Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis

Abstract

The Notch signaling pathway plays a central role in animal growth and patterning, and its deregulation leads to many human diseases, including cancer. Mutations in the tumor suppressor lethal giant discs (lgd) induce strong Notch activation and hyperplastic overgrowth of Drosophila imaginal discs. However, the gene that encodes Lgd and its function in the Notch pathway have not yet been identified. Here, we report that Lgd is a novel, conserved C2-domain protein that regulates Notch receptor trafficking. Notch accumulates on early endosomes in lgd mutant cells and signals in a ligand-independent manner. This phenotype is similar to that seen when cells lose endosomal-pathway components such as Erupted and Vps25. Interestingly, Notch activation in lgd mutant cells requires the early endosomal component Hrs, indicating that Hrs is epistatic to Lgd. These data suggest that Lgd affects Notch trafficking between the actions of Hrs and the late endosomal component Vps25. Taken together, our data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling.

Figure 1. Lgd is a novel cytoplasmic C2 domain protein

(A) Schematic representation of the protein structures of Drosophila lgd and the two human homologs. The arrow labeled ‘d7’ indicates the position at which the lgd^d7 allele has a frameshift mutation: the removal of a single nucleotide results in a new translational frame and a premature stop codon after the addition of 22 new amino acids. The arrow labeled ‘1’ indicates the position at which the lgd¹ allele has a frameshift mutation: two nucleotides are removed resulting in a new translational frame and a premature stop codon after the addition of 15 new amino acids. The arrow labeled ‘J12’ indicates the position at which the EMS-induced allele, lgd^J12, contains a nonsense mutation: Q626 is changed to a stop codon. Green boxes indicate the Lgd homology domains (LH), and orange boxes indicate C2 domains. (B) Western blot to detect Lgd protein in extracts from lgd¹, yw, and lgd^d7 third instar larvae. The lgd¹ and lgd^d7 alleles truncate the protein N-terminally to the epitope used to generate the Lgd antibodies. The band at approximately 120kD contained the majority of Lgd protein, and was missing in both of the lgd mutant lanes. Other specific bands of lower molecular weight were also occasionally seen. (C–C′) Lgd antibodies specifically detect Lgd protein in imaginal discs. lgd mutant clones, indicated by the lack of GFP (green), stained with α–Lgd antibodies (red in C and gray in C′). (D–E) S2 cells stained with Lgd antibodies (green in D and E and gray in D′) and Topro (red). (E) Lgd antibodies incubated with Lgd antigen prior to use for staining. The preabsorbed Lgd antibodies (green) did not display specific staining, demonstrating that the Lgd antibodies specifically detect the Lgd antigen. (F–F″) C5 Gal4 driven UAS-HA-Lgd in the wing pouch of third instar imaginal discs, detected by α-HA (green in F and gray in F″). HA did not specifically colocalize with α-Hrs (red in F and gray in F′).

Figure 2. Lgd acts cells autonomously and restricts Notch activation in a ligand-independent manner

(A and A′) Third instar wing disc with lgd^d10 mutant clones marked by the absence of GFP (Green). The discs were stained for Cut (red in A, gray in A′), a target of the Notch pathway. The arrow in (A) indicates the wing margin. The arrowhead in (A and A′) marks the clone boundary, showing the demarcation between Cut-expressing and non-expressing cells. Only lgd mutant cells (lacking GFP) showed Cut activation outside of the wing margin. (B–B′) Dl^rev10,Ser^RX106 mutant clones in a wild-type wing disc. Cut (red in B and gray in B′) was ectopically expressed in some cells adjacent to the clone boundaries. (C–D′) Dl^rev10,Ser^RX106 mutant clones in an lgd^1/d7 wing imaginal disc. D and D′ show higher magnification of the region boxed in (C). The disc is stained for Cut (red in C and D, shown singly in gray in C′ and D′), and the lack of GFP expression (green in C and D) marks the clone. (D–D′) Cut expression extended throughout the clone. Also, the levels of Cut expression were higher along the boundary of the clone (arrowhead), demonstrating the presence of cis-inhibition in the absence of Lgd.

Figure 3. Mutations in lgd cause disruptions in Notch trafficking

(A–A‴) An lgd^d7 mutant clone in a third instar wing disc, indicated by the absence of ubiGFP (blue), stained for α-N^ICD (green in A and A‴, gray in A′) and α-Hrs (red in A and A‴, gray in A″). N^ICD and Hrs accumulated together in lgd mutant cells (arrowhead). (B–B′) An lgd^d7 mutant clone in a third instar wing disc, indicated by the absence of ubiGFP (blue) stained for α-N^ECD (green in B, gray in B′). The arrowhead indicates one of the subcellular puncta that shows accumulation of the extracellular part of the Notch protein. (C–C′) vps25, UAS-P35 MARCM mutant clones in the third instar wing disc, indicated by the presence of UAS-GFP (blue), stained for α-Hrs (red in C, gray in C′). The arrowhead indicates one of the subcellular puncta that expressed Hrs. (D–D‴) vps25 MARCM mutant clones in a third instar wing disc, indicated by the presence of UAS-GFP (blue) stained for α-N^ICD (green in D and D‴, gray in D′) and α-Lgd (red in D and D‴, gray in D″). Both N^ICD and Lgd protein accumulate in the vps25 mutant cells. (E–E′) An hrs^D28 mutant clone in a third instar wing disc, indicated by the absence of ubiGFP (blue), stained for α-N^ICD (green in E and E‴ and gray in E′) and α-Lgd (red in E and E‴ gray in E″). N^ICD staining accumulated in the mutant cells, but there was no change in Lgd protein levels or localization within the clone compared to wild-type cells.

Figure 4. Hrs is required for Notch activation in lgd mutant cells

(A–A′) lgd^d7 mutant clones in a third instar imaginal wing disc, indicated by the absence of ubiGFP (green), stained for Cut (red in A and gray in A′). Cut, a target gene activated by the Notch pathway, was induced in the lgd mutant tissue in the area surrounding the wing margin. The ectopic Cut induction does not appear to be autonomous in these clones only because this image and all panels in this figure are extended focus views of the whole disc, instead of single focal planes as in Fig. 1D. (B–B′) hrs^D28 mutant clones in a third instar imaginal wing disc, indicated by the absence of ubiGFP (green), stained for α-Cut (red in B and gray in B′). Unlike in lgd mutant clones, Cut was not induced in the hrs mutant tissue. (C–C′) hrs^D28, lgd^d7 double mutant clones in a third instar imaginal wing disc, indicated by the absence of ubiGFP (green), stained for α-Cut (red in C and gray in C′). Cut was not induced in the cells lacking both Lgd and Hrs, suggesting that the hrs mutant phenotype is epistatic over the lgd mutant phenotype. (D–D′) vps25 MARCM mutant clones in the third instar wing disc, indicated by the presence of nuclear UAS-GFP (green), stained for α-Cut (red in D and gray in D′). Cut was not induced in the mutant tissue.