JCS042267 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Uba1B1 clones show pnt expression anterior to the MF and an increase in the MF. pnt expression (red), monitored using an pnt-lacZ reporter is seen in the posterior region of eye discs. (A-F) Increased pnt expression is observed in some Uba1B1 mutant clones anterior, in, and just posterior to the MF (arrows). Clonal boundaries, as indicated by GFP were traced and overlaid onto A,D. Two examples are shown (A-C, D-F). Merge of A,B shown in C. Merge of D,E shown in F. The approximate location of the MF is shown by an arrowhead. Discs are of the genotype yweyFLP; FRT42D PW+, UbiGFP/FRT42D Uba1B1; pnt-lacZ. Scale bars: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Pupal lethality of E1 hypomorphic mutants is sensitive to the gene dosage of Ras but not of Egfr or drk. Bar graph representing the percentage of Uba1B1 homozygous adults to eclose from a cross of balanced Uba1B1 heterozygous parents compared to the percentage to emerge in the presence of mutation in Egfr, drk, Ras. Mutation in two different alleles of Ras dominantly suppresses the pupal lethality, whereas mutation in Egfr, drk and sos do not (sos not shown). Similar numbers of males of w; FRT42D Uba1B1/SM6-TM6B w; FRT42D Uba1B1; Ras85De1B/SM6-TM6B, w; FRT42D Uba1B1 Egfrk05115/SM6-TM6B, w; FRT42D Uba1B1 drkk02401/SM6-TM6B, and w; sose4g, FRT42D Uba1B1/SM6-TM6B were crossed to an equal number of Uba1B1/SM6- TM6B female virgins on normal food at room temperature. Crosses were tapped to new vials every day until egg-laying declined. Upon eclosion, adults were collected daily and scored for Cy and Humeral. Percentage of homozygotes was determined by calculating the number of flies lacking SM6-TM6B markers out of the total number of flies. Subtle variations in temperature, humidity and food preparation affected the percentage of mutants to reach adulthood in separate trials. The graph reflects a representative experiment. Alleles of Ras suppressed Uba1B1/Uba1B1 lethality over several independent experiments.

• Supplemental Figure S3 -

  Fig. S3. Ras is mono- and di-ubiquitinated in Drosophila S2 cells. (A) S2 cells were transfected with FLAG-6His-Ras and HA-Ub constructs and Ras was isolated from cell lysates on nickel beads as in Figure 5. Darker exposure of a different western blot (from that shown in Figure 5) using anti-HA antibodies (upper panel) and anti-FLAG antibodies (lower panel) indicates primarily di-ubiquitinated forms of Ras but also mono-ubiquitinated Ras at much lower levels. When cells are transfected with FLAG-6His-Ras only (right lane), higher molecular weight forms of Ras, which are slightly smaller than the FLAG-6His-Ras/HA-ubiquitin species, are observed, which probably correspond to FLAG-6His-Ras conjugated to endogenous (untagged) ubiquitin, which is slightly smaller than HA-Ubiquitin. Molecular weight markers are indicated to the left. (B) Western blot of wild-type and E1 mutant larvae. Ras levels do not accumulate in E1 mutants compared to a tubulin loading control. (C) We attempted to detect Ras-ubiquitin conjugates in wild-type larvae. Mammalian Ras antibodies weakly detect Drosophila Ras but do not immunopurify Drosophila Ras. These antibodies do recognize a faint band of higher molecular weight than unconjugated Ras (arrow), but it is unclear if this band represents Ras-ubiquitin conjugates or other cross-reacting proteins.

• Supplemental Figure S4 -

  Fig. S4. Increases in pnt and aos in and/or surrounding Uba1A1 clones. (A-F) pnt expression (red) increases in Uba1A1 clones in and anterior to the MF. An enlarged view of the boxed region in A-C is shown in D-F. The disc pictured is of the genotype ywhsFLP; FRT42D PW+, UbiGFP/FRT42D Uba1A1; pnt-lacZ. (G-I) Due to high expression of aos posterior to the MF, distinguishing changes in the staining pattern is difficult; however, by adjusting the laser intensity at scanning, increased aos expression can be seen surrounding some large Uba1A1 clones (arrows) posterior to the MF. The disc pictured is of the genotype ywhsFLP; FRT42D PW+, UbiGFP/FRT42D Uba1A1; aos-lacZ. Clonal boundaries, as indicated by GFP in E,H, were traced and overlaid onto D,G. Scale bar: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. E1 non-autonomous overgrowth resembles oncogenic activation of Ras signaling through ERK, not other downstream effectors. (A,B) Depiction of Ras activation (‘R’) in eye discs generated by creating homozygous Uba1A1 mutant (white) and wild-type (red) cells early in eye development (A) or by overexpressing activated forms of Ras (brown) (B). In a mosaic eye (A), Uba1A1 mutant cells promote Ras activation in neighboring wild-type cells. Because this happens early, it is likely that all wild-type cells have activated Ras. As development proceeds, mostly wild-type cells with activated Ras populate the eye. Similarly, when RasV12 is overexpressed early in eye development (B), these cells with activated Ras populate the eye. (C-F) The gross phenotype observed in Uba1A1 mosaic eyes (left eye, C-F, genotype yweyFLP; FRT42D PW+, UbiGFP/FRT42D Uba1A1) is sensitive to the gene dosage of Ras (right eye, C; also Figure 7A, genotype yweyFLP; FRT42D PW+, UbiGFP/FRT42D Uba1A1; Ras85De1B/+) and also resembles that of overexpressing in the early eye the effector loop mutant RasV12S35 (which primarily signals through RAF/ERK) (right eye, D, genotype w; ey gal4/UAS RasV12S35) and RasV12 (Figure 7D), both of which encode the oncogenic activating V12 mutation. By contrast, overexpressing the effector loop mutants RasV12G37 (right eye, E, genotype w; ey gal4/UAS RasV12G37) and RasV12C40 (right eye, F, genotype w, UAS RasV12C40; ey gal4) early in the eye produces no obvious overgrowth. Male eyes are shown in C-D and female eyes are shown in E-F.

• Supplemental Figure S6 -

  Fig. S6. Mutation in Ras suppresses the non-autonomous decrease in Yan adjacent to Uba1A1 clones and RasV12 overexpression promote non-autonomous decrease in Yan. (A-C) Yan staining (red) in a Uba1A1 mosaic eye where one copy of Ras was removed is normal pattern. No decrease is seen surrounding clones of Uba1A1, confirming that effects on Yan were Ras-dependent. Disc is of the genotype ywhsFLP; FRT42D PW+, UbiGFP/FRT42D Uba1A1; Ras85De1B/+. (D-F) Posterior to the MF, no difference in Yan (red) is seen between FRT42D-tubpGal80 tissue (GFP-negative) and FRT42D clones labeled with a cell-surface GFP (MARCM techniques). (GI) By contrast, RasV12 overexpressing clones (labeled with a cell-surface GFP) exhibit a clear decrease in Yan. Moreover, a decrease in Yan is seen in FRT42D-tubpGal80 tissue (GFP-negative) adjacent to RasV12 clones (arrows). Clonal boundaries indicated by GFP in E,H were traced and overlaid onto D,G. Scale bars: 50 µm.