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First published online 14 April 2009
doi: 10.1242/jcs.042267

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Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo

¹ Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, NY 10029, USA
² Department of Developmental and Regenerative Biology, The Mount Sinai School of Medicine, New York, NY 10029, USA
³ Harvard Medical School, Boston, MA 02115, USA

View larger version (71K): [in this window] [in a new window]   Fig. 1. E1 hypomorphic tissue demonstrates increased proliferation and rare tissue outgrowths. (A,B) Anti-pHH3 staining of Drosophila third instar larval eye discs (red). In wild-type discs (A), anti-pHH3 stains the anterior region (left), mostly just anterior to the MF where cells divide more synchronously. Anti-pHH3 also stains the SMW. No significant staining is seen in the MF or posterior to the SMW (MF indicated by arrowhead). (B) In Uba1B1/Uba1B2 eye discs, extensive anti-pHH3 cells are seen posterior to the MF. Increased anti-pHH3 staining is seen both anterior to the MF and in the most posterior region. Anterior is to the left and posterior is to the right for this and all eye disc figures unless otherwise indicated. (C) Individual spots positive for anti-pHH3 were counted in FRT42D control discs and Uba1B1/Uba1B12 discs and the average calculated per genotype. The bar graph indicates mean ± s.e.m. (D) Adult flies homozygous for Uba1B1 occasionally exhibit protruding outgrowths such as in the humeral region. Genotypes for eye discs and adults for this and all subsequent figures are listed in the Materials and Methods section. Scale bars: 100 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 2. E1 hypomorphic tissue and larvae demonstrate increased dpERK activity. (A) Schematic indicating important components of the Ras pathway, including upstream activation by RTKs and downstream effectors. Tools used in all figures to analyze the pathway (antibodies, reporters, activated Ras constructs) are indicated. (B-J) Activation of ERK is visualized with antibodies to dpERK (red). (B-D) dpERK staining in a larval eye disc containing clones homozygous for the FRT42D control chromosome (GFP-negative) and clones homozygous for FRT42D P[W+, UbiGFP] (GFP-positive). GFP-positive tissue appears green; GFP-negative tissue appears dark. The size of dpERK clusters in the MF does not change in FRT42D clones (dark) compared to GFP-labeled clones (green). Merge of B,C shown in D. (E-J) In eye discs containing clones homozygous for Uba1B1 (GFP-negative, dark), larger dpERK-positive clusters are seen in mutant tissue (arrows) compared with clusters in wild-type clones (GFP-positive, green). Subtle increases in intensity of dpERK may occur in posterior mutant clones (arrowhead). Merge of E,F shown in G. Enlargement of the boxed region in E-G is shown in H-J. Tracing of clonal boundaries (white) indicated by GFP is overlaid onto the dpERK panel. (K) Tracings of representative dpERK clusters in FRT42D control (+/+, black) or GFP wild-type tissue (+/+, green) are to the left; tracings of mutant (-/-, black) or GFP wild-type (+/+, green) are to the right. Below the tracings, average area ± s.e.m. is indicated. dpERK cluster size does not differ between FRT42D and FRT42D P[W+, UbiGFP] tissue, but Uba1B1 dpERK clusters are substantially larger than both FRT42D and FRT42D P[W+, UbiGFP] clones. Statistical analysis using Graphpad online software indicates this increase is statistically significant: P=0.0003 (comparison to FRT42D clones) and P=0.0015 (comparison to FRT42D P[W+, UbiGFP] clones) in paired t-tests. Wild-type tissue is GFP-positive and appears green; mutant tissue is GFP-negative and appears dark in E-J in this figure and all subsequent figures containing mosaic tissues unless otherwise indicated. (L-M) Western blots of third instar larvae homozygous for w; FRT42D, w; FRT42D Uba1B1, or w; FRT42D Uba1B2. (L) Western blot using anti-E1 antibodies. (M) Western blot of individual larvae using dpERK antibodies (upper panel) or stripping and reprobing for total ERK (lower panel). Scale bars: 50 µm.

View larger version (144K): [in this window] [in a new window]   Fig. 3. E1 hypomorphic tissues show inappropriate and increased aos expression. (A-I) aos expression (red), monitored using an aos-lacZ reporter in eye discs containing clones homozygous for Uba1B1 (GFP-negative, dark) and clones homozygous for the wild-type chromosome P[W+, UbiGFP] (GFP-positive, green). (A-F) Increased aos expression is observed in Uba1B1 mutant clones in and anterior to the MF (arrows). Merge of A,B shown in C. Enlargement of the boxed region in A-C is shown in D-F. (G-I) A large Uba1B1 clone shows increased aos (red in D,F) in the posterior region compared with the wild-type clone it surrounds. Measuring staining intensity using Adobe Photoshop indicates an increase in staining by almost 60% for the clone indicated. Tracing of clonal boundaries (white) indicated by GFP was overlaid onto aos panels. Scale bars: 50 µm in A-C; 25 µm in D-I.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Increased proliferation of E1 hypomorphic mutants is sensitive to the gene dosage of Ras but not of Egfr, drk and sos. Uba1B2 homozygous mutant tissue (white) is over-represented in a mosaic eye compared to wild-type FRT42D P[W+, UbiGFP] tissue (red) (B), but a mosaic eye composed of wild-type FRT42D tissue (unpigmented, white) and FRT42D P[W+, UbiGFP] tissue (pigmented, red) shows approximately equal white and red tissue (A). Removing one functional copy of Ras suppresses the over-representation of Uba1B2 (C). (D-F) Expressing eiger in differentiating cells in the eye (GMR>eiger) results in almost no adult eye (D); however, eye size is partially restored in Uba1B1/UbaB2 eyes (E). Removing one copy of Ras in Uba1B1/UbaB2 eyes suppresses the resistance to eiger-induced cell death (F). (G-L) Anti-pHH3 staining of larval eye discs (red) shows the normal pattern in a wild-type disc (G), and increased mitoses in a Uba1B1/UbaB2 disc (H). Removing one copy of Ras dramatically suppresses the increased proliferation (I), particularly posterior to the MF. By contrast, mutation in Egfr, Egfrk05115 (J), drk, drkk02401 (K), or sos, sose4g (L) does not. An arrowhead indicates the MF. (M) Bar graph representing total anti-pHH3 positive spots of FRT42D control discs (`+/+' gray bar) and Uba1B1/Uba1B2 discs in the absence of additional mutations or in the presence of mutation in Egfr, drk, sos and Ras. Uba1B1/Uba1B2 discs show twice as much anti-pHH3 staining as wild-type FRT42D discs. Mutation in Ras (gray bar) dominantly suppresses this increase by more than 50%, whereas mutations in Egfr, drk and sos (dark gray bars) do not. Scale bars: 100 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Ras is ubiquitylated in Drosophila S2 cells. S2 cells were transfected with FLAG-6His-Ras and HA-Ub. Ras was isolated from cell lysates on nickel beads. Western blot using anti-HA antibodies (upper panel) and anti-FLAG antibodies (lower panel) indicates primarily di-ubiquitylated forms of Ras (arrows). Unconjugated Ras is indicated by an arrowhead. Similar bands are not seen in nickel pulldowns from a non-transfected control (left lane) or a control transfected with HA-Ubiquitin only (middle lane). Molecular weight markers, left.

View larger version (84K): [in this window] [in a new window]   Fig. 6. Increased and/or inappropriate Ras-ERK activation occurs in and adjacent to E1 homozygous null clones. (A-F) dpERK staining (red) is altered (arrows) in an eye disc containing clones homozygous for null mutation Uba1A1. Uba1A1 clones show dpERK staining, and dpERK clusters are seen in tissue anterior to the MF in Uba1A1 clones (lower arrows). Occasionally, increased dpERK staining is seen surrounding null clones (upper arrow). Merge of A,B shown in C. Enlargement of the boxed region in A-C is shown in D-F. (G-L) A decrease in Yan staining (red) is seen in tissue adjacent to Uba1A1 clones posterior to the MF (arrows). Merge of G,H shown in I. Enlargement of the boxed region in G-I is shown in J-L. (M-R) aos expression (red) is monitored by staining with anti-β-gal antibodies using an aos-lacZ reporter. (M-O) A ring of aos is seen anterior to the MF in tissue adjacent to Uba1A1 clones (arrows). Merge of M,N shown in O. Enlargement of the boxed region in M-O is shown in P-R. Scale bars: 50 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 7. Non-autonomous overgrowth caused by E1 null mutation is sensitive to the gene dosage of Ras and resembles oncogenic Ras. The dramatic overgrowth of wild-type tissue in an eye containing Uba1A1 clones (left eye, A,B,D) is dramatically suppressed by removing one copy of Ras (right eye in A). (B) By contrast, mutation in AKT (right eye in B) has no obvious effect. (C) When Uba1A1 homozygous mutant cells signal to cells homozygous for mutation in PTP-ER in a mosaic eye (right eye in C), additional outgrowths are seen, primarily around the periphery of the eye (arrows). In comparison, a mosaic eye composed almost entirely of PTP-ER mutant cells (left eye, C) generated by creating an eye mosaic for clones homozygous for PTP-ER mutation and clones homozygous for a cell lethal mutation is of normal size and contains no outgrowths. Darker tissue in the PTP-ER mutant eye (left) represents a small percentage of tissue that did not undergo mitotic recombination. (D) The gross phenotype observed in mosaic eyes containing Uba1A1 homozygous clones and wild-type FRT42D clones resembles expression of oncogenic Ras, RasV12 in the early eye (ey>RasV12, right eye in D). A schematic above each pair of heads depicts the genotypes of cells in the mosaic eyes (A-C, left eye in D) or RasV12 expressing eyes (D, right eye). Eyes in A-D are female. The same Uba1A1 mosaic is shown in A-D. (E-J) Yan staining (red) posterior to the MF in mosaic eye discs containing clones homozygous for expression of RasV12S35. RasV12S35 cells are GFP-positive (traced in white), and non-expressing cells are GFP-negative. Decreased Yan staining surrounding RasV12S235 clones is not due to clones in a different focal plane; white-dashed tracings indicate RasV12S35 cells at a different focal plane. Merge of E,F shown in G. Enlarged view of the boxed region is shown in H-J. Anterior is to the lower left, posterior to the upper right. Scale bar: 50 µm.

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