title: Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.
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.
authors.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.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Obata
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo, 184-8588, Japan. shiho_9099@yahoo.co.jp.
authors.forename: Shiho
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. soshirokashio@gmail.com.
authors.forename: Soshiro
authors.initials: S
authors.lastname: Kashio
authors.affiliation: Developmental Biology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, 183-8509, Japan. tsujmr@cc.tuat.ac.jp.
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Tsujimura
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo, 184-8588, Japan. ryoichi@cc.tuat.ac.jp.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
authors.affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. miura@mol.f.u-tokyo.ac.jp.|CREST, Japan Agency for Medical Research and Development, 20F Yomiuri Shimbun Bldg. 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan. miura@mol.f.u-tokyo.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Miura
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000235:genetics* / Q000494:pharmacology*; D012831:Bombyx / Q000235:genetics*; D004331:Drosophila melanogaster / Q000166:cytology* / Q000187:drug effects / Q000235:genetics; D004731:Endotoxins / Q000008:administration & dosage / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000008:administration & dosage / Q000494:pharmacology*; D019476:Insect Proteins; D020935:MAP Kinase Signaling System; D009336:Necrosis; D061848:Optical Imaging; D011956:Receptors, Cell Surface / Q000235:genetics*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D015854:Up-Regulation; D014921:Wings, Animal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis