves the c-Jun N-Terminal Kinase Pathway and the Induction of Fas Ligand , 2001, The Journal of Neuroscience.

[21]  K. Taira,et al.  Detection of Undegraded Oligonucleotides in Vivo by Fluorescence Resonance Energy Transfer , 1996, The Journal of Biological Chemistry.

[22]  C. Milliman,et al.  BID: a novel BH3 domain-only death agonist. , 1996, Genes & development.

[23]  Junying Yuan,et al.  Solution Structure of BID, an Intracellular Amplifier of Apoptotic Signaling , 1999, Cell.

[24]  S. Korsmeyer,et al.  Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c , 2000, Cell Death and Differentiation.

[25]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[26]  Junying Yuan,et al.  Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β , 2000, Nature.

[27]  B. Roizman,et al.  Herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell-type-dependent manner. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Xiaodong Wang,et al.  Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors , 1998, Cell.

[29]  S. Korsmeyer,et al.  BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. , 2001, Molecular cell.

[30]  Jean-Claude Martinou,et al.  Bid-induced Conformational Change of Bax Is Responsible for Mitochondrial Cytochrome c Release during Apoptosis , 1999, The Journal of cell biology.

[31]  H. Li,et al.  Deciphering the pathways of life and death. , 1999, Current opinion in cell biology.

[32]  S. Korsmeyer,et al.  Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. , 2000, Science.

[33]  S. Barik,et al.  An endoplasmic reticulum‐specific stress‐activated caspase (caspase‐12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus , 2001, Journal of cellular biochemistry.