title: Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate.
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.
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Fan
authors.initials: F
authors.lastname: Sun
authors.forename: Shintaro
authors.initials: S
authors.lastname: Mikuni
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D017209:Apoptosis; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D053148:Caspase 3 / Q000032:analysis* / Q000096:biosynthesis; D053453:Caspase 9 / Q000032:analysis* / Q000096:biosynthesis; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D053148:Caspase 3; D053453:Caspase 9; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000097573:Red Fluorescent Protein; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria