期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 324, 期 2, 页码 849-854出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2004.09.126
关键词
fluorescence cross-correlation spectroscopy; green fluorescent protein; monomeric red fluorescent protein; apoptosis-induced protease activation
Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells. (C) 2004 Elsevier Inc. All rights reserved.
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