The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously

Abstract

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.

Fig. 1. Uba1 mutants are autonomous suppressors of GMR-hid-induced apoptosis

(A) The unmodified small eye phenotype caused by GMR-hid ey-FLP (GheF). (B,C) The GheF phenotype is moderately strongly suppressed by Uba1 alleles at 25°C. Genotype: GheF; FRT42D Uba1^H33 and Uba1^H42/FRT42 P[w⁺], respectively. (D) The unmodified small eye phenotype caused by a GMR-hid transgene that lacks the white⁺ marker gene (GMR-hid[w⁻]). Note, the GMR-hid transgene in (A) carries the white⁺ minigene. Genotype: ey-FLP; GMR-hid[w⁻]. (E,F) The suppression of GMR-hid[w⁻] is mediated by Uba1 mutant tissue which is phenotypically white^–. Flies were incubated at 25°C. Genotype: ey-FLP; FRT42D Uba1^H33 and Uba1^H42/FRT42D P[w⁺]; GMR-hid[w⁻], respectively. (G) Outline of the Uba1 protein and locations of the mutations of the Uba1 alleles isolated in this study. The red box indicates the internal deletion of Uba1^H42. The yellow boxes mark the Thif domains, and the blue boxes highlight the location of the catalytic Ube domains. (H) A highly conserved stretch of residues, incorporating Pro884 is present in range of species from yeast to humans. Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Mm, Mus musculus; XI, Xenopus laevis; Hs, Homo sapiens. (I) RT-PCR of mRNA isolated from wild-type and Uba1^H42 mutant larvae revealing an aberrantly spliced product. This results in an in frame deletion from amino acid residue 162 to 255 of Uba1^H42 (see G). Primers were directed against the N-terminal third of Uba1.

Fig. 2. Strong Uba1 alleles cause non-autonomous overgrowth

(A) Control mosaics: ey-FLP-induced clones in wild-type background. This phenotype is identical at 25°C and 29°C (shown is 25°C). Genotype: ey-FLP; FRT42D+/FRT42D P[w⁺]. (B,C) Clones of weak Uba1 mutants (phenotypically white⁻) develop normally. Genotype: ey-FLP; FRT42D Uba1^H33 and Uba1^H42/FRT42D P[w⁺], respectively. (D) Eye mosaics of the strong Uba1^s3484 allele are overgrown with bulges (arrow). Genotype: ey-FLP;FRT42D Uba1^s3484/FRT42D P[w⁺]. This phenotype was obtained at 25°C, and is similar at 29°C. (E) Eye mosaic of the ts allele Uba1^H33 at 29°C. Uba1^H33 clones, which would be phenotypically white⁻, are not detectable. The eyes appear rough, irregular and are often enlarged compared to wild-type eyes. Identical results were obtained for Uba1^D6. Genotype as in B. (F) Eye mosaic of the weak Uba1^H42 allele at 29°C. This allele does not behave as a ts allele. Phenotype and genotype are identical to those at 25°C (C). (G) Mosaic head of an Uba1^D6 animal raised at 29°C. The left half of the Uba1^H33 mosaic head is larger than the right half, suggesting that the overgrowth phenotype depends on the number and location of the Uba1 clones. Genotype as in B. (H,I) Eye-antennal imaginal discs of wild-type (H) and Uba1^D6 (I) mosaic animals raised at 29°C. These discs were labeled with Elav antibody which labels photoreceptor neurons to identify discs of comparable age. Photoreceptor differentiation does occur in strong Uba1 clones, but is weakly delayed (see also Fig. S1 in the supplementary material). Genotype as in A and B, respectively. Size bar: 100 µm.

Fig. 3. Uba1 mutant clones affect ubiquitin conjugation

(A–D) The left panels indicate the positions of the Uba1 clones by absence of GFP, the middle panels show the experiment and the right panels are the merged images of left and middle panels. Genotype in all panels: ey-FLP; FRT42D Uba1/FRT42D P[ubi-GFP]. The allele is indicated in the panels. (A–C) Uba1 mutant discs were labeled with the FK2 antibody which specifically recognizes mono- and poly-ubiquitinated conjugates, but not unconjugated ubiquitin (Fujimuro et al., 1994). The strong alleles Uba1^s3484 (A) and Uba1^D6 (B, incubated at 29°C) significantly reduce FK2 immunoreactivity (see arrows). Clones of the weak allele Uba1^H42 (C) mildly reduce FK2 immunoreactivity (arrows). (D) Uba1^D6 mosaics incubated at 29°C. The overall protein levels of ubiquitin are unchanged in Uba1 mutant clones.

Fig. 4. Weak Uba1 alleles suppress apoptosis

(A) GMR-hid eye disc labeled with cleaved Caspase-3 (Cas3*) antibodies. Yellow arrows indicate two zones of dying cells in GMR-hid. (B,B′) Suppression of Cas3*-positive cell death in GMR-hid discs. The yellow arrow indicates the position of the first zone of Cas3*-positive cell death in GMR-hid. Uba1^D6 clones suppress Cas3*-positive cell death (white arrows). However, this suppression is not uniform and occurs in a region-specific manner. Clones in an area of the disc marked by the red arrow are still apoptotic. Genotype: GMR-hid ey-FLP; FRT42D Uba1^D6/FRT42D P[ubi-GFP]; (C–C″) Uba1^D6 clones suppress most normal developmental cell death in 28 hours APF pupal discs. Genotype as in B. (D–D″) Uba1^H33 clones contain additional IOCs in 42 hours APF pupal discs. Genotype: ey-FLP; FRT42D Uba1^H33/FRT42D P[ubi-GFP]. (E–E″) Diap1 protein levels are increased in Uba1^H33 mutant clones (arrows). Genotype as in D. (F–F″) Dronc protein levels are decreased in Uba1^D6 mutant clones (arrows). Genotype as in B.

Fig. 5. The apoptotic phenotype of strong Uba1 alleles

All discs were incubated at 29°C. (A,C,D) ey-FLP; FRT Uba1^D6/FRT P[ubi-GFP]; (B) ey-FLP; FRT Uba1^H42/FRT P[ubi-GFP]. (A) Uba1^D6 clones are highly Cas3*-apoptotic (see arrows). (B) Uba1^H42 clones lack apoptotic labeling. (C) Uba1^D6 clones (outlined by dashed line) contain increased levels of Diap1 protein. (D) Uba1^D6 clones (outlined by dashed line) contain increased levels of Dronc protein.

Fig. 6. Cell cycle arrest and non-autonomous proliferation in Uba1 mosaics

(A,B) Third instar mosaic Uba1^D6 (A) and Uba1^H42 (B) wing discs incubated at 29°C. Note lack of BrdU labeling in Uba1^D6 mutant clones, and increased BrdU labeling in wild-type tissue surrounding Uba1^D6 clones. Proliferation in Uba1^H42 mosaics is homogenous within and outside the clones (B). (C,D) Cyclin A labeling of eye discs from third instar larval eye discs containing Uba1^D6 (C) and Uba1^H42 (D) mutant clones. Cyclin A levels are increased in Uba1^D6 clones spanning the morphogenetic furrow (arrows in C). Clones of the weak allele Uba1^H42 contain slightly increased Cyclin A levels (D, arrows). However, this increase does not affect cell proliferation (B).

Fig. 7. Notch signaling induces non-autonomous overgrowth in Uba1 mosaics

All discs were incubated at 30°C, except C which was incubated at 25°C. (A) Uba1^H33 mosaic eye discs labeled with Notch antibody C17.9C6 which was raised against the intracellular domain of Notch. Arrows indicate increase of Notch protein in Uba1 clones. This is most prominent immediately anterior to the morphogenetic furrow (arrowhead). Genotype: ey-FLP; FRT Uba1^D6/FRT P[ubi-GFP]. (B,C) Notch activity as visualized by the Notch reporter E(spl)m8 2.61-lacZ is increased in Uba1^D6 clones at 30°C (B) and 25°C (C). Genotype: ey-FLP; E(spl)m8 2.61-lacZ FRT uba^D6/FRT P[ubi-GFP]. (D) pSTAT labeling is increased in a non-autonomous manner in wild-type tissue immediately adjacent to Uba1 clones (arrow). Genotype as in A. (E) Eyes of Uba1^D6 mosaic animals are overgrown with bulges (arrow). Genotype as in A. (F,G) Heterozygosity of Notch (F) and stat92 (G) dominantly suppresses the Uba1^D6 overgrowth phenotype. Genotype: Notch^264-39/ey-FLP; FRT42 Uba1^D6/FRT42 P[ubi-GFP] (F) and ey-FLP; FRT42 Uba1^D6/FRT42 P[ubi-GFP]; stat92^85c9 (G).