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

Reiko Onuki, Akira Nagasaki, Hiroaki Kawasaki, Tadashi Baba, Taro Q.P. Uyeda, Kazunari Taira*

*この研究の対応する著者

研究成果: Article査読

102 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)14716-14721
ページ数6
ジャーナルProceedings of the National Academy of Sciences of the United States of America
99
23
DOI
出版ステータスPublished - 2002 11 12
外部発表はい

ASJC Scopus subject areas

  • 一般

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