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Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis

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Abstract

Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.

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References

  1. Tracey KJ, Cerami A (1993) Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 9:317–343

    Article  PubMed  CAS  Google Scholar 

  2. Stanger BZ, Leder P, Lee TH, Kim E, Seed B (1995) RIP: a novel protein containing a death domain that interacts with Fas/APO-l (CD95) in yeast and causes cell death. Cell 81:513–523

    Article  PubMed  CAS  Google Scholar 

  3. Medema JP, Scaffidi C, Kischkel FC et al (1997) FLICE is activated by association with CD95 death-inducing signaling complex (DISC). EMBO J 16:2794–2804

    Article  PubMed  CAS  Google Scholar 

  4. Li H, Zhou H, Xu C-j Yuan J (1998) Cleavage of Bid by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501

    Article  PubMed  CAS  Google Scholar 

  5. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481–490

    Article  PubMed  CAS  Google Scholar 

  6. Gross A, Yin X-M, Wang K et al (1999) 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. J Biol Chem 274:1156–1163

    Article  PubMed  CAS  Google Scholar 

  7. Roucou X, Rostovtseva T, Montessuit S, Martinou J-C, Antonsson B (2002) Bid induces cytochrome c-impermeable Bax channels in liposomes. Biochem J 363:547–552

    Article  PubMed  CAS  Google Scholar 

  8. Zheng TS, Hunot S, Kuida K, Flavell RA (1999) Caspase knockouts: matters of life and death. Cell Death Differ 6:1043–1053

    Article  PubMed  CAS  Google Scholar 

  9. Yin X-M (2006) Bid, a BH3-only multi-functional molecule, is at the cross road of life and death. Gene 369:7–19

    Article  PubMed  CAS  Google Scholar 

  10. Eskes R, Desagher S, Antonsson B, Martinou J-C (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20:929–935

    Article  PubMed  CAS  Google Scholar 

  11. Wei MC, Lindsten T, Mootha VK et al (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14:2060–2071

    PubMed  CAS  Google Scholar 

  12. Desagher S, Osen-Sand A, Nichols A et al (1999) Bid-induced conformational change of Bax is responsible for mitochondrial Cytochrome c release during apoptosis. J Cell Biol 144:891–901

    Article  PubMed  CAS  Google Scholar 

  13. Kim T-H, Zhao Y, Barber MJ, Kuharsky DK, Yin X-M (2000) Bid-induced cytochrome c release is mediated by a pathway independent of mitochondrial permeability transition pore and Bax. J Biol Chem 275:39474–39481

    Article  PubMed  CAS  Google Scholar 

  14. Shimizu S, Tsujimoto Y (2000) Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity. Proc Natl Acad Sci U S A 97:577–582

    Article  PubMed  CAS  Google Scholar 

  15. Grinberg M, Sarig R, Zaltsman Y et al (2002) tBID homooligomerizes in the mitochondrial membrane to induce apoptosis. J Biol Chem 277:12237–12245

    Article  PubMed  CAS  Google Scholar 

  16. Walensky LD, Pitter K, Morash J et al (2006) A stapled BID BH3 helix directly binds and activates BAX. Molecular Cell 24:199–210

    Article  PubMed  CAS  Google Scholar 

  17. Terrones O, Antonsson B, Yamaguchi H et al (2004) Lipidic pore formation by the concerted action of proapoptotic BAX and tBID. J Biol Chem 279:30081–30091

    Article  PubMed  CAS  Google Scholar 

  18. Nechushtan A, Smith CL, Lamensdorf I, Yoon S-H, Youle RJ (2001) Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 153:1265–1276

    Article  PubMed  CAS  Google Scholar 

  19. Ward MW, Rehm M, Duessmann H, Kacmar S, Concannon CG, Prehn JHM (2006) Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis. J Biol Chem 281:5837–5844

    Article  PubMed  CAS  Google Scholar 

  20. Miyawaki A (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell 4:295–305

    Article  PubMed  CAS  Google Scholar 

  21. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918

    Article  PubMed  CAS  Google Scholar 

  22. Wu Y-X, Xing D, Chen WR (2006) Single cell FRET imaging for determination of pathway of tumor cell apoptosis induced by photofrin-PDT. Cell Cycle 5:729–734

    PubMed  CAS  Google Scholar 

  23. Onuki R, Nagasaki A, Kawasaki H, Baba T, Uyeda TQP, Taira K (2002) Confirmation by FRET in individual living cells of the absence of significant amyloid β-mediated caspase 8 activation. Proc Natl Acad Sci U S A 99:14716–14721

    Article  PubMed  CAS  Google Scholar 

  24. Valentijn AJ, Metcalfe AD, Kott J, Streuli CH, Gilmore AP (2003) Spatial and temporal changes in Bax subcellular localization during anoikis. J Cell Biol 162:599–612

    Article  PubMed  CAS  Google Scholar 

  25. Tsuruta F, Gotoh NMY (2002) The phosphatidylinositol 3-Kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J Biol Chem 277:14040–14047

    Article  PubMed  CAS  Google Scholar 

  26. Gao X-J, Chen T-S, Xing D, Wang F, Pei Y-H, Wei X-B (2006) Single cell analysis of pkc activation during proliferation and apoptosis induced by laser irradiation. J Cell Physiol 206:441–448

    Article  PubMed  CAS  Google Scholar 

  27. Adams JM (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev 17:2481–2495

    Article  PubMed  CAS  Google Scholar 

  28. Jänicke RU, Sprengart ML, Wati MR, Porter AG (1998) Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273:9357–9360

    Article  PubMed  Google Scholar 

  29. Luo KQ, Yu VC, Pu Y, Chang DC (2003) Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFα-induced apoptosis. Biochem Biophys Res Commun 304:217–222

    Article  PubMed  CAS  Google Scholar 

  30. Grinberg M, Schwarz M, Zaltsman Y et al (2005) Mitochondrial carrier homolog 2 is a target of tBID in cells signaled to die by tumor necrosis factor alpha. Mol Cell Biol 25:4579–4590

    Article  PubMed  CAS  Google Scholar 

  31. Green DR (2000) Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1–4

    Article  PubMed  CAS  Google Scholar 

  32. Wolter KG, Hsu Yi-Te, Smith CL, Nechushtan A, Xi Xu-Guang, Youle RJ (1997) Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139:1281–1292

    Article  PubMed  CAS  Google Scholar 

  33. Stoka V, Turk B, Schendel SL et al (2001) Lysosomal protease pathways to apoptosis. Cleavage of Bid, not pro-caspases, is the most likely route. J Biol Chem 276:3149–3157

    Article  PubMed  CAS  Google Scholar 

  34. Desagher S, Osen-Sand A, Montessuit S et al (2001) Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase-8. Mol Cell 8:601–611

    Article  PubMed  CAS  Google Scholar 

  35. Tafani M, Karpinich NO, Hurster KA et al (2002) Cytochrome c release upon fas receptor activation depends on translocation of full-length Bid and the induction of the mitochondrial permeability transition. J Biol Chem 277:10073–10082

    Article  PubMed  CAS  Google Scholar 

  36. Krysko DV, Roels F, Leybaert L, Herde K (2001) Mitochondrial transmembrane potential changes support the concept of mitochondrial heterogeneity during apoptosis. J Histochem Cytochem 49:1277–1284

    PubMed  CAS  Google Scholar 

  37. Wei QQ, Alam MM, Wang MH, Yu F, Dong Z (2004) Bid activation in kidney cells following ATP depletion,in vitro and ischemia in vivo. Am J Physiol Renal Physiol 286:F803–F809

    Article  PubMed  CAS  Google Scholar 

  38. Slee EA, Keogh SA, Martin SJ (2000) Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release. Cell Death Differ 7:556–565

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This research is supported by the National Natural Science Foundation of China (60378043; 30470494), and the Natural Science Foundation of Guangdong Province (015012; 04010394; 2004B10401011). We thank Dr. R. Onuki (National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan) for providing FRET-Bid and Bid-CFP plasmids. We thank Professor Andrew P. Gilmore (Wellcome Trust Centre for Cell Matrix Research, School of Biological Sciences, University of Manchester, UK) for providing the YFP-Bax plasmid. We also thank Professor Yukiko Gotoh (the Japan Science and Technology Corporation, Japan) for providing DsRed-Mit.

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  1. MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou, 510631, China

    Yihui Pei, Da Xing, Xuejuan Gao, Lei Liu & Tongsheng Chen

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  1. Yihui Pei
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  2. Da Xing
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  3. Xuejuan Gao
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  5. Tongsheng Chen
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Correspondence to Da Xing.

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Pei, Y., Xing, D., Gao, X. et al. Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis. Apoptosis 12, 1681–1690 (2007). https://doi.org/10.1007/s10495-007-0091-7

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  • DOI: https://doi.org/10.1007/s10495-007-0091-7

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