A mathematical model on the propagation of CASP8 activation.

<p>(<b>A</b>–<b>C</b>) The propagation of CASP8 activation was simulated by an one-dimensional diffusion model. The vertical axis (<i>u</i>) indicates the concentration of the activated CASP8. The horizontal axis (<i>x</i>) indicates the distance from the input source of the apoptotic signal and time zero means the starting point of the cell-cell interaction. CASP8 activation propagates from the left to the right in the graph. Each colored line in the figures gives the spatial distribution of activated CASP8 for different time, where time zero is the starting point of the cell-cell interaction. A blue line indicates the start time when the apoptotic signal is inputted. A yellow line indicates 2000 unit time that has passed from the start time. The duration (<i>τ</i>) of the input signal and was set to 100 (A), 500 (B), and 1000 (C) and <i>f</i>₀ = 10. Here, diffusion coefficient is set to <i>D</i> = 1 and the cell size is set to <i>L</i> = 200.</p

Estimation of the diffusion coefficient of CASP8CS/mKikGR.

<p>Estimation of the diffusion coefficient of CASP8CS/mKikGR.</p

Monitoring of caspase activation with the CYR83 in single cells.

<p>(<b>A</b>) 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>B</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s003" target="_blank">Figure S3</a>, 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. (<b>C</b>) 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. (<b>D</b>, <b>E</b>) 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. <b>(F</b>, <b>G)</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s005" target="_blank">Figure S5A–C and S5E–G</a>, three bands corresponding to each seCFP, Venus and mRFP1 peptide fragments were chosen and represented as (F) and (G).</p

Effects of the downregulation of CASP8 on the processing of the SCAT8.1 biosensor.

<p>(<b>A</b>) Immunoblot analyses of CASP8 proteins downregulated by RNA interference. Cell extracts from HeLa cells carrying either pCSIIU6Tet-Neo or pCSIIU6Tet-sh<i>CASP8</i>-Neo were prepared and analyzed by SDS-PAGE, followed by immunoblotting with anti-CASP8 and anti-actin antibodies, respectively. The numbers indicate the ratio of endogenous CASP8 between two stable lines by calculating the measurements of CASP8 and actin using an image analyzer. (<b>B</b>) Immunoblot analyses of CASP8-knockdown cells subjected to intrinsic apoptotic stimuli. Cell extracts from parental HeLa cells (lanes 1–4) or HeLa/CASP8-KD cells (lanes 5–8) expressing SCAT8.1 were prepared at the indicated times after UV-irradiation. Endogenous CASP3, PARP, actin and exogenous SCAT8.1 were examined with indicated specific antibodies. A representative of four independent experiments is shown. Arrows indicate intact proteins while arrowheads identify the processed peptide fragments. (<b>C</b>) The static analysis of the immunoblot data. The relative ratio of processed peptide fragments to total SCAT8.1 proteins was estimated by measuring the intensity of immunoblot bands in four independent experiments using an image analyzer. The ratio of the processed form relative to total CASP3 proteins was also shown as percentage. The both graphs indicate the means and standard deviations of the estimated ratios. Significant differences between the two groups were evaluated by Student’s <i>t</i>-test. An asterisk shows p < 0.05. (<b>D</b>) Immunoblot analyses of HeLa cells overexpressing CASP6 or its mutant. HeLa cells were transiently transfected either with plasmids carrying CASP6 (lanes 5–8), CASP6CS (lanes 9–12) or control vector (lanes 1–4), and cell extracts were prepared at indicated times after UV-irradiation. Endogenous CASP3, PARP, actin and exogenous CASP6 and SCAT8.1 were examined with indicated specific antibodies. A representative of four independent experiments is shown. The arrow and arrowhead indicate the full-length and the cleaved form of proteins examined, respectively. (<b>E</b>) The static analysis of the immunoblot data. Both ratio and percentage of processed fragments to total SCAT8.1 and processed CASP3 to total CASP3 were estimated and shown as the bar graph. Statistical validation was performed by Student’s <i>t</i>-test. *p < 0.05, **p < 0.01.</p

Characterization of the dual-FRET biosensor, CYR83.

<p>(<b>A</b>) A schematic structure of a FRET-based biosensor, CYR83. CYR83 consists of three fluorescent proteins, seCFP (CFP), Venus and mRFP1 (RFP); the linker portion contains two distinct caspase cleavage sequences, IETD and DEVD. (<b>B</b>) Induction of a conformational change of CYR83 by active caspases. The emission spectra of CYR83 were measured by exciting at 440 nm (left panels) or 500 nm (right panels) with a spectrophotometer before or at indicated time of incubation with recombinant active CASP8 (upper panels) or active CASP3 (lower panels). Arrows indicate the major peaks of fluorescent proteins (seCFP, Venus and mRFP1), and the direction reveals the dynamics of the fluorescence intensity. The numbers in the inset box indicate the emission ratio on 528 nm/476 nm and 608 nm/476 nm or 608 nm/528 nm before and after 120 min of incubation with active caspases. (<b>C</b>, <b>D</b>) <i>In vitro</i> cleavage assay of CYR83 by active caspases. Fluorescence data shown in panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s001" target="_blank">Figure S1A</a>–F were merged and represented as (C) and (D). Lower-case characters indicate full length CYR83 (a) and the cleaved peptide fragments (b-f) during incubation with active caspases. Numbers indicate incubation time. Abbreviations; M, FITC-conjugated molecular weight markers; C, seCFP; V, Venus; R, mRFP1. (<b>E</b>) Profiles of the products cleaved by active caspases. Fluorescent bands corresponding to the processed seCFP, Venus, mRFP1, seCFP-Venus and Venus-mRFP1 and the intact CYR83 were extracted from the fluorescence images shown in (C) and (D).</p

Visualization of the diffusional behavior of CASP8 fused with a photoconvertible fluorescent protein.

<p>(<b>A</b>) A schematic structure of a fusion protein, CASP8CS/mKikGR. CASP8CS/mKikGR encodes 735 amino acids and consists of human CASP8CS and a photoconvertible fluorescent protein, mKikGR, which were linked with triple GGGS repeats. CASP8CS is a mutant with a cysteine-to-serine (C360S) substitution in the active site of the protease domain. (<b>B</b>) Immunoblot analysis. Exogenous CASP8CS/mKikGR and endogenous CASP8 in cell extracts of HEK293 transfected cells were detected by SDS-PAGE, followed by immunoblotting with anti-CASP8 antibody. (<b>C</b>) Confocal images showing diffusion of photoconverted CASP8CS/mKikGR. Images for original green (upper panels) and photoconverted red (lower panels) fluorescence of CASP8CS/mKikGR were taken every 0.953 sec. A red arrow indicates the irradiation timing by 405 nm laser beam. Scale bars, 10 µm. (<b>D</b>) A magnified view of the green fluorescence image. An orange circle (diameter 4.140 µm) represents the irradiated area (IA). The black, red, and blue circles (diameter 1.656 µm) represent ROIs marked for the measurement of fluorescence intensity. ROI 1 was set in the photoconverted region and ROI 2 and 3 were placed outside of the photoconverted region. The difference between the center-to-center distance of IA-ROI 2 and IA-ROI 3 is 1.445 µm. (<b>E</b>) Time course of the photoconverted red fluorescence intensity of CASP8CS/mKikGR (left) and the red to green emission ratio (right) in the ROIs 1–3.</p

Graded activation of CASP8 in response to a focal apoptotic stimulus.

<p>(<b>A</b>) A schematic illustration indicating the propagation of apoptotic signals associated with CASP8 activation. Through focal stimulation by cell-to-cell interactions between an effector WD4 cell (expressing FasL), and a recipient HeLa cell, CASP8 activation occurs at the DISC, and then expands to the whole cell via the caspase cascade and the reaction-diffusion system, as illustrated in the steps (1) to (5). Spreading of active CASP8 was indicated by a red zone. (<b>B</b>) Serial images of a HeLa cell undergoing apoptosis induced by cell-to-cell interactions with an effector cell. The HeLa cell (green) expressing a membrane-bound FRET-based biosensor, GsSCAT8, was induced to undergo cell death through contact with a single WD4 cell (red). Numbers indicate time after taking the first image. (<b>C</b>) A FRET image of a HeLa cell expressing GsSCAT8. A red circle indicates the position of an overlaid WD4 cell. (<b>D</b>) The dynamics of CASP8 activation in dying HeLa cells, observed using GsSCAT8. The fluorescence intensity on the cell surface of a HeLa cell shown in (C) was captured over time after overlaying a WD4 cell. Serial images are displayed using pseudo colors based on calculation of the emission ratio. Numbers indicate time after taking the first image. A representative cell from 24 cells examined in seven independent experiments is shown. Scale bars, 10 µm. (<b>E</b>) The graphic patterns of the FRET ratio on seven points marked with a cross shown in (C). An arrow indicates onset of the drop of the FRET ratio in the point “g”.</p

Functional reconstitution of the caspase cascade <i>in vitro</i>.

<p>(<b>A</b>) A schematic structure of FRET-based probes SCAT3.1 and SCAT8.1. Both SCAT3.1 and SCAT8.1 are fusion molecules consisting of seCFP and Venus and containing either the CASP3 recognition sequence (DEVD) or the CASP8 recognition sequence (IETD) in the linker portion. (<b>B</b>) <i>In vitro</i> cleavage assay of the FRET-based probes with recombinant proteins. Various reaction mixtures containing either SCAT3.1 or SCAT8.1 combined with several caspases were incubated at 37°C for 2 h, and the cleaved products were analyzed by SDS-PAGE followed by scanning of the fluorescence in the gel. The arrow and arrowheads indicate the full-length and cleaved form of the probes, respectively. (<b>C</b>) Immunoblot analyses for monitoring the processing profile of procaspases in the reaction mixtures. After incubation for 2 h, the processing pattern of procaspases in the mixture was analyzed by SDS-PAGE following immunoblotting with antibodies against CASP3, CASP6 or CASP8. The arrow indicates the full-length procaspase and the white and black arrowheads indicate the completely and partially cleaved fragments, respectively. The star indicates active CASP8 included in the mixture in advance and the asterisk shows a non-specific reaction. Abbreviations: W, wild-type pro-CASP3; M, the protease-defect pro-CASP3 mutant.</p