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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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debcl is required for developmental cell death in embryos. RNAi was used to ablate debcl function in embryos. Precellularized embryos were injected with double-stranded debcl RNA and aged to stage 16 before fixation and TUNEL labeling. A, An uninjected control embryo showing the normal pattern of TUNEL labeling. B–E, Typical examples of injected embryos from the debcl RNAi experiment showing that the number of TUNEL positive cells is dramatically reduced compared with the control (A; see Fig. 3 H). F, A buffer-injected control shows that the injection procedure does not inhibit apoptosis, but instead an increase in TUNEL positive cells is observed (compare A and F).
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Figure 8: debcl is required for developmental cell death in embryos. RNAi was used to ablate debcl function in embryos. Precellularized embryos were injected with double-stranded debcl RNA and aged to stage 16 before fixation and TUNEL labeling. A, An uninjected control embryo showing the normal pattern of TUNEL labeling. B–E, Typical examples of injected embryos from the debcl RNAi experiment showing that the number of TUNEL positive cells is dramatically reduced compared with the control (A; see Fig. 3 H). F, A buffer-injected control shows that the injection procedure does not inhibit apoptosis, but instead an increase in TUNEL positive cells is observed (compare A and F).

Mentions: Currently, no specific debcl mutants are available. Therefore, to examine the in vivo function of Debcl, we carried out RNAi studies to inhibit debcl gene function. RNAi is a powerful technique to disrupt the function of specific genes (Hunter 1999). Additionally, RNAi has the advantage of ablating maternally contributed mRNA that is difficult to achieve genetically. It was originally used in C. elegans, but recently has been successfully applied to Drosophila (Bhat et al. 1999; Misquitta and Paterson 1999). Precellularized embryos were injected with debcl double-stranded RNA, aged until stage 16, and analyzed by TUNEL staining. As shown in Fig. 8B–E, debcl RNAi resulted in a large reduction in TUNEL positive cells in the embryos. We analyzed at least 200 embryos injected with the debcl double-stranded RNA and, in all cases, a 50–90% reduction in TUNEL positive cells was evident. In control experiments, embryos injected with the injection buffer did not show any inhibition of apoptosis (as in Fig. 8 F). In fact, most of the buffer-injected embryos showed slightly higher numbers of TUNEL positive cells, as compared with uninjected embryos (compare Fig. 8A and Fig. F). These RNAi results indicate that debcl gene function is required for programed cell death in the embryos. Additionally, these data suggest that maternally deposited debcl mRNA may be required for normal cell death in fly embryos.


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)

debcl is required for developmental cell death in embryos. RNAi was used to ablate debcl function in embryos. Precellularized embryos were injected with double-stranded debcl RNA and aged to stage 16 before fixation and TUNEL labeling. A, An uninjected control embryo showing the normal pattern of TUNEL labeling. B–E, Typical examples of injected embryos from the debcl RNAi experiment showing that the number of TUNEL positive cells is dramatically reduced compared with the control (A; see Fig. 3 H). F, A buffer-injected control shows that the injection procedure does not inhibit apoptosis, but instead an increase in TUNEL positive cells is observed (compare A and F).
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Related In: Results  -  Collection

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Figure 8: debcl is required for developmental cell death in embryos. RNAi was used to ablate debcl function in embryos. Precellularized embryos were injected with double-stranded debcl RNA and aged to stage 16 before fixation and TUNEL labeling. A, An uninjected control embryo showing the normal pattern of TUNEL labeling. B–E, Typical examples of injected embryos from the debcl RNAi experiment showing that the number of TUNEL positive cells is dramatically reduced compared with the control (A; see Fig. 3 H). F, A buffer-injected control shows that the injection procedure does not inhibit apoptosis, but instead an increase in TUNEL positive cells is observed (compare A and F).
Mentions: Currently, no specific debcl mutants are available. Therefore, to examine the in vivo function of Debcl, we carried out RNAi studies to inhibit debcl gene function. RNAi is a powerful technique to disrupt the function of specific genes (Hunter 1999). Additionally, RNAi has the advantage of ablating maternally contributed mRNA that is difficult to achieve genetically. It was originally used in C. elegans, but recently has been successfully applied to Drosophila (Bhat et al. 1999; Misquitta and Paterson 1999). Precellularized embryos were injected with debcl double-stranded RNA, aged until stage 16, and analyzed by TUNEL staining. As shown in Fig. 8B–E, debcl RNAi resulted in a large reduction in TUNEL positive cells in the embryos. We analyzed at least 200 embryos injected with the debcl double-stranded RNA and, in all cases, a 50–90% reduction in TUNEL positive cells was evident. In control experiments, embryos injected with the injection buffer did not show any inhibition of apoptosis (as in Fig. 8 F). In fact, most of the buffer-injected embryos showed slightly higher numbers of TUNEL positive cells, as compared with uninjected embryos (compare Fig. 8A and Fig. F). These RNAi results indicate that debcl gene function is required for programed cell death in the embryos. Additionally, these data suggest that maternally deposited debcl mRNA may be required for normal cell death in fly embryos.

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