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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00855

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Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis

HHMI/Molecular Biology Department, Princeton University, Princeton, NJ 08544, USA

View larger version (87K): [in a new window]   Fig. 1. Cyclin A protein expression in the germarium. (A, left) The four mitotic divisions of the cystoblast and its stereotypic relationship with each other and with the fusome (red). (A, right) The different regions of the Drosophila germarium. The germline stem cells, and dividing cystoblast comprise region 1. Region 2A contains post-mitotic 16 cell-cysts (green). The boundary between regions 2A and 2B is marked by the follicle cells migrating inwards to envelop the cyst and to form the egg chamber. The red lines represent the spectrosome in the germline stem cells and the fusome connecting all the cells in the dividing cystoblast and postmitotic cyst. Region 3 of the germarium is characterized by the budding of newly formed egg chambers. (B-G) Expression of Cyclin A (red) and the mitotic marker phosphohistone 3 (green) in wild-type and in encore mutant germaria. (B-D) In wild-type ovaries, Cyclin A is expressed in the stem cells and dividing cystoblast in region 1 of the germarium. It rapidly declines in post-mitotic cysts. (E-G) In encore mutant germaria, Cyclin A expression persists after the end of mitosis. The arrow shows a cyst positive for Cyclin A in a posterior area of the germarium.

View larger version (85K): [in a new window]   Fig. 2. Cyclin A promotes the encore extra division phenotype. (A-C) In wild-type germaria Cyclin A (red) associates with the fusome (green stained with phalloidin for actin) in dividing cystoblasts (arrow and inset). (D-F) Association of Cyclin A with the fusome in encore mutant germaria persists in posterior regions of the germarium (arrow and inset). (G) encore mutant females raised at 25°C produce 55% of the egg chambers with 32 cells. Reducing Cyclin A gene dose has no effect on the encore cell division phenotype. However overexpression of the HS-rux transgene in an encore mutant background reduces the 32 cells egg chamber phenotype from 55 to 25%. Additional reduction of Cyclin A gene dose results in 20% of the egg chambers containing 32 cells. (H) Western blot showing that the amount of Cyclin A protein in encore mutant extract is increased compared with the wild-type extract. Equivalent amounts of germaria-enriched extract were loaded in both lines as seen with the protein dye ponceau (pink).

View larger version (67K): [in a new window]   Fig. 3. Expression of Cyclin E in the germarium. (A,B) Spectrin (red) marks the spectrosome in the germline stem cell and the fusome in the developing cyst. (A) Cyclin E protein expression (green) in the wild-type ovary is observed in region 1 of the germarium and sharply decreases in regions 2A and 2B. Asynchronous expression of Cyclin E resumes in region 3 and persists in the vitellarium. In wild-type germaria, Cyclin E expression oscillates strongly (inset): some cysts show Cyclin E expression (arrow), while other cysts are depleted of Cyclin E protein (arrowhead). (B) In encore mutant germaria, Cyclin E expression is observed throughout the germarium. There is no downregulation in region 2A/B. In encore mutant germaria, all cysts express some Cyclin E protein all the time (inset). (C) Reducing cyclin E gene dose in an encore mutant background suppresses the encore 32-cell egg chamber phenotype from 70 to 10% in females raised at 29°C. (D-F) Western blots of germaria-enriched extracts. (D) The accumulation of Cyclin A observed in encore mutant females can be partly restored to more normal levels by reducing cyclin E gene dose. The blot was stained with the protein dye ponceau (pink) as loading control. (E) The inhibition of proteolysis (Pase Inh.) or/and 19S-RP isopeptidase activity (NEM/Llnl) reveals that in encore mutant extracts Cyclin E accumulates as a high molecular form protein (arrowheads). The blot was stained with the protein dye ponceau (pink) as loading control. (F) Immunoprecipitation assays using anti-Drosophila Cyclin E antibodies followed by IB using anti-Ubiquitin antibodies shows more Cyclin E-Ubn in the encore mutant extract lane compared with wild type. The reciprocal immunoprecipitation assay shows more Cyclin E-Ubn in the encore mutant lane. Extract concentration of the starting material is shown stained with Coloidal blue Coomassie. The control lane (co) represents immunoprecipitation using wild-type extracts and an unrelated antibody raised in the same animal as the immunoprecipitation test.

View larger version (69K): [in a new window]   Fig. 4. Expression of Cul1 in the Drosophila germarium. (A-C) The Cul1 protein (red) is a nuclear protein but in the wild-type germarium mostly associates with the spectrosome (arrow) and the fusome (stained with antibodies against the Hts protein, green). (D-F) In encore mutant germaria, Cul1 association with the fusome is much-reduced (inset) and the Cul1 staining is prominently nuclear. Wild-type and encore mutant flies were raised at 29°C. (G) Genetic interactions between encore and cul1, UbcD2 and effete alleles. Reduction of cul1 gene dose in an encore mutant background enhances the 32-cell egg chamber phenotype from 27 to 65% at 25°C and from 3 to 44% at room temperature (RT). Enhancement of the phenotype from 27 to 60% at 25°C and from 3 to 17% at room temperature is also seen by reducing the gene dose of UbcD2. More dramatic enhancement from 27 to 82% at 25°C and from 3 to 75% at room temperature is observed when effete gene dose is reduced. Similar results were obtained using different cul1 and effete alleles.

View larger version (83K): [in a new window]   Fig. 5. Encore associates with Cyclin E, Cul1, the proteasome 19S-RP subunit S1 and the proteasome 20S-CP LMP7. (A) Immunoblot (IB) using antibodies against Cyclin E and Ubiquitin showing that Cul1 and Encore can immunoprecipitate Cyclin E-Ubn. Immunoprecipitation with S1 and LMP7 precipitates several polyubiquitinated Cyclin E forms. The control lane (co) refers to immunoprecipitations that use unrelated antibodies raised in the same animal as the antibody used for the immunoprecipitation test. (B) IB using antibodies against Encore shows that Cyclin E, Cul1, S1 and LMP7 associate with Encore in the ovary. Notice that the amounts of immunoprecipitated protein in the Cul1 panel are comparable in wild-type and encore mutant extracts. The S1 and LMP7 immunoprecipitation lanes show that several forms of Encore associates with S1 and LMP7. The wild-type extract lane indicates the position of Encore in the gel. The extracts were run in 7%Tris-Acetate Nupage gel (Invitrogen).

View larger version (123K): [in a new window]   Fig. 6. Expression of the 19S-RP subunit S1 and P-Cyclin E in the Drosophila germarium. (A-C) 19S-S1 (red) localizes to the fusome (green, stained with Hts). Some fusomes are free of 19S-S1 staining (arrowhead). S1 is also accumulated in a granular manner in the rest of the germarium. (D-F) 19S-S1 expression in encore mutants is reduced; however, some S1 protein can still by observed in the mutant germarium. (G-I) In wild-type germaria, P-Cyclin E (red) localizes to the fusome (green). The expression is transient as some fusomes express some P-Cyclin E and others do not. (J-L) In encore mutant germaria, P-Cyclin E is more often observed localized to the fusome.

View larger version (34K): [in a new window]   Fig. 7. Encore is required for proteolysis. (A) Proteasome assays measuring the hydrolysis of the fluorogenic peptide Suc-LLVYMCA. The 20S-CP proteolysis activity is not significantly compromised in encore mutant germarium-enriched extracts at 29°C (yellow) or room temperature (light blue) compared with wild-type extracts at 29°C (pink). Control assays contain no substrate peptide (dark blue). (B) Western blot using anti p27 antibodies show that Ubiquitination reactions produce the same polyubiquitinated p27 forms (arrowheads) in wild-type and encore mutant extracts. The deubiquitination and degradation reaction is much slower when encore mutant extract was used as a source of the degradation machinery. (C) Model of Encore function. Cul1 (pink) is localized to the fusome (green) in an Encore (blue)-dependent manner (Encore may directly or indirectly modify Cul1 and thus influence its subcellular localization). Cul1 may serve as an anchor where the SCF E3 complex is assembled and a phosphorylated substrate (yellow) is recognized and ubiquitinated. The polyubiquitinated substrate is then recognized by the 19S-recognition particle. 19S-RP and presumably the 20S-core particle are recruited to the fusome where the substrate is de-ubiquitinated, unfolded and degraded by the 26S-subunit of the proteasome.

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