Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate |
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Authors: | Sun Fan Mikuni Shintaro Kinjo Masataka |
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Institution: | Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Kita-21 Nishi-11 Kita-ku, Sapporo 001-0021, Japan |
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Abstract: | 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. |
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Keywords: | Abbreviations: FCS fluorescence correlation spectroscopy FCCS fluorescence cross-correlation spectroscopy TNF-α tumor necrosis factor-α CHX cycloheximide LSM laser scanning fluorescence microscopy RCA relative cross-correlation amplitude |
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