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

October 30, 2002
99 (23) 14716-14721

Abstract

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.

Continue Reading

Acknowledgments

We thank Prof. Yukiko Gotoh at the University of Tokyo and Dr. Laura Nelson at the National Institute of Advanced Industrial Science and Technology for helpful comments on the manuscript. This research was supported by grants from the Ministry of Economy, Trade and Industry (METI) of Japan, a grant from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, a grant from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Culture (MEXT) of Japan.

References

1
Li H. & Yuan, J. (1999) Curr. Opin. Cell Biol.11,261-266.
2
Korsmeyer S. J., Wei, M. C., Saito, M., Weiler, S., Oh, K. & Schlesinger, P. H. (2000) Cell Death Differ.7,1166-1173.
3
Wang K., Yin, X. M., Chao, D. T., Milliman, C. L. & Korsmeyer, S. J. (1996) Genes Dev.10,2859-2869.
4
Wei M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson, C. B. & Korsmeyer, S. J. (2000) Genes Dev.14,2060-2071.
5
Gross A., Yin, X. M., Wang, K., Wei, M. C., Jockel, J., Milliman, C., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S. J. (1999) J. Biol. Chem.274,1156-1163.
6
Cheng E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T. & Korsmeyer, S. J. (2001) Mol. Cell8,705-711.
7
Luo X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. (1998) Cell94,481-940.
8
Li H., Zhu, H., Xu, C. J. & Yuan, J. (1998) Cell94,491-501.
9
Zha J., Weiler, S., Oh, K. J., Wei, M. C. & Korsmeyer, S. J. (2000) Science290,1761-1765.
10
Desagher S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B. & Martinou, J. C. (1999) J. Cell Biol.144,891-901.
11
Li P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S. & Wang, X. (1997) Cell91,479-489.
12
Pollok B. A. & Heim, R. (1999) Trends Cell Biol.9,57-60.
13
Sorkin A., McClure, M., Huang, F. & Carter, R. (2000) Curr. Biol.10,1395-1398.
14
Harpur A. G., Wouters, F. S. & Bastiaens, P. I. (2001) Nat. Biotechnol.19,167-169.
15
Luo K. Q., Yu, V. C., Pu, Y. & Chang, D. C. (2001) Biochem. Biophys. Res. Commun.283,1054-1060.
16
Uchiyama H., Hirano, K., Kashiwasake-Jibu, M. & Taira, K. (1996) J. Biol. Chem.271,380-384.
17
Xu X., Gerard, A. L., Huang, B. C., Anderson, D. C., Payan, D. & Luo, Y. (1998) Nucleic Acids Res.26,2034-2035.
18
Ota N., Hirano, K., Warashina, M., Andrus, A., Mullah, B., Hatanaka, K. & Taira, K. (1998) Nucleic Acids Res.26,735-743.
19
Liu L., Chahroudi, A., Silvestri, G., Wernett, M. E., Kaiser, W. J., Safrit, J. T., Komoriya, A., Altman, J. D., Packard, B. Z. & Feinberg, M. B. (2002) Nat. Med.8,185-189.
20
Kawasaki H., Onuki, R., Suyama, E. & Taira, K. (2002) Nat. Biotechnol.20,376-380.
21
Kawasaki H. & Taira, K. (2002) EMBO Rep.3,443-450.
22
Kawasaki H. & Taira, K. (2002) Nucleic Acids Res.30,3609-3614.
23
Khwaja A. & Tatton, L. (1999) J. Biol. Chem.274,36817-36823.
24
Galvan V. & Roizman, B. (1998) Proc. Natl. Acad. Sci. USA95,3931-3936.
25
Nakagawa T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A. & Yuan, J. (2000) Nature403,98-103.
26
Imaizumi K., Miyoshi, K., Katayama, T., Yoneda, T., Taniguchi, M., Kudo, T. & Tohyama, M. (2001) Biochim. Biophys. Acta1536,85-96.
27
Morishima Y., Gotoh, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., Davis, R. J., Shirasaki, Y. & Greenberg, M. E. (2001) J. Neurosci.21,7551-7560.
28
Estus S., Tucker, H. M., van Rooyen, C., Wright, S., Brigham, E. F., Wogulis, M. & Rydel, R. E. (1997) J. Neurosci.17,7736-7745.
29
Imaizumi K., Morihara, T., Mori, Y., Katayama, T., Tsuda, M., Furuyama, T., Wanaka, A., Takeda, M. & Tohyama, M. (1999) J. Biol. Chem.274,7975-7981.
30
Chou J. J., Li, H., Salvesen, G. S., Yuan, J. & Wagner, G. (1999) Cell96,615-624.
31
McDonnell J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J. & Cowburn, D. (1999) Cell96,625-634.
32
Yuan J. & Yankner, B. A. (2000) Nature407,802-809.
33
Nakagawa T. & Yuan, J. (2000) J. Cell Biol.150,887-894.
34
Bitko V. & Barik, S. (2001) J. Cell Biochem.80,441-454.

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 99 | No. 23
November 12, 2002
PubMed: 12409609

Classifications

Submission history

Received: March 25, 2002
Published online: October 30, 2002
Published in issue: November 12, 2002

Acknowledgments

We thank Prof. Yukiko Gotoh at the University of Tokyo and Dr. Laura Nelson at the National Institute of Advanced Industrial Science and Technology for helpful comments on the manuscript. This research was supported by grants from the Ministry of Economy, Trade and Industry (METI) of Japan, a grant from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, a grant from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Culture (MEXT) of Japan.

Authors

Affiliations

Reiko Onuki
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan
Akira Nagasaki
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan
Hiroaki Kawasaki
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan
Tadashi Baba
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan
Taro Q. P. Uyeda
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan
Kazunari Taira
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan; and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan

Notes

R.O. and A.N. contributed equally to this work.
To whom correspondence should be addressed. E-mail: [email protected].
Edited by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved September 19, 2002

Metrics & Citations

Metrics

Note: The article usage is presented with a three- to four-day delay and will update daily once available. Due to ths delay, usage data will not appear immediately following publication. Citation information is sourced from Crossref Cited-by service.


Citation statements

Altmetrics

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

Cited by

    Loading...

    View Options

    View options

    PDF format

    Download this article as a PDF file

    DOWNLOAD PDF

    Get Access

    Login options

    Check if you have access through your login credentials or your institution to get full access on this article.

    Personal login Institutional Login

    Recommend to a librarian

    Recommend PNAS to a Librarian

    Purchase options

    Purchase this article to get full access to it.

    Single Article Purchase

    Confirmation by FRET in individual living cells of the absence of significant amyloid β-mediated caspase 8 activation
    Proceedings of the National Academy of Sciences
    • Vol. 99
    • No. 23
    • pp. 14613-15244

    Media

    Figures

    Tables

    Other

    Share

    Share

    Share article link

    Share on social media