The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish-5

<p><b>Copyright information:</b></p><p>Taken from "The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish"</p><p>http://www.biomedcentral.com/1471-2164/8/141</p><p>BMC Genomics 2007;8():141-141.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903365.</p><p></p>tracellular region. The double line and the dotted line indicate the transmembrane region and the death domain (DD) within the cytoplasmic region, respectively. (B) Alignment of Medaka and human FADD. The bold and dotted lines under the sequence indicate the death effector domain (DED) and the DD, respectively. (C) Alignment of Medaka and human caspase-8 (CASP8). The two bold lines and a box indicate the DEDs and the protease domain, respectively. Asterisks represent the amino acids essential for catalytic activity. Identical and similar amino acids between Medaka and human in all alignments are indicated in black and shaded boxes, respectively

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish-3

<p><b>Copyright information:</b></p><p>Taken from "The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish"</p><p>http://www.biomedcentral.com/1471-2164/8/141</p><p>BMC Genomics 2007;8():141-141.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903365.</p><p></p>ains. Human, chicken and zebrafish TRADD were used as the outgroup proteins for rooting the tree. The number noted at branches indicates the percentage of times that a node was supported in 1000 bootstrap pseudoreplications and are shown only greater than 50% for the bootstrap value. The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position. All of known or identified Fas and FADD proteins in ascidian (), catfish (), chicken(), human (), Medaka (), mouse (), stickleback (), West African clawed frog () and zebrafish () were included in this tree. (B) Multialignment of the C-terminus of FADD. Amino acids in a DD are indicated in shaded boxes. A phosphorylated serine residue in human and mouse FADD is indicated by a black box. Analyzed animals: human, mouse, chicken, frog, catfish, stickleback, zebrafish and Medaka

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish-1

<p><b>Copyright information:</b></p><p>Taken from "The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish"</p><p>http://www.biomedcentral.com/1471-2164/8/141</p><p>BMC Genomics 2007;8():141-141.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903365.</p><p></p>on-coding regions are indicated by black and white boxes, respectively. The majority of the exons of the Medaka gene were identified in the genomic sequence from scaffold1622 (exon 1: 72, 135-72, 214), scaffold28293 (exons 3 and 4: 5, 721-5, 396; exons 7–9: 4, 964-2, 872) and scaffold100324 (exons 5 and 6: 405-121). (B) Comparison of the splice junction sites in Medaka, human and chicken Fas. Arrowheads on amino acid alignment indicate the splice junction sites in the Medaka, human and chicken genes, respectively. (C) Genomic organization of the Medaka and human genes. Both the Medaka and human genes contain two exons. The black and white boxes correspond to the coding and non-coding regions, respectively. The gene was detected from the genomic sequence (23, 960-15, 238) of scaffold7231. (D) Comparison of the splice junction sites of FADD from several vertebrates. An arrowhead indicates the splice junction site, defined by the comparison of the cDNA and the genomic sequences of human (), mouse (), chicken(), frog (), zebrafish (), stickleback () and Medaka (). (E, F) Genomic organization of the Medaka and human genes. The Medaka gene was identified in the genomic sequence (1, 102, 834-1, 096, 083) of scaffold169 (E). The Medaka gene is composed of 12 exons, while the human gene is 9 exons. The coding and non-coding regions are indicated by black and white boxes, respectively. Comparison of the splice junction sites in Medaka, stickleback, zebrafish and human caspase-8 is shown in (F). Arrowheads on amino acid alignment indicate the splice junction sites in the Medaka, stickleback, zebrafish and human genes, respectively. Abbreviations: CRD, cysteine repeat domain; DD, death domain; DED, death effector domain; TM, transmembrane domain

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish-0

<p><b>Copyright information:</b></p><p>Taken from "The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish"</p><p>http://www.biomedcentral.com/1471-2164/8/141</p><p>BMC Genomics 2007;8():141-141.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903365.</p><p></p>tracellular region. The double line and the dotted line indicate the transmembrane region and the death domain (DD) within the cytoplasmic region, respectively. (B) Alignment of Medaka and human FADD. The bold and dotted lines under the sequence indicate the death effector domain (DED) and the DD, respectively. (C) Alignment of Medaka and human caspase-8 (CASP8). The two bold lines and a box indicate the DEDs and the protease domain, respectively. Asterisks represent the amino acids essential for catalytic activity. Identical and similar amino acids between Medaka and human in all alignments are indicated in black and shaded boxes, respectively

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish-4

<p><b>Copyright information:</b></p><p>Taken from "The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish"</p><p>http://www.biomedcentral.com/1471-2164/8/141</p><p>BMC Genomics 2007;8():141-141.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903365.</p><p></p> and EGFP/oCasp8 proteins. The chimeric h/oFas consists of the extracellular domain of human FAS and the transmembrane and cytoplasmic regions of Medaka Fas. The Flag/oFADD-EGFP construct translates both Flag-tagged Medaka FADD and EGFP molecules from a bicistronic mRNA. The EGFP/oCasp8 is a fusion of Medaka caspase-8 with EGFP at the N-terminus. (B) Cytotoxicity assays of chimeric Fas introduced into mouse NIH3T3 cells. Empty pME18S vector (panels a and b), pME18S-h/oFas (panels c, d and e) or pME18S-hFAS (panels f and g) was cotransfected transiently with pEGFP-C1 into NIH3T3 cells. After culture for 48 h, these transfectants were incubated for 14 h in the presence (panels b, d, e and g) or absence (panels a, c and f) of 500 ng/ml anti-human Fas antibody CH11. Cell viability was measured by detecting EGFP-positive cells by fluorescent microscopy. Arrows indicate dead cells. The typical dead cell exhibiting apoptotic bodies was magnified (panel e). (C) Immunocytochemical analysis of transfectants expressing h/oFas. pME18S-h/oFas (panels a and b) or pME18S-hFAS (panels c and d) were cotransfected transiently with phLBR1TM-EGFP into NIH3T3 cells. After culturing for 48 h, transfectants were incubated for 12 h in the presence (panels b and d) or absence (panels a and c) of CH11. Activated Casp3 in cells expressing EGFP in the nucleus was visualized by staining with anti-cleaved Casp3 and fluorescently-labeled secondary antibodies. After counterstaining with DAPI, cells were photographed by fluorescent microscopy. Arrows indicate transfectants. (D) Cytotoxicity assays of Medaka FADD-expressing mammalian cell lines. The pME18S-Flag/oFADD-EGFP plasmid was transfected into HeLa cells (panels a and b) and wild-type (panel c) or -deficent (panel d) MEF cells. Half of the HeLa transfectants were cultured in the presence of 100 μM zVAD-fmk (panel b). After 24 h of culture, cells were washed, fixed, and examined by fluorescence microscopy. Viable cells were defined as EGFP-positive cells, while typical dead cells are shown by arrows. Abbreviations: WT, wild-type; Casp8-KO, -deficient. (E) Cytotoxicity assays of Medaka Casp8-expressing HeLa cells. The pCMV-EGFP/oCasp8 construct, encoding EGFP/oCasp8, was transfected into HeLa cells alone (panels a, b and d) or in conjunction with pCX-CrmA that encoded CrmA (panel c). Half of transfectants expressing EGFP/oCasp8 alone were incubated with 100 μM zVAD-fmk (panels b and d). After 24 h of culture, transfectants were washed, fixed, and examined by fluorescence microscopy. Viable cells were defined as EGFP-positive cells. Surviving cells expressing EGFP/oCaspa8 were examined by confocal laser scanning microscopy (panel d). In panel d, a dotted line demarks the edge of a single cell. (F) The DNA content of transfectants expressing Medaka FADD or caspase-8 was assessed by flow cytometry. Twenty-four hours after transfection, the DNA content of cells transfected with pME18S (panel a), pME18S-Flag/oFADD-EGFP (panels b and c), pCMV-EGFP/oCasp8 (panels d and e) together with pCX-p35 (panels c and e) was analyzed by staining with PI. The percentage indicates the cellular population with sub-GDNA content

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

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

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

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

Estimation of the diffusion coefficient of CASP8CS/mKikGR.

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