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First published online 6 June 2007
doi: 10.1242/dev.02859

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A Drosophila ortholog of the human cylindromatosis tumor suppressor gene regulates triglyceride content and antibacterial defense

¹ Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Street, 16672 Vari, Greece.
² Institute of Molecular Biology and Genetics, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Street, 16672 Vari, Greece.
³ Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
⁴ Department of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Reverse transcriptase-PCR analysis of CYLD expression in Drosophila melanogaster. (A) Five transcripts of Drosophila CYLD have been described, and are shown here. The positions of the CYLD primers used in B (DCYF3 and DCYR6) are shown. (B) CYLD (DmCYLD) expression was detected by reverse transcriptase (RT)-PCR using the primers DMCYLDF3 (DCYF3) and DMCYLDR6 (DCYR6), and RNAs were isolated at the indicated developmental stages and in the indicated body compartments of adult w1118 flies (upper panel). RP49 (also known as RpL32) RNA was used as a control for the amount of cDNA used in the RT-PCR reaction (lower panel).

View larger version (99K): [in this window] [in a new window]   Fig. 2. Distribution of CYLD in Drosophila embryos and adults. (A-D) Detection of CYLD in embryos by immunohistochemistry. (A) At stage 7, CYLD is expressed in the anterior midgut rudiment (amg), in the posterior midgut rudiment (pmg) and in the mesoderm (ms). (B) At stage 12, CYLD is localized in the developing brain area (br), in the forming midgut (amg and pmg) and in the ventral nerve cord (vnc). (C) At stage 14, CYLD is detected at the brain (br), the ventral nerve cord (vnc), the midgut (mg) and the hindgut (hg). (D) Stage 12 embryos homozygous for the Df(2L)J21 deficiency, which deletes the CYLD locus, did not stain with the anti-CYLD antibody. (E-J) Detection of CYLD in adult tissues by immunofluorescence. (E) Negative control using a non-specific rabbit polyclonal antibody (anti-GFP) as primary antibody and the same secondary antibody used in F-J. Using the same settings as in E, endogenous CYLD levels were detected with a rabbit polyclonal anti-CYLD antibody on adult w1118 sagittal paraffin sections (8 µm). CYLD was detected in ovaries (F), in testes (G), in the fat body (H), in the antenna (I) and in the eye (J). amg, anterior midgut rudiment; br, brain; hg, hindgut; mg, midgut; ms, mesoderm; pmg, posterior midgut rudiment; vnc, ventral nerve cord.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Drosophila (Dm)CYLD is a deubiquitylating enzyme. E. coli expressing a ubiquitin-ß-galactosidase (Ub-ßgal) fusion were transformed with the indicated expression constructs for glutathione-S-transferase (GST): GST-HCYLD(538-956), GST-DmCYLD or GST-DmCYLD(C284S). (A) After induction of the GST-domain-containing proteins, whole extracts from an approximately equal number of bacteria were analyzed by immunoblotting with a polyclonal anti-ß-galactosidase antibody. The positions of Ub-ßgal and ß-galactosidase (ß-gal) are indicated. (B) The GST-domain-containing proteins were expressed at similar levels, as detected by immunoblotting of whole-cell lysates of an equal number of bacteria with polyclonal anti-GST antibody.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Molecular characterization of P-element insertion lines. (A) The positions of P-element insertions analyzed and the primers that were used [DMCYLDF3 (DCYF3), DMCYLDR6 (DCYR6), DMCYLDF7 (DCYF7) and DMCYLDR9 (DCYR9)] are shown relative to the five transcripts of Drosophila CYLD that have been identified. (B) The P-element-insertion lines f00135, f00814, f02492 and d10472 were analyzed by reverse transcriptase (RT)-PCR for CYLD mRNA. The results were compared with wild-type flies (w1118). rp49 (also known as RpL32) RNA was used as a control for the amount of cDNA used in the RT-PCR reaction (lower panel).

View larger version (132K): [in this window] [in a new window]   Fig. 5. Impaired Drosophila CYLD expression alters fat body morphology. (A-C) Adult female abdomens from wild-type (A), CYLDf00814 homozygotes (B) and CYLD-rescue (C) flies were analyzed by hematoxylin and Eosin staining of paraffin sections (8 µm). (D-F) Oocyte morphology in wild-type (D), CYLDf00814 homozygotes (E) and CYLD-rescue (F) flies. (G,H) Nile red staining for the presence of lipid droplets in cryosections from adult wild-type flies (G) or CYLDf00814 homozygotes (H).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Impaired Drosophila CYLD expression results in elevated triglyceride content and increased survival under starvation. (A) Triglyceride (TAG) content (expressed as µg TAG per µg protein) of 2-3-day-old male flies after hatching. The following strains were analyzed: wild type (WT) w1118, CYLDf00814 homozygotes (f00814), UAS-CYLD/Y;CYLDf00814;hs-Gal4/MKRS (DmCYLD rescue) after heat-shock induction of UAS-CYLD transcription and CYLD-rescue flies that were not subjected to heat-shock [DmCYLD rescue(NO HS)]. Mean values (±s.d.) from at least three independent experiments are shown. (B) Survival under starvation conditions of 2-3-day-old male flies. Representative data from at least three independent experiments are shown.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Drosophila CYLD (DmCYLD) interacts with Kenny (dIKK). (A) Quantitative yeast two-hybrid assay with all of the components of the IMD pathway (PGRP-LC, PGRP-LE, PGRP-LA, IMD, dTAK1, dFADD, DREDD, IRD5 and Kenny). CYLD interacts specifically with Kenny, the Drosophila ortholog of the mammalian protein NEMO (IKK). (B) Anti-V5 western blot of anti-c-Myc-immunoprecipitated material from lysates of Drosophila S2 cells transfected with plasmids expressing the proteins indicated on the top of the panel.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Antimicrobial peptide expression in flies with impaired Drosophila CYLD expression. (A) Diptericin expression [expressed as a ratio of Diptericin:rp49 (also known as RpL32) mRNA levels] in wildtype w1118 flies (w) and in CYLDf00814 homozygotes (f00814) was determined before and after infection with E. coli. (B) Diptericin expression (expressed as a ratio of Diptericin:rp49 mRNA levels) in wildtype w1118 (w) and flies overexpressing CYLD in the fat body (DmCYLD/yolk) before and after E. coli infection. (A,B) Mean values (±s.d.) from three independent experiments are shown.

View larger version (14K): [in this window] [in a new window]   Fig. 9. A Drosophila strain with impaired CYLD expression is susceptible to infection by Gram-negative and Gram-positive bacteria. (A,B) A total of 25 flies were infected with either Gram-negative (E. coli; A) or Gram-positive (E. faecalis; B) bacteria. Survival was monitored for 7 days. Representative data from one out of four independent experiments are shown in each panel. WT, w1118 wild type; relish/Dif, positive controls; f00814: CYLDf00814 homozygotes.