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Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson H, Kumar S - J. Cell Biol. (2000)

Bottom Line: Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects.RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos.These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

View Article: PubMed Central - PubMed

Affiliation: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia.

ABSTRACT
Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

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Genetic interactions of GMR-p35, the Df(3L)H99 genes, diap1 and dark with GMR-GAL4; UAS-debcl#26. The effects of GMR-p35 and reducing the dosage of the Df(3L)H99 genes, diap1 and dark genes on the eye phenotype of heterozygous GMR-GAL4; UAS-debcl#26 flies, were examined after crossing GMR-GAL4/CyO; UAS-debcl#26/TM6B flies to the relevant stocks. A, Scanning electron micrograph of a wild-type adult eye (Canton S). B, Scanning electron micrograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye. C, Photograph of a wild-type eye (Canton S). D, Photograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye and patches of reduced pigmentation. E, Photograph of GMR-GAL4; UAS-debcl#26/GMR-p35 adult eye, showing strong suppression of the ablated eye phenotype. F, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)H99 (removing rpr, hid, and grim) adult eye, showing little effect on the ablated eye phenotype. G, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)brm11 (removing diap1) adult eye, showing enhancement of the ablated eye phenotype. Similar results were obtained using another diap1 deficiency, (Df(3L)stf-13). H, Photograph of GMR-GAL4/darkCD8 (hypomorphic allele); UAS-debcl#26 showing suppression.
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Figure 6: Genetic interactions of GMR-p35, the Df(3L)H99 genes, diap1 and dark with GMR-GAL4; UAS-debcl#26. The effects of GMR-p35 and reducing the dosage of the Df(3L)H99 genes, diap1 and dark genes on the eye phenotype of heterozygous GMR-GAL4; UAS-debcl#26 flies, were examined after crossing GMR-GAL4/CyO; UAS-debcl#26/TM6B flies to the relevant stocks. A, Scanning electron micrograph of a wild-type adult eye (Canton S). B, Scanning electron micrograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye. C, Photograph of a wild-type eye (Canton S). D, Photograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye and patches of reduced pigmentation. E, Photograph of GMR-GAL4; UAS-debcl#26/GMR-p35 adult eye, showing strong suppression of the ablated eye phenotype. F, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)H99 (removing rpr, hid, and grim) adult eye, showing little effect on the ablated eye phenotype. G, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)brm11 (removing diap1) adult eye, showing enhancement of the ablated eye phenotype. Similar results were obtained using another diap1 deficiency, (Df(3L)stf-13). H, Photograph of GMR-GAL4/darkCD8 (hypomorphic allele); UAS-debcl#26 showing suppression.

Mentions: To characterize further the biological activity of Debcl, we expressed debcl in Drosophila SL2 cells under the control of an inducible insect promoter. Within 16 h of transfection, Debcl induced apoptosis in a majority of the transfected SL2 cells (Fig. 5 A). By 48 h, all debcl transfected cells had been lost (not shown). This cell death was partially inhibited by the cell permeable peptide caspase inhibitor zVAD-fmk and much more effectively by baculovirus caspase inhibitor P35, indicating that Debcl-induced apoptosis is, at least in part, mediated by caspases. While zVAD-fmk is an efficient inhibitor of many mammalian caspases, it is not known whether it can inhibit Drosophila caspases as effectively. Therefore, the partial inhibition of Debcl-induced cell death by zVAD-fmk may reflect its inability to efficiently inhibit all Drosophila caspases. To confirm that Debcl's cell killing function is dependent on caspase activity, we crossed debcl transgenic flies with GMR-p35 flies. As discussed below and shown in Fig. 6, in the resulting flies the effect of Debcl in eye ablation was significantly reduced.


Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson H, Kumar S - J. Cell Biol. (2000)

Genetic interactions of GMR-p35, the Df(3L)H99 genes, diap1 and dark with GMR-GAL4; UAS-debcl#26. The effects of GMR-p35 and reducing the dosage of the Df(3L)H99 genes, diap1 and dark genes on the eye phenotype of heterozygous GMR-GAL4; UAS-debcl#26 flies, were examined after crossing GMR-GAL4/CyO; UAS-debcl#26/TM6B flies to the relevant stocks. A, Scanning electron micrograph of a wild-type adult eye (Canton S). B, Scanning electron micrograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye. C, Photograph of a wild-type eye (Canton S). D, Photograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye and patches of reduced pigmentation. E, Photograph of GMR-GAL4; UAS-debcl#26/GMR-p35 adult eye, showing strong suppression of the ablated eye phenotype. F, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)H99 (removing rpr, hid, and grim) adult eye, showing little effect on the ablated eye phenotype. G, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)brm11 (removing diap1) adult eye, showing enhancement of the ablated eye phenotype. Similar results were obtained using another diap1 deficiency, (Df(3L)stf-13). H, Photograph of GMR-GAL4/darkCD8 (hypomorphic allele); UAS-debcl#26 showing suppression.
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Figure 6: Genetic interactions of GMR-p35, the Df(3L)H99 genes, diap1 and dark with GMR-GAL4; UAS-debcl#26. The effects of GMR-p35 and reducing the dosage of the Df(3L)H99 genes, diap1 and dark genes on the eye phenotype of heterozygous GMR-GAL4; UAS-debcl#26 flies, were examined after crossing GMR-GAL4/CyO; UAS-debcl#26/TM6B flies to the relevant stocks. A, Scanning electron micrograph of a wild-type adult eye (Canton S). B, Scanning electron micrograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye. C, Photograph of a wild-type eye (Canton S). D, Photograph of GMR-GAL4; UAS-debcl#26 adult eye, showing severe ablation of the eye and patches of reduced pigmentation. E, Photograph of GMR-GAL4; UAS-debcl#26/GMR-p35 adult eye, showing strong suppression of the ablated eye phenotype. F, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)H99 (removing rpr, hid, and grim) adult eye, showing little effect on the ablated eye phenotype. G, Photograph of GMR-GAL4; UAS-debcl#26/Df(3L)brm11 (removing diap1) adult eye, showing enhancement of the ablated eye phenotype. Similar results were obtained using another diap1 deficiency, (Df(3L)stf-13). H, Photograph of GMR-GAL4/darkCD8 (hypomorphic allele); UAS-debcl#26 showing suppression.
Mentions: To characterize further the biological activity of Debcl, we expressed debcl in Drosophila SL2 cells under the control of an inducible insect promoter. Within 16 h of transfection, Debcl induced apoptosis in a majority of the transfected SL2 cells (Fig. 5 A). By 48 h, all debcl transfected cells had been lost (not shown). This cell death was partially inhibited by the cell permeable peptide caspase inhibitor zVAD-fmk and much more effectively by baculovirus caspase inhibitor P35, indicating that Debcl-induced apoptosis is, at least in part, mediated by caspases. While zVAD-fmk is an efficient inhibitor of many mammalian caspases, it is not known whether it can inhibit Drosophila caspases as effectively. Therefore, the partial inhibition of Debcl-induced cell death by zVAD-fmk may reflect its inability to efficiently inhibit all Drosophila caspases. To confirm that Debcl's cell killing function is dependent on caspase activity, we crossed debcl transgenic flies with GMR-p35 flies. As discussed below and shown in Fig. 6, in the resulting flies the effect of Debcl in eye ablation was significantly reduced.

Bottom Line: Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects.RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos.These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

View Article: PubMed Central - PubMed

Affiliation: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia.

ABSTRACT
Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

Show MeSH
Related in: MedlinePlus