Common and distinct genetic properties of ESCRT-II components in Drosophila

Abstract

Background: Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner.

Principal findings: Here, we genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and--when the tissue is predominantly mutant--show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity.

Conclusions/significance: The ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. Our data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation.

Figure 1. Mutant clones of ESCRT-II components display endosomal defects and accumulate ubiquitinated proteins.

Shown are eye imaginal discs of 3rd instar larvae mosaic for ESCRT-II mutants. Mutant clones are marked by the absence of GFP. Mutant clones of ESCRT-II components show abnormal accumulation of the early endosomal marker Hrs and accumulation of ubiquitin-conjugated proteins as visualized by the FK1 antibody. Hrs and ubiquitin-conjugated proteins accumulate in foci which frequently co-localize. Scale bars represent 50 µm. (A,B,C) GFP/Hrs/FK1 (green/red/blue) co-labelings of (A) vps22^5F8-3, (B) vps25^N55 and (C) vps36^L5212 eye mosaics. (A′,B′,C′) Hrs/FK1 (red/blue) co-labelings of (A′) vps22^5F8-3, (B′) vps25^N55 and (C′) vps36^L5212 eye mosaics. (A″,B″,C″) Hrs labeling of (A″) vps22^5F8-3, (B″) vps25^N55 and (C″) vps36^L5212 eye mosaics. (A′″,B′″,C′″) FK1 labeling of (A′″) vps22^5F8-3, (B′″) vps25^N55 and (C′″) vps36^L5212 eye mosaics. Genotypes: (A) eyFlp ; FRT82B vps22^5F8-3/FRT82B P[ubi-GFP]. (B) eyFlp; FRT42D vps25^N55/FRT42D P[ubi-GFP]. (C) eyFlp ; vps36^L5212 FRT2A/P[ubi-GFP] FRT2A.

Figure 2. Accumulation of Notch and Delta proteins in clones of ESCRT-II mutants.

(A,B,C) Areas of increased Ubiquitin (Ub) immunoreactivity frequently co-localize with areas containing accumulated levels of the Notch receptor (antibody against the extracellular domain of Notch, N[extra]) in ESCRT-II mutant tissue. GFP/Ubiquitin/N^extra (green/red/blue) co-labelings of (A) vps22^5F8-3, (B) vps25^N55 and (C) vps36^L5212 eye mosaics. Scale bars represent 50 µm. (A′,B′,C′) Ubiquitin/N^extra (red/blue) co-labeling of (A′) vps22^5F8-3, (B′) vps25^N55 and (C′) vps36^L5212 eye mosaics. (A″,B″,C″) Ubiquitin labeling of (A″) vps22^5F8-3, (B″) vps25^N55 and (C″) vps36^L5212 eye mosaics. (A′″,B′″,C′″) N^extra labeling of (A′″) vps22^5F8-3, (B′″) vps25^N55 and (C′″) vps36^L5212 eye mosaics. (D,E,F) Accumulation of Notch protein using an antibody detecting the intracellular domain of Notch, N[intra]). GFP/N^intra (green, red) co-labelings of (D) vps22^5F8-3, (E) vps25^N55 and (F) vps36^L5212 eye mosaics. Scale bars represent 100 µm (D′,E′,F′) N^intra (red) labeling of (D′) vps22^5F8-3, (E′) vps25^N55 and (F′) vps36^L5212 eye mosaics. (G,H,I) The Notch ligand Delta accumulates in clones of ESCRT-II mutants. GFP/Delta (green/magenta) co-labeling of (G) vps22^5F8-3, (H) vps25^N55 and (I) vps36^L5212 eye mosaics. Scale bars represent 100 µm (G′,H′,I′) Delta labeling of (G′) vps22^5F8-3, (H′) vps25^N55 and (I′) vps36^L5212 eye mosaics. Genotypes: (A,D,G) eyFlp; FRT82B vps22^5F8-3/FRT82B P[ubi-GFP]. (B,E,H) eyFlp ; FRT42D vps25^N55/FRT42D P[ubi-GFP]. (C,F,I) eyFlp ; vps36^L5212 FRT2A/P[ubi-GFP] FRT2A.

Figure 3. Apoptosis phenotype of ESCRT-II mutants.

Labeling of vps22, vps25 and vps36 eye-antennal imaginal discs with cleaved Caspase-3 antibody as apoptotic marker. Arrows in (A′), (B′) and (C′) point to one representative clone in each panel containing increased caspase-3 activity. (A–C) GFP/Caspase-3 (green/red) co-labelings of (A) vps22^5F8-3, (B) vps25^N55 and (C) vps36^L5212 eye mosaics. (A′–C′) Caspase-3 labeling (red) of (A′) vps22^5F8-3, (B′) vps25^N55 and (C′) vps36^L5212 eye mosaics. Genotypes: (A) eyFlp ; FRT82B vps22^5F8-3/FRT82B P[ubi-GFP]. (B) eyFlp ; FRT42D vps25^N55/FRT42D P[ubi-GFP]. (C) eyFlp ; vps36^L5212 FRT2A/P[ubi-GFP] FRT2A.

Figure 4. Eye discs predominantly mutant for ESCRT-II are overgrown and lose cellular architecture.

All discs are labeled for phalloidin and were obtained with the eyFlp-cell lethal system. Scale bars represent 100 µm. (A) Control eye-antennal imaginal disc. (B–D) Eye-antennal imaginal discs predominantly mutant for vps22 (B), vps25 (C) and vps36 (D). Genotypes: (A) eyFlp ; FRT82B/FRT82B cl GMR-hid. (B) eyFlp ; FRT82B vps22^5F8-3/FRT82B cl GMR-hid. (C) eyFlp ; FRT42 vps25^N55/FRT42 cl GMR-hid. (D) eyFlp ; vps36^L5212 FRT80/GMR-hid cl FRT80.

Figure 5. Adult phenotypes of ESCRT-II mosaics.

vps22 and vps25 mosaics display strong overgrowth phenotypes of the adult eyes and heads, and the larval eye imaginal discs. In contrast, vps36 mutants show no or only a mild proliferation phenotype and cause a roughening of the adult eye. (A–D) Side view of genetic eye mosaics of (A) control flies, (B) vps22^5F8-3, (C) vps25^N55 and (D) vps36^L5212 mutants. (E–H) Eye mosaics of (E) control (heterozygous Notch), (F) vps22^5F8-3, (G) vps25^N55 and (H) vps36^L5212 in heterozygous Notch (N) background. The Notch allele used is N^264-39. (I–L) Top view of genetic mosaics of (I) control flies, (J) vps22^5F8-3, (K) vps25^N55 and (L) vps36^L5212 mutants. (M–P) Head mosaics of (M) control (heterozygous Notch), (N) vps22^5F8-3, (O) vps25^N55 and (P) vps36^L5212 in heterozygous Notch (N) background. The Notch allele used is N^264-39. (Q–T) Size comparison of (Q) control, (R) vps22^5F8-3, (S) vps25^N55 and (T) vps36^L5212 mosaic eye imaginal discs. Green: GFP; red: BrdU labeling. The scale bars represent 100 µm. Genotypes: (A) eyFlp ; FRT82B/FRT82B P[w⁺]. (B,J) eyFlp ; FRT82B vps22^5F8-3/FRT82B P[w⁺]. (C,K) eyFlp ; FRT42D vps25^N55/FRT42D P[w⁺]. (D,L) eyFlp ; vps36^L5212 FRT2A/P[w⁺] FRT2A. (E,M) N^264-39/+. (F–H) and (N–P): same as (B–D) and (J–L) except they also carry N^264-39 as heterozygous mutation. (Q–T) same as in corresponding panels A–D except they carry P[ubi-GFP] instead of P[w⁺].

Figure 6. Proliferation phenotype of ESCRT-II mosaics.

Non-autonomous regulation of proliferation in vps22 and vps25 eye mosaics as depicted by BrdU incorporation (red). Arrows in (B) and (C) point to areas of increased BrdU density next to mutant clones. Compared to control discs, vps36 mutations do not affect the proliferation pattern significantly. The scale bar represents 50 µm. (A–D) GFP/BrdU (green/red) co-labelings of (A) control, (B) vps22^5F8-3, (C) vps25^N55 and (D) vps36^L5212 eye mosaics. (A′–D′) BrdU labeling of (A′) control, (B′) vps22^5F8-3, (C′) vps25^N55 and (D′) vps36^L5212 eye mosaics. Genotypes: (A) eyFlp; FRT42B/FRT42B P[ubi-GFP]. (B) eyFlp; FRT82B vps22^5F8-3/FRT82B P[ubi-GFP]. (C) eyFlp; FRT42D vps25^N55/FRT42D P[ubi-GFP]. (D) eyFlp; vps36^L5212 FRT2A/P[ubi-GFP] FRT2A.

Figure 7. Notch activity in ESCRT-II mutants.

Notch activity in ESCRT-II mutants was assessed using the reporter transgene E(spl)m8 2.61-lacZ and β-Gal immunohistochemistry. In wild-type discs, this reporter is turned on posterior to the morphogenetic furrow (see bar in A″). Note that in vps22^5F8-3 and vps25^N55 mutant clones located anterior to the morphogenetic furrow ectopic reporter activity is detectable (arrows in A″ and B″). vps36^L5212 clones do not or only weakly (arrow) induce reporter activity (C″). Genotypes: (A) eyFlp; E(spl)m8 2.61-lacZ ; FRT82B vps22^5F8-3/FRT82B P[w⁺]. (B) eyFlp ; FRT42D vps25^N55 E(spl)m8 2.61-lacZ/FRT42D E(spl)m8 2.61-lacZ. (C) eyFlp ; E(spl)m8 2.61-lacZ; vps36^L5212 FRT2A/P[w⁺] FRT2A.

Figure 8. Suppression of the GMR-hid-eye ablation phenotype by ESCRT-II mosaics.

(A) Expression of the pro-apoptotic gene hid under control of the eye-specific GMR enhancer (GMR-hid) gives rise to a strong eye ablation phenotype due to excessive apoptosis. (B–D) vps25^N55 (C) and vps36^L5212 (D) eye mosaics are strong suppressors of the GMR-hid-induced eye ablation phenotype in adult flies. vps22^5F8-3 mosaics (B) do not suppress the GMR-hid-eye ablation phenotype. Genotypes: (A) eyFlp ; GMR-hid; FRT82B/FRT82B P[w⁺]. (B) eyFlp; GMR-hid; FRT82B vps22^5F8-3/FRT82B P[w⁺]. (C) GMR-hid eyFlp; FRT42D vps25^N55/FRT42D P[w⁺]. (D) eyFlp; GMR-hid; vps36^L5212 FRT2A/P[w⁺] FRT2A.