A Hedgehog-Responsive Region in the Drosophila Wing Disc Is Defined by Debra- Mediated Ubiquitination and Lysosomal Degradation of Ci  Ping Dai, Hiroshi Akimaru, Shunsuke Ishii  Developmental Cell  Volume 4, Issue 6, Pages 917-928 (June 2003) DOI: 10.1016/S1534-5807(03)00158-8

Figure 1 Dbr Is a Ci Binding Protein (A) Binding of in vitro-translated Ci to Dbr-resin. The relative binding activities of Dbr are designated ++, +, and −, which indicate 10%–20%, 4%, and <1% binding of the input protein, respectively. (Lower left) Clone-8 cells were transfected with the Flag-Dbr expression plasmid, and Dbr proteins were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting with anti-Dbr antibody. (Lower middle and right) In vitro-translated Ci (input) and Ci bound to Dbr-resin were analyzed by SDS-PAGE followed by autoradiography. (B) Binding of in vitro-translated Dbr to the GST-Ci fusions. GST-Ci containing the N-terminal 440 amino acids (GST-Ci-R) or amino acids 441–683 of Ci (GST-Ci-ZF) was used. Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 2 Expression of Dbr in Wing Discs (A–D) Expression of Dbr and Ci-155 in the wing disc. Wing disc stained with the anti-Dbr antibody ([A], green). Immunostainings of Dbr ([B], monochrome) and Ci-155 ([C], red), and the superimposition of both stainings (D) are shown in the wing pouch. Anterior is to the left; dorsal is up. Bars indicate the A/P boundary. (E–J′) Location of Dbr expression in the A/P compartment in the wing disc. Dbr immunostaining ([E], red), expression of ptcZ-Gal4 UAS-GFP ([F], monochrome), and hh-lacZ ([G], green) are merged in (H). Expression of dpp-lacZ ([I], green) and En immunostaining ([J], green) are superimposed with Dbr expression (red) in (I′) and (J′), respectively. (K) Schematic expression pattern of Dbr in the wing disc. Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 3 Dbr Decreases Ci-155 Levels (A) Molecular map of the dbr locus. The open and closed bars indicate the untranslated and protein-coding regions of the dbr cDNA, respectively. The EP element in dbrEP9 is shown by an open triangle. The basal promoter downstream of UAS at the 3′ end is oriented such that it directs the expression of dbr. B, BamHI; E, EcoRI; H, HindIII; X, XhoI. (B) Loss of Dbr leads to the upregulation of dpp. Clones of dbrEP9 mutant cells are marked by the absence of the green GFP marker (left). Expressions of dpp are monitored by the dpp-lacZBS3.0 transgene (dppZ) in red (center). The merged images are shown to the right. (C and D) Overexpression of Dbr leads to downregulation of Hh target genes. Clones of cells expressing dbr marked by GFP (green) are induced by combining Act5C>y+>Gal4 UAS-GFP with dbrEP9 in which the UAS of the EP element directs the dbr transcription. Expressions of dpp (C) and ptc (D) are shown by the dpp-lacZP1552 reporter (dppZ) and ptc-lacZ reporter (ptcZ) in red (right). The merged images are shown to the left. (E) Loss of Dbr leads to increased levels of Ci-155. dbrEP9 mutant clones marked by the absence of GFP (left, green), Ci detected by the 2A1 antibody (center, red), and the merged images (right) are shown. (F) Overexpression of Dbr reduces the Ci-155 levels. Clones overexpressing dbr (Act>dbr) visualized by GFP (left, green), Ci detected by the 2A1 antibody (center, red), and the merged images (right) are shown. Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 4 PKA Phosphorylation Sites in Ci and Slimb Are Required for Ci Degradation by Dbr (A and B) Coexpression of Dbr with Ci-155 blocks the accumulation of Ci-155. Clones overexpressing ci-155 (Act>ci-155) alone (B) or with dbr (Act>dbr) (A) are visualized by GFP (left, green). Ci is detected by 2A1 antibody (right, red). (C and D) Dbr does not induce the Ci-75 degradation. Clones overexpressing ci-75 (Act>ci-75) alone (D) or with dbr (Act>dbr) (C) are visualized by GFP (left, green). The reporter gene activity of hh-lacZ (hhZ) (right, red) and the superimposition of both signals (left) are shown. (E and F) Dbr does not affect Ci−4P levels. Clones of cells expressing ci−4P (Act>ci−4P) alone (F) or with dbr (Act>dbr) (E) are marked by GFP (left, green). Ci-155 is stained by 2A1 antibody (right). (G and H) Dbr does not affect CiU levels. Clones of cells expressing ciU (Act>ciU) alone (H) or with dbr (Act>dbr) (G) are marked by GFP (left, green). Ci-155 is stained by 2A1 antibody (right). (I–L) Dbr overexpression does not downregulate Ci-155 in the slimb1/slimb2 wing disc. The Ci-155 levels detected by 2A1 antibody (red) in the C765-Gal4 driver strain are similar to that in the wild-type disc (I). Ci-155 levels in anterior compartment cells overexpressing Dbr under the control of C765-Gal4 are dramatically reduced (J). Ci-155 processing is blocked and thus Ci-155 (red) accumulates in the anterior compartment cells of the slimb1/2 wing disc (K). Ci-155 levels are not reduced by Dbr overexpressed by the C765-Gal4 driver in the anterior compartment cells of the slimb1/2 transheterozygous wing disc (L). Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 5 Dbr and Slimb Cooperatively Induce the Lysosomal Degradation of Ci-155 by First Ubiquitinating It (A) Downregulation of Ci-155 levels by Dbr. Clone-8 cells were transfected with the HA-Ci-155 expression vector (10 μg) with the Dbr expression plasmid (0, 1, 4, 6, and 10 μg). Whole cell lysates were used for the Western blotting with the anti-HA antibody. To confirm the amounts of proteins used, the proteins were analyzed by SDS-PAGE followed by Coomassie staining (lower). (B) Half-life of Ci-155. Clone-8 cells were transfected with the HA-Ci-155 expression plasmid (8 μg) with the Dbr expression plasmid (8 μg) or the empty vector, and pulse-chase labeled with [35S]methionine. At various times after the chase, Ci-155 protein levels were examined by immunoprecipitation and plotted below. (C) Dbr does not induce Ci-75 degradation. Transfection and Western blotting were performed using HA-Ci-75 expression vector (10 μg) and Dbr expression plasmid (10 μg) as described in (A). (D) E64 blocks the Dbr-induced degradation of Ci-155. Clone-8 cells were transfected with the HA-Ci-155 expression vector (10 μg) with or without the Dbr expression plasmid (10 μg). In some cases, the transfected cells were treated with MG132 or E64. Ci-155 proteins were detected by Western blotting with anti-HA antibody. (E) Requirement of the PKA phosphorylation sites for Dbr-induced Ci-155 degradation. Transfection and Western blotting were performed using the plasmid expressing Ci−4P (10 μg) and the Dbr expression plasmid (10 μg) as described in (A). (F) Slimb is required for Dbr-induced Ci-155 degradation. Clone-8 cells were transfected with the HA-Ci-155 expression vector (4 μg) with or without the Dbr expression vector (4 μg) and increasing amounts of slimb double-stranded RNA (0, 0.01, and 0.1 μg). Western blotting was performed as described above. The amount of endogenous slimb mRNA was examined by RT-PCR analysis. (G) Requirement of both Dbr and Slimb in the ubiquitination of Ci-155. Clone-8 cells were transfected with the plasmids indicated above each lane. The amounts of each plasmid used are: HA-Ci, 8 μg; Myc tag-linked ubiquitin, 4 μg; Dbr, 4 μg; Slimb, 4 μg. In some cases, 0.1 μg of double-stranded Slimb or Dbr RNA was added. Transfected cells were treated with E64 as described in (D). Whole cell lysates were immunoprecipitated with the anti-HA antibody, and the immunocomplexes were analyzed by Western blotting with the anti-Myc antibody. (H) Polyubiquitination of Ci-155 is required for the Dbr-induced degradation of Ci-155. Clone-8 cells were transfected with the plasmid indicated above, and Ci-155, ubiquitinated Ci-155, Slimb, Dbr, and ubiquitin were detected as described above. Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 6 Localization of Dbr in MVBs (A) Subcellular localization of Dbr, Ci-155, and Slimb. Clone-8 cells were transfected with the Dbr expression plasmid with either the Ci-155 or Slimb expression plasmid. The cells were immunostained with the antibodies against the protein indicated and analyzed by confocal microscopy. Ci and Slimb stainings were visualized by rhodamine-conjugated antibodies, while Dbr staining was indicated by FITC-conjugated antibodies. Both signals are superimposed (right). (B) Subcellular localization of Dbr and Ci in the wing discs. (Upper left) The frontal view of the wing disc and the Dbr expression pattern (red) are indicated. The dotted line shows the location where the wing disc was cut to make the cross-section shown to the right. The dotted square points to the region examined by electron microscopy. Anterior is to the left; apical is up. (Ba–Bc) Immunoelectron micrographs. (Ba) Dbr is localized in the membranes of the MVB/late endosome structure (arrow) away from the apical plasma membrane. Dbr is not seen in the membranes of earlier endosomes (arrowhead). Ci can be seen on the membranes of the early endosome (arrow in [Bb]) adjacent to the apical plasma membrane and the membranes of the MBV/late endosome structure (arrow in [Bc]). Ci is also localized in some small particles in the cytoplasm. The nucleus in each cell is located in the basal region outside these panels. PM, peripordial membrane; L, lumen. Scale bars, 0.5 μm. (Bd–Bg) Higher magnification of the early/late endosome structures. The endosome structures indicated by an arrow in (Ba), (Bb), and (Bc) are shown with higher magnification in (Bd), (Be), and (Bf), respectively. The arrowhead in (Bf) marks the early endosome that has just fused with the MVB/late endosome. (Bg) Ci staining is observed in a number of luminal vesicles in the MVB/late endosome. Scale bars, 0.1 μm. Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)

Figure 7 Schematic Representation of the Role of Dbr in Pattern Formation of the Wing Imaginal Disc Developmental Cell 2003 4, 917-928DOI: (10.1016/S1534-5807(03)00158-8)