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First published online 20 July 2005
doi: 10.1242/dev.01940

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Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (113K): [in a new window]   Fig. 1. Embryos from remnants mutant females arrest in early embryogenesis. (A-C) Embryos labeled for DNA (green), microtubules (red) and Cnn to label centrosomes (blue). (A) Wild-type embryo in metaphase of syncytial cycle. (B,C) Embryos from remHG24/remHG24 arrested with one metaphase spindle (B) and with multiple metaphase spindles (C). Nuclei fail to migrate and accumulate at the anterior of the embryo. Spindles often lack centrosomes and/or are unipolar (insets B,C). (D) Unfertilized egg from remRA74/Df(2L)30AC possessing polar bodies but no mitotic spindles. (E,F) Microtubules (white) and FISH against X chromosome (green), and Y chromosome (red). (E) Wild-type embryo in which all mitotic spindles label for both X and Y chromosomes. (F) remHG24/remHG24 embryo in which mitotic spindles contain both X and Y chromosomes. Scale bars: in A, 20 µm for A,B,C; in D, 50 µm; in E, 10 µm for E,F.

View larger version (52K): [in a new window]   Fig. 2. Female meiosis is disrupted in rem mutants. (A,B,D,F) remHG24/remHG24, (C,E,G) wild type and (H) remRA74/Df(2L)30AC, labeled for DNA (green) and microtubules (red). (A) Metaphase I of meiosis is typically not affected in rem mutants, but 7% display disorganized spindles as in B. (C,D) Metaphase of meiosis II. In wild type, the meiosis II spindles are arrayed in tandem and connected by the spindle mid-body (arrow), while in rem, the spindles are kinked or separated. (E,F) Anaphase II. In wild type, four meiotic products with equal DNA content can be distinguished. rem spindles frequently break down and mis-segregation occurs. In the lower spindle, the two meiotic products are of different sizes, while the upper spindle has not entered anaphase. Pronuclear fusion in wild type (G) and rem (H). All nuclei are in interphase, and the male pronucleus and female pronucleus are in apposition and associated with the male sperm aster (arrows in G,H). The male pronucleus is largely obscured by the sperm aster in H. (I,J) Polar bodies from wild type (I) and remHG24/remRA74 (J) stained for microtubules (red) and the prophase/metaphase marker phospho-histone H3 (blue). Overlap appears pink. In wild type and in rem, DNA labels with phospho-histone H3, although in rem the chromatin is surrounded by microtubules within a shell, while in wild type the chromatin radiates out from a microtubule array. Scale bar: in A, 5 µm for A-H.

View larger version (48K): [in a new window]   Fig. 3. rem encodes one of two Drosophila Cks gene homologs. (A) Alignment of Drosophila Cks30A (Remnants) with Cks from other organisms. * indicates residue P61 altered in Cks30ARA74 and Cks30AHG24. + indicates residues implicated in Skp2 binding (S/T41,N45) for human Cks1. (B) Phylogenetic reconstruction (MacVector Neighbor Building Best Tree) does not permit assignment of Drosophila Cks proteins as orthologs of vertebrate Cks1 and Cks2, respectively. (C) Developmental northern blot probed for Cks30A and Cks85A reveals that Cks30A is mainly expressed in ovaries and embryos. Low concentrations are detected in third instar larvae and pupae. Cks85A is found at all stages examined. (D) Western blot from 0-2 embryos from wild type, Cks30AKO/Cks30AKO and Cks30AHG24/Cks30AHG24 probed with anti-Cks30A antiserum reveals a single band in wild-type and Cks30AHG24 mutants, but no protein in Cks30AKO.

View larger version (139K): [in a new window]   Fig. 4. Cks30AKO homozygous embryos (40-80 minutes after egg-laying) arrest either with a single metaphase spindle (A) or without assembling a spindle (C). The arrow in A indicates a part of a meiotic spindle that has failed to disassemble. Arrows in C point to separated centrosomes. Embryos are labeled for DNA (green), microtubules (red) and Cnn (blue). (B) Early embryo (0-20 minutes after egg-laying) labeled for DNA (green) and microtubules (red), contains two closely apposed centrosomes, one of which is associated with the paternal chromatin (arrow). This embryo also shows the typical delay in metaphase of meiosis II. (D) FISH labeling of the X chromosome (green) and Y chromosome (red) and microtubules (white) reveals that the metaphase spindle (lower) in Cks30AKO homozygous embryos contains only the male DNA complement, indicating that pronuclear fusion has not occurred. Polar bodies with only X chromosome signal (green) are at the top. Scale bars: 20 µm.

View larger version (93K): [in a new window]   Fig. 5. Abdominal cuticle phenotypes of (A) wild type, (B) Cks30AHG24/Cks30ARA74, (C) Cks30AHG24/Cks30ARA74,cdk1E1-23. Arrow in B indicates a minor cuticle defect.

View larger version (93K): [in a new window]   Fig. 6. Drosophila Cks30A interacts with Cdks. Western blots from FLAG-Cks immunoprecipitations probed with antibodies to Cdk1, Cdk2, Cyclin A, Cyclin B and Cyclin B3. The lower band in the Cyclin B lane is antibody heavy chain (HC). FLAG-Cks85A strongly co-immunoprecipitates Cdk1, Cdk2 and the mitotic cyclins, while FLAG-Cks30A co-immunoprecipitates Cdk1 and very weakly Cdk2. Cyclin B and Cyclin B3 are detected only in longer exposures. Cyclin B3 is overexposed here. FLAG-Cks30AP90A fails to co-immunoprecipitate Cdk1 or mitotic cyclins. Sup (last lane) is IP supernatant.

View larger version (68K): [in a new window]   Fig. 7. Cks30A and Cortex mediate Cyclin A destruction. (A) Western blot from Cks30AKO/Cks30AKO and unfertilized wild-type embryos (0-2 hours after egg-laying) and ovaries probed for Cyclin A, B and B3, revealing excess Cyclin A in Cks30A mutants. The upper (active) form is particularly enriched. Far lane, Cks30AKO extract at 1/12 dilution contains approximately equal Cyclin A concentrations to wild type (see Materials and methods), indicating a 12-fold excess of Cyclin A in the mutant. (B) RT-PCR on wild-type and Cks30AKO/Cks30AKO ovaries and embryos (0-2 hours after egg-laying) reveals similar cyclin A mRNA concentrations. (C) Embryo from Cks30ARA74/Cks30AKO;cycAC8LR1/+ female (0-2 hours after egg-laying). Reduction of cyclin A dosage partially suppresses the Cks30A mutant phenotype, allowing some embryos to develop normally through several syncytial divisions. Mitotic spindles in this embryo are bipolar and are evenly spaced (compare to Fig. 1C – both embryos are at a similar stage, with approximately 32 nuclei). (D) Western blot from wild-type and cortQW55 ovaries probed for Cyclin A and B reveals an elevated concentration of Cyclin A. (E) Northern blot from wild-type and Cks30AHG24/Cks30AHG24 ovaries probed for fzy and cortex mRNA shows equal transcript concentrations, and (below) western blot from wild-type and Cks30AKO/Cks30AKO ovaries probed for Fzr protein, showing wild-type concentrations of Fzr protein. The upper band is non-specific and serves as a loading control.

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