1. Cancer Biology
  2. Cell Biology
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Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression

  1. Linda Julian
  2. Gregory Naylor
  3. Grant R Wickman
  4. Nicola Rath
  5. Giovanni Castino
  6. David Stevenson
  7. Sheila Bryson
  8. June Munro
  9. Lynn McGarry
  10. Margaret Mullin
  11. Alistair Rice
  12. Armandodel Del Río Hernández
  13. Michael F Olson  Is a corresponding author
  1. Cancer Research United Kingdom Beatson Institute, Garscube Estate, United Kingdom
  2. Institute of Cancer Sciences, University of Glasgow, United Kingdom
  3. Department of Chemistry and Biology, Ryerson University, Canada
  4. Electron Microscopy Facility, School of Life Sciences, University of Glasgow, United Kingdom
  5. Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, United Kingdom
Research Article
Cite this article as: eLife 2021;10:e61983 doi: 10.7554/eLife.61983
8 figures, 2 videos, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
ROCK1 cleavage is essential for increased myosin light chain (MLC) phosphorylation during apoptosis.

(A) Representative western blot showing Myc-tagged ROCK1 immunoprecipitated from transfected HEK293T cells assayed in vitro for MLC phosphorylation (pMLC) without (-) or with (+) cleavage by recombinant active caspase 3. KD: kinase-dead (K105A; Ishizaki et al., 1997); CA: catalytically active (ROCK1Δ2; Ishizaki et al., 1997). Means ± SEM pMLC/MLC relative to KD levels, n = 3 independent replicates. Pairwise Student’s t-test between indicated conditions; ns: not significant. (B) Homozygous ROCK1wt (green) or ROCK1nc (red) mouse embryo fibroblasts (MEFs) were serum-starved overnight, then left untreated or stimulated for 5 min with medium containing 10% fetal bovine serum (FBS) without or with 10 µM Y27632 as indicated. Fixed cells were stained for pMLC and DNA using DRAQ5, and levels quantified by infrared quantitative imaging. Means ± SEM. pMLC/DRAQ5 levels relative to starved condition for each genotype, n = 3 independent replicates. Pairwise Student’s t-test between indicated conditions. (C) Homozygous ROCK1wt (WT) or ROCK1nc (NC) MEFs were serum-starved overnight, then left untreated or treated with tumour necrosis factor α (TNFα) (50 ng/mL) plus cycloheximide (CHX; 10 µg/mL) for 4 hr. Representative blot shows quantitative western blot of ROCK1, cleaved ROCK1 (ΔROCK1), PARP1, cleaved PARP1 (ΔPARP1) α-tubulin, cleaved caspase 3 and pMLC as indicated, using corresponding infrared fluorophore-conjugated secondary antibodies. Means ± SEM. pMLC/α-tubulin levels relative to starved condition for each genotype, n = 5 independent replicates. Pairwise Student’s t-test between indicated condition; ns: not significant. (D) Homozygous WT or NC MEFs were serum-starved overnight, then left untreated or treated with TNFα plus CHX for 4 hr to induce apoptosis. Phosphatidylserine externalization was measured using FITC-labelled Annexin V flow cytometry. Means ± SEM percentage FITC-labelled Annexin V-positive cells, n = 3 independent replicates. Pairwise Student’s t-test within conditions between genotypes; ns: not significant. (E) Homozygous WT or NC MEFs were serum-starved overnight, then left untreated or treated with TNFα plus CHX or anti-CD95 antibody (2 µg/mL) plus CHX (10 µg/mL) to induce apoptosis. Caspase activity was determined after 4 hr by Caspase-Glo 3/7 assays. Means ± SEM relative luminescence units (RLUs), n = 4 independent replicates. Pairwise Student’s t-test within conditions between genotypes; ns: not significant. (F) Homozygous WT or NC MEFs were serum-starved overnight, then left untreated or treated with TNFα plus CHX or anti-CD95 antibody plus CHX to induce apoptosis. Lactate dehydrogenase (LDH) activity was measured after 8 or 24 hr by colorimetric assay. Means ± SEM percentage LDH activity in media normalized to cell density determined using crystal violet, then relative values calculated using the 8 hr starved WT condition as reference, n = 4 independent replicates. Pairwise Student’s t-test between indicated conditions; ns: not significant. (G) Homozygous ROCK1wt or ROCK1nc MEFs were serum-starved overnight, then left untreated or treated with TNFα plus CHX for 4 or 24 hr. Left panel: representative western blot shows cleaved PARP1 and α-tubulin in cell lysates 4 hr after TNFα plus CHX treatment. Right panel: representative western blot shows recombinant GST-PAK2 used as a control for the efficiency of protein recovery with StrataClean resin, and HMGB1 in conditioned media 24 hr after TNFα plus CHX treatment.

Figure 1—figure supplement 1
Uncropped western blots and in-cell westerns.

(A) Uncropped western blot panels for Figure 1A, boxes with dashed border indicating regions used for the composite. (B) Uncropped in-cell westerns for Figure 1B, boxes with dashed border indicating regions used for the composite. (C) Uncropped western blot panels for Figure 1C, boxes with dashed border indicating regions used for the composite. (D) Uncropped western blot panels for Figure 1G, boxes with dashed border indicating regions used for the composite.

Figure 1—figure supplement 2
Homologous recombination targeting strategy to generate ROCK1 non-cleavable allele.

(A) Line diagram of mouse genomic Rock1 wild-type (ROCK1wt) locus with numbered Rock1 exons indicated by blue boxes (top). Targeting vector homology arms to Rock1 wild-type locus is indicated by black crosses. Targeting vector contains mutations 3338A>C and 3339T>A in Rock1 exon 27 (yellow box, indicated by red asterisk) and a PGK neomycin (neobPA) selection cassette in red flanked by LoxP sequences (blue triangles). Mouse embryonic stem cells (mESCs) were transfected with the targeting vector and then selected for stable vector insertion with neomycin. After homologous recombination, the mutant Rock1 non-cleavable (ROCK1nc) genomic locus is shown on the bottom (ROCK1nc+/- Neo+/-). (B) Line diagram of PCR screening strategy of genomic DNA from neomycin-resistant mESC. 5′ PCR primer is indicated with black arrow and is within the Neo cassette, 3′ primer is outside the targeting vector homology arm. These primers generate a 3.5 kb PCR product from mESCs with correct 3′ recombination, while no product should be evident from wild-type ROCK1 or incorrect targeting vector insertion (in table). Lower panel: representative agarose gel electrophoresis of PCR products from the genomic screening reactions. Each lane represents a separate reaction from individual neomycin-resistant clones. PCR reaction from clone 6b (highlighted in red) yielded the expected reaction product of 3.5 kb, suggesting correct 3′ homologous recombination while the remaining clones were negative. In total, 200 clones were screened with three testing positive. (C) Line diagram of secondary PCR screening strategy of genomic DNA from neomycin-resistant mESCs. 5′ PCR primer (indicated with black arrow) is outside the targeting vector homology arm, and the 3′ primer is within the Neo selection cassette. These primers generate a 5.5 kb PCR reaction product from mESCs with correct 5′ recombination, while no product should be evident from wild-type ROCK1 or incorrect vector insertion (in table). Right panel: representative agarose gel electrophoresis of PCR products from clones with correct 3′ homologous recombination (6b, 7h, and 4g). Clones 6b and 7h both produced a PCR product of expected size while clone 4g had no reaction product. mESC clones 6b and 7h demonstrate correct homologous recombination of the mutant ROCK1nc targeting vector. (D) Summary of offspring genotypes from ROCK1nc heterozygous matings (n = 129 animals from four mating pairs).

Figure 1—figure supplement 3
Example fluorescence-activated cell sorting (FACS) determination of ROCK1wt and ROCK1nc mouse embryo fibroblast (MEF) apoptosis.

Representative FACS dot plots of ROCK1wt and ROCK1nc MEFs that were untreated or treated with tumour necrosis factor α (TNFα) and cycloheximide (CHX) for 4 hr. Cells were stained with FITC-conjugated Annexin V to detect externalization of phosphatidylserine and with propidium iodide (PI) to determine membrane integrity.

Figure 2 with 1 supplement
Apoptotic ROCK1nc mouse embryo fibroblasts (MEFs) do not undergo forceful contraction.

(A) Homozygous ROCK1wt (upper panels) or ROCK1nc (lower panels) MEFs were serum-starved overnight, then treated with tumour necrosis factor α (TNFα) (50 ng/mL) plus cycloheximide (CHX) (10 µg/mL) to induce apoptosis. Differential interference contrast time-lapse microscopy images were acquired at 3 min intervals after the addition of TNFα plus CHX. Scale bar = 50 µm. Right panels: scanning electron microscope images. Scale bar = 10 µm. (B) Schematic diagram of a cell on elastic micropillars. (C) Homozygous ROCK1wt MEFs plated on elastic micropillars were serum-starved overnight, then treated with TNFα plus CHX to induce apoptosis. Brightfield time-lapse microscopy images were acquired at 2 s intervals after the commencement of cell contraction over 10 min. Right panel: force heat map indicating the magnitude of the force applied to each individual pillar. Scale bar = 10 µm. (D) Homozygous ROCK1wt (upper panels) or ROCK1nc (lower panels) MEFs were serum-starved overnight, then treated with TNFα plus CHX to induce apoptosis. Brightfield time-lapse microscopy images were acquired at 2 s intervals after the commencement of cell contraction over 10 min. Right panels: force vector map with direction and magnitude of the forces applied to each individual pillar. Scale bar = 20 nN. Mean maximum force is the average of the maximum forces applied to each pillar over 10 min. (E) Mean maximum force for untreated or TNFα plus CHX-treated homozygous ROCK1wt (green) or ROCK1nc (red) MEFs. Means ± SD n=27–50 individual cells. One-way ANOVA with post-hoc Tukey’s multiple comparison test; ns: not significant.

Figure 2—figure supplement 1
Characterization of ROCK1wt and ROCK1nc mouse embryo fibroblasts (MEFs).

(A) Cell numbers after plating 4 × 103 ROCK1wt and ROCK1nc MEFs at daily intervals up to 4 days. Means ± standard deviation, n = 5 independent replicates. (B) Example images used for high content analysis. F-actin staining with Oregon Green conjugated phalloidin, phosphorylated myosin light chain (MLC) (Ser19) staining with mouse primary antibody and AF594-conjugated (red) goat anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). High content image analysis of ROCK1wt and ROCK1nc MEFs was performed using an Operetta high content imaging system as described in George et al., 2007 to determine (C) nuclear area, (D) nuclear roundness, (E) nuclear width, (F) nuclear length, (G) nuclear width to length ratio, (H) cell area, (I) cell roundness, (J) cell width, (K) cell length and (L) cell width to length ratio. Means ± SEM, n = 3 independent replicates.

Figure 3 with 2 supplements
Elevated chemically induced liver damage in ROCK1nc mice.

(A) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of apoptotic cells in representative liver sections from ROCK1wt or ROCK1nc mice 72 hr after diethylnitrosamine (DEN) injection. TUNEL-positive cells (lower panel, green) were scored as a percentage of the total 4′,6-diamidino-2-phenylindole (DAPI; upper panel, blue) cell numbers from 10 random fields per section per mouse. Scale bar = 100 µm. Right panel: means ± SEM, n = 3–11 mice at each time. Pairwise Student’s t-test at each time. (B) Haematoxylin and eosin (H&E) staining of representative liver sections from ROCK1wt or ROCK1nc mice 72 hr after DEN injection. Scale bar = 100 µm. Liver necrotic areas were scored as a percentage of the total area from 10 random fields per section per mouse. Right panel: means ± SEM, n = 3–5 mice at each time. Pairwise Student’s t-test at each time. (C) Sera from 10-week-old homozygous ROCK1wt (WT) and ROCK1nc (NC) mice treated with DEN (100 mg/kg body weight) were assessed for alanine transaminase and (D) bilirubin levels at indicated times after intraperitoneal injection. Means ± SEM, n = 6–11 mice at each time. Pairwise Student’s t-test at each time.

Figure 3—figure supplement 1
Absence of indications of autoimmunity in aged ROCK1wt or ROCK1nc kidneys or spleens.

(A) Representative periodic acid-Schiff (PAS)-stained kidney sections from ROCK1wt and ROCK1nc mice showing no observable differences in basement membrane thickening (white arrows). Scale bars represent 100 µm. (B) Representative Perls Prussian blue-stained spleen sections showing no observable differences in hemosiderin deposition between the two genotypes. Scale bars represent 500 µm.

Figure 3—figure supplement 2
Ageing does not affect blood cells, liver or spleen masses differently in ROCK1wt or ROCK1nc mice.

Numbers of (A) red blood cells, (B) white blood cells, (C) neutrophils, (D) lymphocytes, (E) monocytes, (F) eosinophils, or (G) platelets in ROCK1wt or ROCK1nc mice 10 weeks (ROCK1wt n = 11 mice; ROCK1nc n = 10 mice) or 1 year (ROCK1wt n = 9 mice; ROCK1nc n = 15 mice) after birth. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles. (H) Spleen to body mass ratios and (I) liver to body mass ratios for ROCK1wt (n = 15 mice) and ROCK1nc (n = 18 mice) 1 year after birth. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles.

Higher neutrophil infiltration in chemically damaged ROCK1nc livers.

(A) Immunohistochemical (IHC) staining with F4/80 antibody of formalin-fixed paraffin-embedded (FFPE) liver sections from homozygous ROCK1wt (WT) or ROCK1nc (NC) mice 72 hr after diethylnitrosamine (DEN) injection. Scale bar = 100 µm. F4/80-positive macrophages were scored as number per mm2 in five random fields per section per mouse. Right panel: means ± SEM, n = 3–11 mice at each time. (B) IHC staining with anti-S100A9 antibody of FFPE liver sections from ROCK1wt or ROCK1nc mice 72 hr after DEN injection. Scale bar = 100 µm. S100A9-positive neutrophils were scored as number per mm2 in five random fields per section per mouse. Right panel: means ± SEM, n = 3–11 mice at each time. Pairwise Student’s t-test at each time. (C) Homozygous WT or NC mice were treated with 1 mg zymosan by intraperitoneal injection. At the indicated times after injection, the numbers of neutrophils, (D) white blood cells, (E) monocytes and (F) lymphocytes in the peritoneal cavity were determined. Boxes indicate minimum to maximum values with median designated by a line, n = 4 WT mice, 3 NC mice. Pairwise Student’s t-test within times between genotypes; ns: not significant.

Inhibition of high-mobility group B1 (HMGB1) signalling reduces diethylnitrosamine (DEN)-induced liver damage.

(A) Immunohistochemical (IHC) staining with anti-HMGB1 antibody of formalin-fixed paraffin-embedded (FFPE) liver sections from homozygous ROCK1wt (WT) or ROCK1nc (NC) mice 72 hr after DEN injection. White box indicates magnified area shown to the right of each image. Scale bar = 100 µm. Right panel: cells showing nucleus to cytoplasm translocation were scored as a percentage of the total cell numbers from five random fields per section per mouse. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, n = 5 mice. Student’s t-test. (B) IHC staining with anti-S100A9 antibody of FFPE liver sections from ROCK1nc mice 72 hr after DEN injection alone or with glycyrrhizin (GLZ; 200 mg/kg) or CLI095 (3 mg/kg). Scale bar = 100 µm. S100A9-positive neutrophils 72 hr after DEN injection alone, or with GLZ or CLI095 (n = 10 mice for each condition), were scored as number per mm2 in five random fields per section per mouse. Right panel: boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise one-tailed Student’s t-test as indicated. (C) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of apoptotic cells in representative liver sections from ROCK1nc mice 72 hr after DEN injection alone, or with GLZ or CLI095. Right panel: TUNEL-positive cells (green) were scored as a percentage of the DAPI-positive cells (blue) from 10 random fields per section per mouse following DEN injection alone or with GLZ or CLI095 (n = 10 mice for each condition). Scale bar = 100 µm. Right panel: boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise one-tailed Student’s t-test as indicated. (D) Serum alanine transaminase levels in ROCK1nc mice 72 hr after DEN injection alone (n = 8 mice) or with GLZ (n = 10 mice) or CLI095 (n = 5 mice). Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise one-tailed Student’s t-test as indicated. (E) F4/80-positive macrophages in ROCK1nc mouse livers 72 hr after DEN injection alone or with GLZ or CLI095 (n = 10 mice for each condition), scored by IHC as number per mm2 in five random fields per section per mouse. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise one-tailed Student’s t-test as indicated; ns: not significant.

Diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) tumour numbers are lower in ROCK1nc mice.

(A) Timeline of DEN-induced HCC protocol. (B) Macroscopic view of representative livers from ROCK1wt and ROCK1nc mice 9 months after HCC initiation with DEN. Scale bars = 1 cm. (C) Tumour numbers were counted in ROCK1wt and ROCK1nc livers 6 months (n = 5 mice for each genotype) or 9 months (12 mice for each genotype) after HCC initiation with DEN. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated. (D) Total tumour volume per ROCK1wt or ROCK1nc liver 6 months (n = 5 mice for each genotype) or 9 months (12 mice for each genotype) after HCC initiation with DEN. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated; ns: not significant. (E) Representative H&E-stained lung sections from ROCK1wt and ROCK1nc mice 9 months after HCC initiation with DEN. Black arrow indicates metastatic nodules. Scale bars = 100 µm. Numbers of lung metastases were counted in ROCK1wt and ROCK1nc mice 6 months (n = 5 mice for each genotype) or 9 months (12 mice for ROCK1wt, 7 mice for ROCK1nc) after HCC initiation with DEN. Right panel: boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated; ns: not significant.

Figure 7 with 1 supplement
Similar liver mass and tumour grades in diethylnitrosamine (DEN)-treated ROCK1wt and ROCK1nc mice.

(A) Liver mass, (B) body mass and (C) liver to body ratios for ROCK1wt and ROCK1nc mice (n = 12 mice for each genotype) 9 months after hepatocellular carcinoma (HCC) initiation with DEN. Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated; ns: not significant. (D) Tumour classification in ROCK1wt (left) and ROCK1nc (right) mice (n = 12 mice for each genotype) 9 months after HCC initiation with DEN. H&E-stained sections liver tumours were classified into three types: hepatic nodule 1 (HN1), hepatic nodule 2 (HN2) and HCC in increasing order of tumour grade as described by Yanagi et al., 1984. HN1 mainly comprised tumour nodules with slight difference from the normal hepatic architecture but produced obvious compression of the surrounding parenchyma. HN2 consisted of two- to three-cell thick irregular trabecular arrangements with well-maintained sinusoidal spaces. HCC exhibited irregular macrotrabecular arrangement with dilated sinusoidal spaces. (E) Photomicrographs of liver sections from ROCK1wt and ROCK1nc mice 9 months after DEN injection stained with Sirius Red and counterstained with Fast Green. Scale bars represent 100 µm. Percentage fibrotic area measured as Sirius Red positive area relative to the total area analysed, showing no significant difference between the two genotypes (ROCK1wt n = 8 mice; ROCK1nc n = 7 mice). Right panel: boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated; ns: not significant.

Figure 7—figure supplement 1
Histopathological features of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC).

Representative H&E-stained liver sections from mice with DEN-induced HCC showing (A) tumour border (dotted line), (B) trabecular pattern (arrow), (C) macrotrabecular structures (arrow), (D) compact tumour mass, (E) necrotic foci (arrow) and (F) inflammatory foci (arrow). Scale bars represent 100 μm.

Figure 8 with 1 supplement
Glycyrrhizin (GLZ) increases diethylnitrosamine (DEN)-induced liver tumour number in ROCK1nc mice.

(A) Timeline of the DEN-induced hepatocellular carcinoma (HCC) and GLZ treatment protocol. (B) The number of liver tumours in ROCK1nc mice 9 months after treatment with GLZ alone (n = 10 mice) or after HCC initiation with DEN alone (n = 14 mice) or with GLZ (n = 10 mice). Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated. (C) Numbers of lung metastases in ROCK1nc mice 9 months after treatment with GLZ alone (n = 10 mice) or after HCC initiation with DEN alone (n = 14 mice) or with GLZ (n = 10 mice). Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles, pairwise Student’s t-test as indicated; ns: not significant. (D) Schematic diagram depicting how acute acting neutrophils influence tumour development. ROCK1wt mice had fewer recruited neutrophils and less tissue damage than ROCK1nc mice 3 days after equivalent DEN treatment. The healthier ROCK1wt liver tissue enabled more DEN-mutated hepatocytes to initiate the HCC tumours that were observed 9 months later. Following GLZ administration, ROCK1nc mice resembled ROCK1wt mice with fewer neutrophils and less tissue damage acutely, and more HCC tumours 9 months later. Figure created with BioRender.com.

Figure 8—figure supplement 1
Similar liver mass in ROCK1nc mice treated with diethylnitrosamine (DEN) and glycyrrhizin (GLZ).

(A) Liver mass, (B) body mass and (C) liver to body mass ratios for ROCK1nc mice 9 months after treatment with GLZ alone (n = 10 mice) or following hepatocellular carcinoma (HCC) initiation with DEN alone (n = 14 mice) or with GLZ (n = 10 mice). Boxes indicate upper and lower quartiles and median, whiskers indicate 5–95% percentiles.

Videos

Video 1
Time lapse of apoptotic ROCKwt mouse embryo fibroblasts (MEFs).

ROCK1wt MEFs were plated on 6-well glass-bottom dishes at a density of 2 × 106 cells per well. Cells were serum-starved overnight prior to induction of apoptosis with tumour necrosis factor α (TNFα) (50 ng/mL) and cycloheximide (10 µg/mL) diluted in serum-free starvation medium. Differential interference contrast (DIC) time-lapse microscopy images were acquired with a 20× DIC objective using a Nikon TE 2000 microscope with a heated stage and 5% CO2 gas line. Immediately after induction of apoptosis the dishes were transferred to the microscope and time-lapse images were taken each minute.

Video 2
Time lapse of apoptotic ROCKnc mouse embryo fibroblasts (MEFs).

ROCK1nc MEFs were plated on 6-well glass-bottom dishes at a density of 2 × 106 cells per well. Cells were serum-starved overnight prior to induction of apoptosis with tumour necrosis factor α (TNFα) (50 ng/mL) and cycloheximide (10 µg/mL) diluted in serum-free starvation medium. Differential interference contrast (DIC) time-lapse microscopy images were acquired with a 20× DIC objective using Nikon Eclipse TI microscope with a heated stage and 5% CO2 gas line. Immediately after induction of apoptosis the dishes were transferred to the microscope and time-lapse images were taken each minute.

Tables

Table 1
Conservation of ROCK1 caspase cleavage site.
Mammals
Homo sapiensQLRAKLLDLSDSTSVASFPSADETDGNLP
ChimpQLRAKLLDLSDSTSVASFPSADETDGNLP
GorillaQLRAKLLDLSDSTSVASFPSADETDGNLP
OrangutanQLRAKLLDLSDSTSVASFPSADETDGNLP
GibbonQLRAKLLDLSDSTSVASFPSADETDGNLP
RhesusQLRAKLLDLSDSTSVASFPSADETDGNLP
Crab-eating macaqueQLRAKLLDLSDSTSVASFPSADETDGNLP
BaboonQLRAKLLDLSDSTSVASFPSADETDGNLP
Green monkeyQLRAKLLDLSDSTSVASFPSADETDGNLP
MarmosetQLRAKLLDLSDSTSVASFPSADETDGNLP
Squirrel monkeyQLRAKLLDLSDSTSVASFPSADETDGNLP
BushbabyQLRAKLLDLSDSTSVASFPSADETDGNLP
Chinese tree shrewQLRAKLLDLSDSTSVASFPSADETDGNLP
SquirrelQLRAKLLDLSDSTSVASFPSADETDGNLP
Lesser Egyptian jerboaQLRAKLLDLSDSTSVASFPSADETDGSLP
Prairie voleQLRAKLLDLSDSTSVASFPSADETDGNLP
Chinese hamsterQLRAKLLDLSDSTSIASFPSADETDGNLP
Golden hamsterQLRAKLLDLSDSTSVASFPSADETDGNLP
MouseQLRAKLLDLSDSTSVASFPSADETDGNLP
RatQLRAKLLDLSDSTSVASFPSADETDGNLP
Naked mole-ratQLRAKLLDLSDSTSVASFPSADETDGNLP
Guinea pigQLRAKLLDLSDSTSVASFPSADETDGNLP
ChinchillaQLRAKLLDLSDSTSVASFPSADETDGNLP
Brush-tailed ratQLRAKLLDLSDSTSVASFPSADETDGNLP
RabbitQLRAKLSDLSDSTSVASFPSADETDGNLP
PikaQLRAKLLDLSDSTSVASFPSTDEaDGNLP
PigQLRAKLLDLSDSTSVASFPSADETDGNLP
AlpacaQLRAKLLDLSDSTSVASFPSADETDGNLP
Bactrian camelQLRAKLLDLSDSTSVASFPSADETDGNLP
DolphinQLRAKLLDLSDSTSVASFPSADETDGNLP
Killer whaleQLRAKLLDLSDSTSVASFPSADETDGNLP
Tibetan antelopeQLRAKLLDLSDSTSVASFPSADETDGNLP
CowQLRAKLLDLSDSTSVASFPSADETDGNLP
SheepQLRAKLLDLSDSTSVASFPSADETDGNLP
Domestic goatQLRAKLLDLSDSTSVASFPSADETDGNLS
HorseQLRAKLLDLSDSTSVASFPSADETDGNLP
White rhinocerosQLRAKLLDLSDSTSVASFPSADETDGNLP
CatQLRAKLLDLSDSTSVASFPSADETDGNLP
DogQLRAKLLDLSDSTSIASFPSADETDGNLP
FerretQLRAKLLDLSDSTSVASFPSADETDGNLP
PandaQLRAKLLDLSDSTSVASFPSADETDGNLP
Pacific walrusQLRAKLLDLSDSTSVASFPSADETDGNLP
Weddell sealQLRAKLLDLSDSTSVASFPSADETDGNLP
Black flying-foxQLRAKLLDLSDSTSVASFPSADETDGNLP
MegabatQLRAKLLDLSDSTSVASFPSADETDGNLP
David’s myotis (bat)QLRAKLLDLSDSTSVASFPSADETDGNLP
MicrobatQLRAKLLDLSDSTSVASFPSADETDGNLP
Big brown batQLRAKLLDLSDSTSVASFPSADETDGNLP
HedgehogQLRAKLLDLADSTSVASFPSADETDGNLP
ShrewQLRSKLLDLSDSTSVASFPSADETDGTLP
Star-nosed moleQLRAKLLDLSDSTSVASFPSADETDGNLP
ElephantQLRAKLLDLSDSTSVASFPSADETDGNLP
Cape elephant shrewQLRAKLLDLSDSTSVASFPSADETDGSLP
ManateeQLRAKLLDLSDSTSVASFPSADETDGNLP
Cape golden moleQLRAKLLDLSDSTSVASFPSADETDGNLP
TenrecQLRAKLLDLSDSTSVASFPSADETDGNLP
AardvarkQLRAKLLDLSDSTSVASFPSADETDGNLP
ArmadilloQLRAKLLDLSDSTSVASFPSADETDGNLP
OpossumQLRAKLLDLSDSTSVTSFPSADETDGNLP
Tasmanian devilQLRAKLLDLSDSTSVTSFPSADETDGNLP
WallabyQLRSKLSDLLDSGSVASL-S-DETDGNLV
platypusQLRRKILDLLDSTSVASLQ--DETDGNLS
Birds
Saker falconQLRRKILDLLDSTSVASLQ--DETDGNLS
Peregrine falconQLRRKIMDLLDSTSVASLP--DEmDGNLP
Collared flycatcherQLRRKIMDLLDSTSVASLP--DETDGNLS
White-throated sparrowQLRRKIMDLLDSTSVASLP--DETDGNLS
Medium ground finchQLRRKIMDLLDSTSVASLP--DETDGNLS
Zebra finchQLRRKIMDLLDSTSVASLP--DETDGNLS
Tibetan ground jayQLRRKILDLLDSTSVASLQ--DETDGNLS
BudgerigarQLRRKILDLLDSTSVASLQ--DETDGNLS
ParrotQLRRKILDLLDSTSVASLQ--DETDGNLS
Scarlet macawQLRRKILDLLDSTSVASLQ--DETDGNLS
Rock pigeonQLRRKILDLLDSTSVASLP--DETDGNLS
Mallard duckQLRRKILDLLDSTSVASLQ--DETDGNLS
ChickenQLRRKILDLLDSTSVSSLQ--DEiDGNLS
TurkeyQLRRKILDLLDSTSVSSLQ--DEiDGNLS
Sarcopterygii
American alligatorQLRSKILDLLDSTSVASLQ--DETDGNVS
Green seaturtleQLRSKILDLLDSTSVASLQ--DETDGNLT
Painted turtleQLRSKLLDLLDSTSVASLQ--DETDGNLT
Chinese softshell turtleQLRSKILDLLDSASVASLQ--DETDGNLT
Spiny softshell turtleQLRSKILDLLDSASVASLQ--DETDGNLT
LizardQLRSKILDLLDSTSVASLP--DETDGNIV
X. tropicalisQLRARLADLLDSTSVASLQ--DdTDGIIG
CoelacanthQLRAKLMDLLDNTSVASLQ--DEvDGNVI
Fish
TetraodonQLREKLNDLLDNSSITSLQ--DETDSNIA
FuguQLREKLNDLLDNSSITSLQ--DETDSNIA
Yellowbelly pufferfishQLREKLNDLLDNSSITSLQ--DETDSNIA
Nile tilapiaQLREKLNDLLDNSSVTSLQ--DETDSNIA
Princess of BurundiQLREKLNDLLDNSSVTSLQ--DETDSNIA
Burton's mouthbreederQLREKLNDLLDNSSVTSLQ--DETDSNIA
Zebra mbunaQLREKLNDLLDNSSVTSLQ--DETDSNIA
Pundamilia nyerereiQLREKLNDLLDNSSVTSLQ--DETDSNIA
MedakaQLREKLNDMLENSSITSLQ--DETDSNIA
Southern platyfishQLREKLSDLLENSSVTSLQ--DETDSNTA
SticklebackQLREKLNDLLDTSSVTSLQ--DETDGNIA
Atlantic codQLREKLNDLLDSSSVTSLL--DETDGNLA
ZebrafishQLREKLNDLLDNSSVTSLQ--DElDSNIA
Spotted garQLREKLNDLLDNSSVASLQ--DEaDGNIA
  1. Protein sequences orthologous to human ROCK1 were aligned in 93 vertebrate species using Multiz Alignment (Blanchette et al., 2004) in the UCSC Genome Browser. These include representative species from the major classes: 62 mammals, 14 birds, 8 Sarcopterygii and 14 fish. The conserved caspase cleavage site indicated with underlined bold typeface, with non-identical amino acids in lowercase.

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