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First published online 12 April 2005
doi: 10.1242/jcs.02312

Journal of Cell Science 118, 1851-1859 (2005)
Published by The Company of Biologists 2005
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Nef induces apoptosis by activating JNK signaling pathway and inhibits NF-{kappa}B-dependent immune responses in Drosophila

Sung Bae Lee1, Jeehye Park1, Jae U. Jung2 and Jongkyeong Chung1,*

1 National Creative Research Initiatives Center for Cell Growth Regulation and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea
2 Department of Microbiology and Molecular Genetics and Tumor Virology Division, New England Primate Research Center, Harvard Medical School, Southborough, MA 01772, USA



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  		Fig. 1. Characterization of Drosophila wing phenotypes induced by nef expression. (A) MS1096/Y. (B) MS1096/Y;; UAS-nef/+. (C) MS1096/Y;; UAS-nef/UAS-nef. (D) MS1096/Y;; UAS-nef-G2A/+. (E) ap-GAL4/bc. (F) ap-GAL4/+; UAS-nef/+. (G) Comparison between tissue sizes of nef-expressing wings and control wings. Wing imaginal discs were obtained from third-instar larvae. The standard deviation is obtained by examining 20 samples of the same genotype. Fly genotypes: MS1096 (MS1096/X). 1xnef (MS1096/X;; UAS-nef/+). 2xnef (MS1096/X;; UAS-nef/UAS-nef). G2A (MS1096/X; UAS-nef-G2A/+). (H) Determination of expression and induction levels of nef transcript using northern-blot analysis. To induce expression of wild-type and G2A nef, third-instar larvae were subjected to 2 hours of heat shock at 37°C. 18S rRNA was used as a control. Fly genotypes were as follows. Cont: Control, hsp70-GAL4/+. nef: hsp70-GAL4/+; UAS-nef/+. G2A: hsp70-GAL4/UAS-nef-G2A.

 


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  		Fig. 2. Caspase-dependent apoptosis in wings expressing nef. (A-F) Nef-induced caspase-dependent apoptosis in wings. Wing imaginal discs were stained with acridine orange (A-C) or antibody against active Drice (D-F). (G-L) Nef did not affect cell proliferation in wings. Wing imaginal discs were examined by BrdU incorporation assay (G-I) or by immunostaining with antibody against phosphospecific histone H3 (J-L).

 


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  		Fig. 3. JNK signaling pathway mediates Nef-induced apoptosis. (A-H) Functional interactions between Nef and the JNK pathway were determined by wing phenotypes. Fly genotypes were as follows. (A) MS1096/X; UAS-bsk/+. (B) MS1096/X; UAS-hep/+. (C) MS1096/X; UAS-bsk/+; UAS-nef/+. (D) MS1096/X; UAS-hep/+; UAS-nef/+. (E) bsk1/+. (F) MS1096/Y; bsk1/+; UAS-nef/+. (G) Basc/Y; ap-GAL4/+; UAS-nef/+. (H) hep1/Y; ap-GAL4/+; UAS-nef/+. (I-K) Suppression of the Nef-induced apoptosis by reducing JNK gene dosage. Wing imaginal discs were stained with acridine orange. Fly genotypes were as follows. (I) bsk1/+. (J) MS1096/X;; UAS-nef/+. (K) MS1096/Y; bsk1/+; UAS-nef/+. (L-N) Enhanced puc expression caused by nef expression. Wing imaginal discs were stained with X-gal to determine ß-galactosidase production. Fly genotypes were as follows. (L) ap-GAL4/+; puc-lacZ/+. (M) ap-GAL4/+; UAS-nef/puc-lacZ. (N) hepr75/Y; ap-GAL4/+; UAS-nef/puc-lacZ. (O) ap-GAL4/UAS-hep; puc-lacZ/+.

 


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  		Fig. 4. JNK activation and apoptosis in the clones expressing nef. (A-F) Cells with active JNK and nef-expressing clones in wing imaginal discs were determined by immunostaining with anti-phosphospecific JNK antibody (green) (A,D) and anti-ß-galactosidase antibody (purple) (B,E), respectively. Merged images are shown (C,F). In this experiment, Nef-expressing cells were marked by cytosolic ß-galactosidase expression. (G-O) Cells with active Drice and nef-expressing clones in wing imaginal discs were determined by immunostaining with antibody against active Drice (green) (G,J,M) and anti-ß-galactosidase antibody (purple) (H,K,N), respectively. Merged images are shown (I,L,O). In this experiment, nef-expressing cells were marked by either cytosolic ß-galactosidase (H,K) or nuclear ß-galactosidase (N). The wing disc images shown represent clones generated either in the columnar cell layer (A-L) or in the peripodial membrane cell layer (M-O) and were taken under a magnification of 400x (A-C,G-I) or 4000x (D-F,J-L,M-O).

 


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  		Fig. 5. Inhibition of immune responses by Nef. (A,B) Induction of Dpt mRNA by bacterial infection was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). (C) Intracellular localization of Relish was determined by immunostaining with anti-Relish antibody (left). BOBO-3 iodide was used to visualize the nuclear structure (middle). Merged images (right) are shown. (D) Induction of Dpt mRNA by DTRAF2 overexpression was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). (E,F) Induction of Dpt mRNA by bacterial infection (E) or DTRAF2 overexpression (F) was determined by northern-blot analysis (top). 18S rRNA was used as a loading control (bottom). Fly genotypes were as follows. C: Control, hsp70-GAL4/+. nef or 1xnef: hsp70-GAL4/+; UAS-nef/+. 2xnef: hsp70-GAL4/+; UAS-nef/UAS-nef. G2A: hsp70-GAL4/UAS-nef-G2A. nef+JNKDN: UAS-JNKDN/+; hsp70-GAL4/+; UAS-nef/+. nef+hep1: hep1/hep1; hsp70-GAL4/+; UAS-nef/+. T: EP(X)1516/X; hsp70-GAL4/+. T+N: EP(X)1516/X; hsp70-GAL4/+; UAS-nef/+. T+N+JNKDN: EP(X)1516/UAS-JNKDN; hsp70-GAL4/+; UAS-nef/+. To induce ectopic gene expression, third-instar larvae were heat shocked for 30 minutes at 37°C and subsequently incubated for 90 minutes at 25°C [A,B,D (left three lanes),E] or heat shocked for 2 hours at 37°C [C,D (right three lanes),F].

 


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  		Fig. 6. Intracellular signals initiated from Nef. Nef localized to the plasma membrane activates MKK7 and JNK to induce caspase-dependent apoptosis, and also inhibits Relish NF-{kappa}B activity to negatively modulate immune responses.

 




© The Company of Biologists Ltd 2005