Monitoring of caspase activation with the CYR83 in single cells.
(A) A schematic structure of CYR83 and its variants. In CYR83(IETA) variant, the IETD sequence was replaced by IETA; in the CYR83(DEVA) variant, DEVD was replaced by DEVA. (B) The graphic pattern of the emission ratio based on the fluorescence intensity of the CYR83 in single cells undergoing apoptosis. The CYR83-expressing HeLa cells were induced to undergo apoptosis with an agonistic anti-Fas antibody and monitored by dual-FRET. As shown in Figure S3, time course was set up before and after 1 h of cell shrinkage. The temporal fluctuations of the emission ratio on Venus/seCFP and mRFP1/Venus in single cells are plotted as red and blue lines, respectively. The IETDase and DEVDase activities are inversely proportional to graphic data. The arrow indicates a rebound detected by monitoring the fluorescence. (C) The CYR83-expressing HeLa cells were induced to undergo apoptosis by UV-irradiation and monitored by dual-FRET. A time course of the emission ratio is indicated. (D, E) HeLa cells expressing CYR83 variants were monitored for fluorescence. Transfected cells expressing CYR83(IETA) (D) or CYR83(DEVA) (E) were treated with an anti-Fas antibody and monitored for fluorescence. (F, G) Fluorescence image analyses on the proteolytic processing profiles of CYR83 and its variants. HeLa cells expressing CYR83 or its variants were subjected to extrinsic (F) and intrinsic (G) apoptotic stimuli at indicated times. Cell extracts prepared from those cells were resolved by SDS-PAGE and scanned for fluorescence in the gel using the imaging analyzer. Among fluorescence bands detected in panels of Figure S5A–C and S5E–G, three bands corresponding to each seCFP, Venus and mRFP1 peptide fragments were chosen and represented as (F) and (G).