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First published online 10 August 2005
doi: 10.1242/dev.01979

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The interplay between DSL proteins and ubiquitin ligases in Notch signaling

¹ Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Greece
² Department of Biology, University of Crete, Heraklion, Greece

View larger version (119K): [in a new window]   Fig. 1. Dl and Ser act redundantly during lateral inhibition. Third instar nota are stained for Senseless (red), to visualize SOPs. Proximal is upwards, anterior is towards the left. Scale bar: 40 µm. (A) Notch mutant cells marked by absence of GFP. (B-F) Mutant cells marked by presence of GFP. (A'-F') Red/Sens channels of A-F, respectively; mutant areas are indicated by arrows. (A) N or (B) Dl Ser clones display a large number of clustered ectopic SOPs. This phenotype probably represents the complete loss of lateral inhibition. By contrast, (C) Dl mutant clones show only a few ectopic SOPs and (D) Ser mutant clones appear wild type. (E) Dl mutant clones simultaneously expressing UAS-fng display a more severe phenotype than Dl (C). (F) Dl Ser mutant clones expressing UAS-Ser appear wild type.

View larger version (103K): [in a new window]   Fig. 2. Neur enhances Dl or Ser loss of function. (A-C) Nota stained for Senseless (red) to visualize SOPs; Sens channel shown separately in A'-C'. Mutant areas are indicated by arrows. Proximal is upwards, anterior is towards the left. Scale bar: 40 µm. Mutant cells express GFP. (A) neur mutant clones display weak/moderate SOP overcommitment. By contrast, (B) neur Ser clones and (C) neur Dl clones display a much more severe SOP overcommitment. In C the outline of the notum is drawn.

View larger version (52K): [in a new window]   Fig. 3. Physical association between Neur and DSL proteins. (A,B) Immunoprecipitations from transfected S2 cells expressing V5-tagged Dl (A) or myc-tagged Ser (B), along with nothing (lanes 1,4), EGFPneur (lanes 2,5) or neurR-GFP (lanes 3,6). Lanes 1-3: cell extract (input). Lanes 4-6: anti-Neur immunoprecipitate. (C,D) Immunoprecipitations from larval disk/CNS complexes. (C) hs-Gal4; UAS-Dl-V5 along with another UAS transgene as follows: nothing (lanes 1,4), UAS-EGFPneur (lanes 2,5) or UAS-neurR-GFP (lanes 3,6). (D) hs-Gal4; UAS-Ser-myc plus another UAS transgene, as in C. In lanes 4 of all panels (no Neur expressed) no DSL protein is detected, showing the specificity of the immunoprecipitation. In lanes 5 and 6, Neur and NeurR, respectively, immunoprecipitate both Dl and Ser. Endogenous Neur protein is present in S2 cells, seen as a doublet in A and B (lane 4, asterisks). Transfected Neur produces higher molecular weight species owing to the GFP tags. Curiously the DlIC fragment was never detected in larval extracts. Molecular mass standards are shown in kDa to the right of each panel. FL, full-length; IC, Dl intracellular fragment; trunc, truncated Ser.

View larger version (88K): [in a new window]   Fig. 4. Neur induces Ser endocytosis. Details of wing pouches are shown; anterior is towards the left, dorsal is upwards and the DV boundary is at the bottom of each panel. Scale bar: 20 µm. (A,B) dpp-Gal4; UAS-Ser with co-expression of UAS-GFP (A) or UAS-EGFPneur (B). GFP (green) marks the domain of overexpression and Ser (red) is shown separately in A' and B'. Neur causes loss of pericellular staining and an increase in intracellular aggregates in B. (C) Effects of mosaic expression of UAS-EGFPneur on endogenous Ser (red, C'). In cells expressing EGFPneur (green), apical Ser staining is lost. The detection sensitivity for Ser is increased in C compared with A,B to image endogenous Ser levels. (D,E) Green (EGFPneur) channels of C,B, respectively. EGFPneur is cortical with little punctate accumulation when no DSL protein is co-overexpressed (D); when Ser is co-overexpressed there is additional accumulation into punctate structures, which also contain Ser (yellow dots in B). All images are projections of the apical-most 1.5-2 µm of the wing epithelium.

View larger version (111K): [in a new window]   Fig. 5. mib1 enhances the lateral inhibition phenotype of neur. (A-C) Nota stained for Sens (red, A'-C') to reveal SOPs. (A,B) Mutant clones marked by GFP expression; (C) mutant clones marked by GFP absence. Arrows indicate mutant areas. (A) mib1EY9780 clones do not produce supernumerary SOPs. (B) neur1 mutant clones show mild defects. (C) neur1 mutant clones in a mib1EY9780 background show severe hyperplasia of SOPs. Scale bar: 40 µm.

View larger version (163K): [in a new window]   Fig. 6. Dl and Ser signaling in the wing pouch needs ubiquitin ligase activity. (A-H) Wg (red) in wing pouches carrying mutant clones marked by the expression of GFP (green). In all panels, ventral is downwards. Scale bar: 40 µm; 60 µm in D,G. (A,B) Clones overexpress UAS-Dl in a wild-type (A) or mib1 (B) background. Whereas ectopic Wg is induced by dorsal and (less) by ventral (arrows) clones in A, no Wg induction is observed in B. The endogenous Wg stripe is abolished (arrowhead); this is also observed in mib1 clones without Dl overexpression (data not shown). (C) mib1 clones overexpressing UAS-Dl together with UAS-neur restore Wg induction; in fact Dl activity in the ventral compartment is enhanced (arrows, compare with A). (D,E) Clones overexpress UAS-Ser in a wild-type (D) or mib1 (E) background. Ser induces Wg exclusively in the ventral compartment (D), but not when expressed in mib1 cells (E). (F) Ser regains its activity to induce Wg in a mib1 background, if it is co-expressed with UAS-neur. Ser cannot induce Wg in the dorsal compartment, irrespective of the presence of ubiquitin ligases (D,F; arrowheads), probably owing to high Fng levels. (G) Co-expression of UAS-Dl with UAS-neurR in a wild-type background efficiently induces ectopic Wg in both compartments, indicative of increased Dl activity (compare with A). (H) This is abolished by loss of mib1. (I) Cut is imaged (red), which is a nuclear marker for the DV boundary. mib1 mutant clones are marked by the absence of GFP. UAS-Dl is overexpressed via ptc-Gal4, which drives expression just anterior of the anteroposterior boundary (white line). In the cells posteriorly adjacent to this expression domain (to the right of the white line), ectopic Cut is detected within mib1 cells if they abut anterior wild-type cells (arrow), but not if they abut mutant anterior cells (arrowhead). Inset shows an enlargement of the boxed area; the Cut-positive nuclei, which do not contain GFP appear red. The presence of these Cut-positive mib1 mutant cells suggests that mib1+ is not needed for signal reception.