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First published online 2 September 2003
doi: 10.1242/jcs.00722

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Mutations in mákos, a Drosophila gene encoding the Cdc27 subunit of the anaphase promoting complex, enhance centrosomal defects in polo and are suppressed by mutations in twins/aar, which encodes a regulatory subunit of PP2A

Péter Deák^*, Mary Donaldson and David M. Glover

Cancer Research UK, Cell Cycle Genetics Research Group, University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK

View larger version (67K): [in a new window]   Fig. 1. Mutations in mks, a Cdc27 APC/C subunit gene at 65E, result in a metaphase arrest with overcondensed chromosomes. (A,E) Squashed preparations of wild-type larval neuroblasts in metaphase and late anaphase, respectively. (B-D,F-H) Overcondensed mitotic chromosomes in preparations of mks1 cells. Most cells are diploid (B,C,F,G), although 1-2% are tetraploid (D,H). (C, arrows) A diploid cell in which sister chromatids are well separated. Anaphase cells were classified as cells that were always elongated and in which the chromosome distribution followed the direction of the elongation. They were distinguished from prometaphase cells, which did not show such elongation and had chromosomes in a random position. The proportion of anaphases is reduced compared to the wild type (Table 1A) and, when these do occur, they show lagging chromatids (F,G, arrowheads). (I) A molecular map of the 65E region indicating the sites of the P-insertions in mks1 and mks2. In situ hybridization shows the P-element responsible for the mks1 mutation is inserted at 65E. Restriction endonuclease cleavage sites for HindIII (H), EcoRI (E), BamHI (B), BglII (Bg), SaccII (S), SpeI (Sp), ScaI (Sc) and XmnI (X) are indicated. Segments of chromosomal DNA inserted into the rescuing transformation plasmid, pR6.2 and its negative control, p76.2Bam (see Materials and Methods) are indicated below the map. (J) Estimation of mks transcripts relative to those of rpL17A in third instar larvae by RT-PCR (see Materials and Methods). Canton S (Can S) larvae were used as the wild type.

View larger version (71K): [in a new window]   Fig. 2. Sister centromere separation occurs in mks1 neuroblasts. (A,B) Hybridization of a dodecasatellite probe to squashed preparations of wild-type and mks1 cells, respectively. Two dots of signal can be detected in wild-type cells (mitotic chromosomes in inset), corresponding to the centromeric regions of the maternal and paternal third chromosome. mks1 cells show four dots of signal, indicating that the sister centromeric regions have separated. DNA is shown in red, hybridization signal in yellow. Scale bar, 50 µm. (C-H) Immunolocalization of Prod (red, C-E) or Bub1 (red, F-H) on condensed chromosomes (blue) associated with mitotic spindles (green) in wild-type (C,F) and mks1 mutant cells (D,E,G,H). The paired monochromatic images show Prod (C-E) or Bub1 (F-H) staining alone. Prod is present at four punctate sites on chromosomes 2 and 3 in wild-type cells (C) and four pairs of sites, separated centromeres, in mks1 (D: one pair is out of the focal plane). (E) A polyploid cell. Bub1 is present on separated kinetochores in mks1 cells (F, 4N; G, 8N; not all kinetochores are in the focal plane). The mks1 cell in (H) has progressed into anaphase and the Bub1 signal can no longer be detected. Scale bars, 10 µm.

View larger version (96K): [in a new window]   Fig. 3. Cyclins A and B are not degraded in mks1 cells. Paired images of mitotic cells showing tubulin (green), DNA (blue) and cyclin A (red and monochrome; A-D) or cyclin B (red and monochrome; E-H). (A) Wild-type cell in prophase showing cyclin A staining. (B) Wild-type cell at onset of metaphase in which cyclin A has been degraded. (C,D) Metaphase-like mks1 cells in which cyclin A is not degraded. (E) Wild-type cell at metaphase showing cyclin B staining. (F) Wild-type cell at anaphase in which cyclin B has been degraded. (G) Metaphase-like mks1 cells in which cyclin B is not degraded. Scale bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 4. mks1 is epistatic to polo1 in respect of mitotic progression and cyclin degradation. (A-D) Orcein-stained squashed preparations of polo1 mks1 cells that display overcondensed chromosomes (A,C,D), polyploidy (B,C) and circular (A,B) or bar-like figures (D). The mitotic index, proportions of metaphase:anaphase cells and proportion of polyploids are similar to that in the mks1 mutant alone (Table 1B). (E) Western blots showing levels of cyclins A and B in wild-type, polo1, mks1 and polo1 mks1 brains. This reflects cyclin levels in the cycling cells of this tissue. Cyclin A is substantially reduced in, but not absent from, polo1 cells, suggesting that the APC/C is functional. Cyclin B is still present, indicative of checkpoint delay to mitosis. Both mitotic cyclins are present at equivalent levels in the double mutant combination, indicating loss of APC/C function. Actin was used as a protein-loading control.

View larger version (97K): [in a new window]   Fig. 5. mks1 enhances polo1 centrosomal defects. The columns display mitotic spindles from neuroblasts of the respective genotypes wild-type, polo1, mks1 and polo1 mks1 stained to reveal DNA (blue) and microtubules (green). The first three rows show immunostaining to reveal the indicated centrosomal antigens CNN, -tubulin and CP190 (all in red). The final row displays immunostaining of the mitotic phosphoepitope MPM2 (red). In all the indicated genotypes, the core centrosomal antigen CNN is localized in a wild-type manner to the centrosomes. -Tubulin and CP190 are localized to centrosomes and have a wild-type pattern of localization in both polo1 and mks1. However, in the double mutant, they are dispersed throughout the cell. The MPM2 epitope is present throughout the cell, at kinetochores and spindle poles in all indicated genotypes except polo1 mks1, in which it is absent from the spindle poles. Scale bar, 10 µm.

View larger version (128K): [in a new window]   Fig. 6. tws618/5 mks1 cells show abnormal anaphases. Orcein stained squashed preparations of mks1(A,D), tws618/5 (B,E) and tws618/5 mks1 (C,F) cells show that tws618/5 is epistatic to mks1 with respect to abnormal anaphase. The proportion of anaphase figures is similar in tws618/5 mks1 and tws618/5 cells (Table 1C). However, mks1 is epistatic to tws618/5 with respect to chromosome condensation.

View larger version (111K): [in a new window]   Fig. 7. Cyclin B is not degraded in the abnormal anaphase tws618/5 mks1 cells. Typical mitotic figures from mks1 (A,D,G), tws618/5 (B,E,H) and tws618/5mks1 (C,F,I) cells stained to reveal microtubules (green), DNA (blue) and cyclin B (red). The panels of monochrome staining show chromosomes (D-F) and cyclin B (G-I) alone. Cyclin B is not degraded in mks1, tws618/5 or tws618/5 mks1 double mutant cells. We use the same criteria to define the abnormally resolved anaphases of the twins/aar mutants as Gomes et al. (Gomes et al., 1993) and Mayer-Jaekel et al. (Mayer-Jaekel et al., 1993).

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