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Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm
Fumiaki Obata1, Shiho Tanaka2, Soshiro Kashio3
1Department 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.
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.
Insights
We developed a novel method for selective cell necrosis using the bacterial toxin Cry1Aa. This system effectively ablates target cells by inducing osmotic lysis, independent of apoptosis pathways, offering a versatile tool for biological research.
Area of Science:
- Cell biology
- Molecular biology
- Genetics
Background:
- Genetic cell ablation is crucial for studying cell function, regeneration, and disease models.
- Existing methods rely on apoptosis, limiting their use to cells with intact apoptotic machinery.
Purpose of the Study:
- To establish a novel method for rapid and selective cell necrosis induction.
- To utilize the Cry1Aa bacterial toxin and its receptor (CryR) for targeted cell death.
Main Methods:
- Overexpression of the silkworm-derived Cry1Aa receptor (CryR) in Drosophila melanogaster tissues.
- Application of the pore-forming bacterial toxin Cry1Aa to induce cell membrane damage and osmotic lysis.
- Live imaging to observe cell death dynamics and in vivo injection for systemic ablation.
Main Results:
- Cry1Aa toxin specifically induced rapid cell necrosis in CryR-expressing cells via osmotic lysis.
- Cell death occurred independently of apoptosis, JNK activation, or autophagy.
- The Cry/CryR system effectively ablated both proliferating and postmitotic cells in vivo.
Conclusions:
- A new method for genetically inducing cell necrosis using Cry toxins from Bacillus thuringiensis was developed.
- This system acts as a 'proteinous drill,' causing physical membrane injury for cell death.
- The method is potentially applicable to any cell type, including those resistant to apoptosis or JNK-dependent programmed cell death.
