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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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Expression of ArmD88A interferes with redistribution of DE-cadherin during apoptosis. (A, B) th109 homozygous embryo (stage 9, 60 min post morphogenetic block) expressing ArmD88A stained for TUNEL (A, A') and expression of the Flag-tagged ArmD88A transgene (B, B' anti-Flag). (C, D) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A and stained for Arm (C) and DE-cadherin (D); note persistent localization of DE-cadherin at the plasma membrane. (E-H) DE-cadherin localization in th109 homozygous embryos with or without expression of ArmD88A. (E, F) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A stained for DE-cadherin (E, E'; red in F, F') and TUNEL (green in F, F'). (G, H) th109 homozygous embryo (60 min post cephalic furrow formation) stained for DE-cadherin (red in H) and TUNEL (green in H). Scale bar in (B) and (F) 40 μm, scale bar in B') and F') 10 μm.
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Figure 6: Expression of ArmD88A interferes with redistribution of DE-cadherin during apoptosis. (A, B) th109 homozygous embryo (stage 9, 60 min post morphogenetic block) expressing ArmD88A stained for TUNEL (A, A') and expression of the Flag-tagged ArmD88A transgene (B, B' anti-Flag). (C, D) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A and stained for Arm (C) and DE-cadherin (D); note persistent localization of DE-cadherin at the plasma membrane. (E-H) DE-cadherin localization in th109 homozygous embryos with or without expression of ArmD88A. (E, F) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A stained for DE-cadherin (E, E'; red in F, F') and TUNEL (green in F, F'). (G, H) th109 homozygous embryo (60 min post cephalic furrow formation) stained for DE-cadherin (red in H) and TUNEL (green in H). Scale bar in (B) and (F) 40 μm, scale bar in B') and F') 10 μm.

Mentions: Our results showed that in apoptotic cells Arm is cleaved by the caspase drICE and that this cleavage generates a stable fragment of Arm. Upon over-expression of ArmD88A in DIAP1 mutant embryos the execution of apoptosis occurred slightly delayed, but remained otherwise unimpaired. For example, DIAP1 mutant embryos expressing ArmD88A show massive appearance of TUNEL positive cells in stage 9 similar to DIAP1 mutant embryos without ArmD88A transgene expression (Fig. 6A, A'). While endogenous full length Arm protein is no longer associated with the surface of apoptotic cells, the Flag-tagged ArmD88A transgenic protein remains associated with the plasma membrane of late apoptotic cells (Fig. 6B, B'). This result suggests that cleavage of Arm at DQVD88 is required for removal of membrane associated full length Arm in apoptosis. Expression of ArmD88A also resulted in a more persistent localisation of DE-cadherin at the membrane in a phase during apoptosis when it is normally redistributed to the cytoplasm (Fig. 6C–H). This result indicates that drICE-mediated cleavage of Arm at the N-terminal domain is required for the observed redistribution of DE-cadherin and might contribute to the breakdown of cell-cell adhesion in apoptosis.


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

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

Expression of ArmD88A interferes with redistribution of DE-cadherin during apoptosis. (A, B) th109 homozygous embryo (stage 9, 60 min post morphogenetic block) expressing ArmD88A stained for TUNEL (A, A') and expression of the Flag-tagged ArmD88A transgene (B, B' anti-Flag). (C, D) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A and stained for Arm (C) and DE-cadherin (D); note persistent localization of DE-cadherin at the plasma membrane. (E-H) DE-cadherin localization in th109 homozygous embryos with or without expression of ArmD88A. (E, F) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A stained for DE-cadherin (E, E'; red in F, F') and TUNEL (green in F, F'). (G, H) th109 homozygous embryo (60 min post cephalic furrow formation) stained for DE-cadherin (red in H) and TUNEL (green in H). Scale bar in (B) and (F) 40 μm, scale bar in B') and F') 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657781&req=5

Figure 6: Expression of ArmD88A interferes with redistribution of DE-cadherin during apoptosis. (A, B) th109 homozygous embryo (stage 9, 60 min post morphogenetic block) expressing ArmD88A stained for TUNEL (A, A') and expression of the Flag-tagged ArmD88A transgene (B, B' anti-Flag). (C, D) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A and stained for Arm (C) and DE-cadherin (D); note persistent localization of DE-cadherin at the plasma membrane. (E-H) DE-cadherin localization in th109 homozygous embryos with or without expression of ArmD88A. (E, F) th109 homozygous embryo (60 min post cephalic furrow formation) expressing ArmD88A stained for DE-cadherin (E, E'; red in F, F') and TUNEL (green in F, F'). (G, H) th109 homozygous embryo (60 min post cephalic furrow formation) stained for DE-cadherin (red in H) and TUNEL (green in H). Scale bar in (B) and (F) 40 μm, scale bar in B') and F') 10 μm.
Mentions: Our results showed that in apoptotic cells Arm is cleaved by the caspase drICE and that this cleavage generates a stable fragment of Arm. Upon over-expression of ArmD88A in DIAP1 mutant embryos the execution of apoptosis occurred slightly delayed, but remained otherwise unimpaired. For example, DIAP1 mutant embryos expressing ArmD88A show massive appearance of TUNEL positive cells in stage 9 similar to DIAP1 mutant embryos without ArmD88A transgene expression (Fig. 6A, A'). While endogenous full length Arm protein is no longer associated with the surface of apoptotic cells, the Flag-tagged ArmD88A transgenic protein remains associated with the plasma membrane of late apoptotic cells (Fig. 6B, B'). This result suggests that cleavage of Arm at DQVD88 is required for removal of membrane associated full length Arm in apoptosis. Expression of ArmD88A also resulted in a more persistent localisation of DE-cadherin at the membrane in a phase during apoptosis when it is normally redistributed to the cytoplasm (Fig. 6C–H). This result indicates that drICE-mediated cleavage of Arm at the N-terminal domain is required for the observed redistribution of DE-cadherin and might contribute to the breakdown of cell-cell adhesion in apoptosis.

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