Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

• Sun, Fan

• Mikuni, Shintaro

• Kinjo, Masataka

抄録

Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.

収録刊行物

• Biochemical and Biophysical Research Communications

  Biochemical and Biophysical Research Communications 404 (2), 706-710, 2011-01-14

  Elsevier

キーワード

• FCCS
• Caspase inhibition
• Multi color
• Morphological change

詳細情報

• CRID: 1050282813983920256
• NII論文ID: 120002834708
• DOI: 10.1016/j.bbrc.2010.12.047
• HANDLE: 2115/44926
• ISSN: 0006291X
• Web Site: https://api.elsevier.com/content/article/PII:S0006291X10022850?httpAccept=text/xml
• Web Site: https://api.elsevier.com/content/article/PII:S0006291X10022850?httpAccept=text/plain
• 本文言語コード: en
• 資料種別: journal article
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