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Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells.

Kessler T, Müller HA - BMC Dev. Biol. (2009)

Bottom Line: During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases.To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein.Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich Heine Universität, Düsseldorf, Germany. kessler@molgen.mpg.de

ABSTRACT

Background: During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.

Results: We define early and late apoptotic stages and find that early in apoptosis Dalpha-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis.

Conclusion: This study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

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A stable Arm cleavage product persists in apoptotic cells. (A) ArmCentral antibody immunoprecipitates full length Arm; Full length Arm (Mr 110 kDa; indicated as ArmFull) is immunoprecipitated (IP) by the ArmNterm or the ArmCentral antibody. In Western blots of these immunoprecipitates ArmFull is detected by the ArmNterm antibody. DE-cadherin or Arm antibodies were used to immunoprecipitate proteins from embryo extracts (IP) as indicated. Immunoprecipitates were then subjected to Western blotting using ArmCentral antiserum. Anti ArmCentral detects full length Arm in the input lane (in) and in immunoprecipitates by all three antibodies. (B) Detection of ArmΔN in protein extracts of th109 homozygous embryos. Western blot of protein extracts from th109 heterozygous (wt) and th109 homozygous embryos. Levels of ArmFull are decreased in th109 homozygous embryos and a short form of Arm with Mr ~90 kDa is detected corresponding to ArmΔN. Actin was used as loading control. (C-F) Localization of ArmΔN in th109 homozygous embryos (45 min after onset of morphogenesis arrest) stained for ArmNterm (C), ArmCentral (D) and Dα-Cat (E). Arrows in (C-E) point to an apoptotic cell where full length Arm is absent from the plasma membrane but ArmCentral is still present (F) Merged image with ArmNterm in green, ArmCentral in red and Dα-Cat in blue. (G-J) th109 homozygous embryo (60 min after onset of morphogenesis arrest) (inset in J) stained for (G) TUNEL, (H) ArmCentral and (I) Dα-Cat; (J) Overlay with TUNEL in green, ArmCentral in red and Dα-Cat in blue.
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Figure 5: A stable Arm cleavage product persists in apoptotic cells. (A) ArmCentral antibody immunoprecipitates full length Arm; Full length Arm (Mr 110 kDa; indicated as ArmFull) is immunoprecipitated (IP) by the ArmNterm or the ArmCentral antibody. In Western blots of these immunoprecipitates ArmFull is detected by the ArmNterm antibody. DE-cadherin or Arm antibodies were used to immunoprecipitate proteins from embryo extracts (IP) as indicated. Immunoprecipitates were then subjected to Western blotting using ArmCentral antiserum. Anti ArmCentral detects full length Arm in the input lane (in) and in immunoprecipitates by all three antibodies. (B) Detection of ArmΔN in protein extracts of th109 homozygous embryos. Western blot of protein extracts from th109 heterozygous (wt) and th109 homozygous embryos. Levels of ArmFull are decreased in th109 homozygous embryos and a short form of Arm with Mr ~90 kDa is detected corresponding to ArmΔN. Actin was used as loading control. (C-F) Localization of ArmΔN in th109 homozygous embryos (45 min after onset of morphogenesis arrest) stained for ArmNterm (C), ArmCentral (D) and Dα-Cat (E). Arrows in (C-E) point to an apoptotic cell where full length Arm is absent from the plasma membrane but ArmCentral is still present (F) Merged image with ArmNterm in green, ArmCentral in red and Dα-Cat in blue. (G-J) th109 homozygous embryo (60 min after onset of morphogenesis arrest) (inset in J) stained for (G) TUNEL, (H) ArmCentral and (I) Dα-Cat; (J) Overlay with TUNEL in green, ArmCentral in red and Dα-Cat in blue.

Mentions: Because the specificity of the anti-ArmNterm antibody might prevent detection of an N-terminally truncated Arm cleavage product, we raised a polyclonal antibody against the central domain of Arm (amino acids 172–755) referred to as anti-ArmCentral antibody (Fig. 4A). The specificity of the anti-ArmCentral antibody was determined by immunofluorescence as well as by immunoprecipitation experiments (Fig. 5). In early embryos, anti-ArmCentral immunoreactivity always co-localised with the staining obtained with anti-ArmNterm (data not shown). Furthermore, anti-ArmCentral detects full length Arm, which was immunoprecipitated either with anti-ArmCentral, anti-ArmNterm, or anti-DE-cadherin antibodies (Fig. 5A). We conclude that the anti-ArmCentral antibody recognizes full length Arm protein in Drosophila embryos.


Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells.

Kessler T, Müller HA - BMC Dev. Biol. (2009)

A stable Arm cleavage product persists in apoptotic cells. (A) ArmCentral antibody immunoprecipitates full length Arm; Full length Arm (Mr 110 kDa; indicated as ArmFull) is immunoprecipitated (IP) by the ArmNterm or the ArmCentral antibody. In Western blots of these immunoprecipitates ArmFull is detected by the ArmNterm antibody. DE-cadherin or Arm antibodies were used to immunoprecipitate proteins from embryo extracts (IP) as indicated. Immunoprecipitates were then subjected to Western blotting using ArmCentral antiserum. Anti ArmCentral detects full length Arm in the input lane (in) and in immunoprecipitates by all three antibodies. (B) Detection of ArmΔN in protein extracts of th109 homozygous embryos. Western blot of protein extracts from th109 heterozygous (wt) and th109 homozygous embryos. Levels of ArmFull are decreased in th109 homozygous embryos and a short form of Arm with Mr ~90 kDa is detected corresponding to ArmΔN. Actin was used as loading control. (C-F) Localization of ArmΔN in th109 homozygous embryos (45 min after onset of morphogenesis arrest) stained for ArmNterm (C), ArmCentral (D) and Dα-Cat (E). Arrows in (C-E) point to an apoptotic cell where full length Arm is absent from the plasma membrane but ArmCentral is still present (F) Merged image with ArmNterm in green, ArmCentral in red and Dα-Cat in blue. (G-J) th109 homozygous embryo (60 min after onset of morphogenesis arrest) (inset in J) stained for (G) TUNEL, (H) ArmCentral and (I) Dα-Cat; (J) Overlay with TUNEL in green, ArmCentral in red and Dα-Cat in blue.
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Related In: Results  -  Collection

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Figure 5: A stable Arm cleavage product persists in apoptotic cells. (A) ArmCentral antibody immunoprecipitates full length Arm; Full length Arm (Mr 110 kDa; indicated as ArmFull) is immunoprecipitated (IP) by the ArmNterm or the ArmCentral antibody. In Western blots of these immunoprecipitates ArmFull is detected by the ArmNterm antibody. DE-cadherin or Arm antibodies were used to immunoprecipitate proteins from embryo extracts (IP) as indicated. Immunoprecipitates were then subjected to Western blotting using ArmCentral antiserum. Anti ArmCentral detects full length Arm in the input lane (in) and in immunoprecipitates by all three antibodies. (B) Detection of ArmΔN in protein extracts of th109 homozygous embryos. Western blot of protein extracts from th109 heterozygous (wt) and th109 homozygous embryos. Levels of ArmFull are decreased in th109 homozygous embryos and a short form of Arm with Mr ~90 kDa is detected corresponding to ArmΔN. Actin was used as loading control. (C-F) Localization of ArmΔN in th109 homozygous embryos (45 min after onset of morphogenesis arrest) stained for ArmNterm (C), ArmCentral (D) and Dα-Cat (E). Arrows in (C-E) point to an apoptotic cell where full length Arm is absent from the plasma membrane but ArmCentral is still present (F) Merged image with ArmNterm in green, ArmCentral in red and Dα-Cat in blue. (G-J) th109 homozygous embryo (60 min after onset of morphogenesis arrest) (inset in J) stained for (G) TUNEL, (H) ArmCentral and (I) Dα-Cat; (J) Overlay with TUNEL in green, ArmCentral in red and Dα-Cat in blue.
Mentions: Because the specificity of the anti-ArmNterm antibody might prevent detection of an N-terminally truncated Arm cleavage product, we raised a polyclonal antibody against the central domain of Arm (amino acids 172–755) referred to as anti-ArmCentral antibody (Fig. 4A). The specificity of the anti-ArmCentral antibody was determined by immunofluorescence as well as by immunoprecipitation experiments (Fig. 5). In early embryos, anti-ArmCentral immunoreactivity always co-localised with the staining obtained with anti-ArmNterm (data not shown). Furthermore, anti-ArmCentral detects full length Arm, which was immunoprecipitated either with anti-ArmCentral, anti-ArmNterm, or anti-DE-cadherin antibodies (Fig. 5A). We conclude that the anti-ArmCentral antibody recognizes full length Arm protein in Drosophila embryos.

Bottom Line: During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases.To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein.Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich Heine Universität, Düsseldorf, Germany. kessler@molgen.mpg.de

ABSTRACT

Background: During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.

Results: We define early and late apoptotic stages and find that early in apoptosis Dalpha-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis.

Conclusion: This study defines the specificity of caspase cleavage of Arm in Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.

Show MeSH
Related in: MedlinePlus