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Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

Obata F, Tanaka S, Kashio S, Tsujimura H, Sato R, Miura M - BMC Biol. (2015)

Bottom Line: Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body.With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis.Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. obataf@mol.f.u-tokyo.ac.jp.

ABSTRACT

Background: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.

Results: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.

Conclusions: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

No MeSH data available.


Related in: MedlinePlus

Cry1Aa induces cell death even in cells with inhibited apoptosis or JNK-dependent cell death. (a–c) Propidium iodide (PI) staining of cultured wing discs from third instar larvae. En > GFP, CryR was crossed with LacZ (a), inhibitor of apoptosis, p35 (b), and JNK inhibitor, puc (c), and wing discs from F1 progeny were incubated for 1 h with 100 nM Cry1Aa. Scale bar, 100 μm
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Fig5: Cry1Aa induces cell death even in cells with inhibited apoptosis or JNK-dependent cell death. (a–c) Propidium iodide (PI) staining of cultured wing discs from third instar larvae. En > GFP, CryR was crossed with LacZ (a), inhibitor of apoptosis, p35 (b), and JNK inhibitor, puc (c), and wing discs from F1 progeny were incubated for 1 h with 100 nM Cry1Aa. Scale bar, 100 μm

Mentions: One of the limitations of conventional cell ablation systems is their dependency on the cell’s ability to activate programmed cell death. In contrast, Cry1Aa directly forms a pore on the plasma membrane, suggesting that cellular context does not affect toxicity. To test this, we overexpressed p35, a baculoviral inhibitor of apoptosis that inactivates caspases, and tested whether Cry1Aa could still induce cell death (Fig. 5a, b). As expected, PI signals were not attenuated by p35 overexpression. In addition, Cry1Aa-induced cell death was not blocked by treatment with z-VAD-fmk, a pan-caspase inhibitor (Additional file 1: Figure S3A). We further confirmed that knock down of either dronc, an initiator caspase, or of pro-apoptotic RHG genes, did not inhibit cell death (Additional file 1: Figure S3B, C), indicating that apoptosis is not required for the Cry/CryR system.Fig. 5


Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

Obata F, Tanaka S, Kashio S, Tsujimura H, Sato R, Miura M - BMC Biol. (2015)

Cry1Aa induces cell death even in cells with inhibited apoptosis or JNK-dependent cell death. (a–c) Propidium iodide (PI) staining of cultured wing discs from third instar larvae. En > GFP, CryR was crossed with LacZ (a), inhibitor of apoptosis, p35 (b), and JNK inhibitor, puc (c), and wing discs from F1 progeny were incubated for 1 h with 100 nM Cry1Aa. Scale bar, 100 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495774&req=5

Fig5: Cry1Aa induces cell death even in cells with inhibited apoptosis or JNK-dependent cell death. (a–c) Propidium iodide (PI) staining of cultured wing discs from third instar larvae. En > GFP, CryR was crossed with LacZ (a), inhibitor of apoptosis, p35 (b), and JNK inhibitor, puc (c), and wing discs from F1 progeny were incubated for 1 h with 100 nM Cry1Aa. Scale bar, 100 μm
Mentions: One of the limitations of conventional cell ablation systems is their dependency on the cell’s ability to activate programmed cell death. In contrast, Cry1Aa directly forms a pore on the plasma membrane, suggesting that cellular context does not affect toxicity. To test this, we overexpressed p35, a baculoviral inhibitor of apoptosis that inactivates caspases, and tested whether Cry1Aa could still induce cell death (Fig. 5a, b). As expected, PI signals were not attenuated by p35 overexpression. In addition, Cry1Aa-induced cell death was not blocked by treatment with z-VAD-fmk, a pan-caspase inhibitor (Additional file 1: Figure S3A). We further confirmed that knock down of either dronc, an initiator caspase, or of pro-apoptotic RHG genes, did not inhibit cell death (Additional file 1: Figure S3B, C), indicating that apoptosis is not required for the Cry/CryR system.Fig. 5

Bottom Line: Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body.With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis.Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. obataf@mol.f.u-tokyo.ac.jp.

ABSTRACT

Background: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.

Results: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.

Conclusions: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

No MeSH data available.


Related in: MedlinePlus