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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01602

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The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis

¹ Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
² Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
³ Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar n° 2, 4099-003 Porto, Portugal

View larger version (58K): [in a new window]   Fig. 1. Mutations in bub3 result in premature sister chromatid separation, abnormal anaphase and aneuploidy. (A) Western blot of total protein extracts from wild-type (Oregon R), bub31 homozygous and bub31/Df(3R)Dr-rv1 neuroblasts with anti-Bub3 antibody. -tubulin was detected as a loading control. (B) Immunolocalization of Bub3 in wild-type and bub31 mutant neuroblasts (n=354) showing that the Bub3 mutant protein fails to localize to kinetochores in 46% of the mitotic cells. In 45% of the prometaphases of bub31 mutant cells, Bub3 can localize to some kinetochores, whereas in only 9% of prometaphases is Bub3 detected with reduced intensity at all kinetochores. Polo was used to label kinetochores. DNA is shown in blue, polo in red and bub3 in green in the merged image. (C) Mitotic index of third instar larvae neuroblasts from Oregon R, bub31/bub31 and bub31/Df(3R)Dr-rv1, after a 1 hour incubation in colchicine. bub31 mutant cells fail to sustain mitotic arrest upon spindle damage. (D) Squashed preparations of wild-type and bub31 neuroblasts after incubation in colchicine. Upon spindle damage, bub31 mutant cells fail to maintain sister chromatid cohesion. (E) Squashed preparations of wild-type and bub31 mutant larvae neuroblasts at different stages of mitosis. bub31 mutant neuroblasts show aneuploidy, PSCS and anaphases with lagging chromatids. (F) Quantification of mitotic parameters in Oregon R, bub31 homozygous and bub31/Df(3R)Dr-rv1 third instar larvae neuroblasts, showing that homozygous and hemizygous cells behave differently during the initial stages of mitosis. Bar, 5 µm.

View larger version (33K): [in a new window]   Fig. 2. Depletion of Bub3 in Drosophila S2 cells results in slower progression through prophase and premature exit from mitosis. (A) Western blot showing depletion of Bub3 after RNAi at different times. -tubulin antibody was used as loading control. (B) Immunolocalization of Bub3 in control and Bub3 dsRNA-treated cells, after 96 hours in culture. Polo was used to label kinetochores. Merged images are shown with DNA in blue, polo in red and bub3 in green. (C) Mitotic index of control and S2 cells treated with Bub3 dsRNA for 96 hours after incubation in colchicine for different times. Mitotic index was determined as the number of phospho-histone H3 (PH3)-positive cells over the total cell population. (D) Percentage of mitotic cells showing sister chromatid separation (SCS, anaphases plus PSCS) after incubation with colchicine, in control and Bub3-depleted cells. (E) RNAi-treated Bub3 cells showing SCS after incubation with colchicine. DNA is shown in red and PH3 in green. (F) Proliferation rate of control and Bub3 RNAi-treated cells. (G) Time course analysis of the mitotic index after Bub3 RNAi, assessed by PH3 staining. (H-J) Analysis of mitotic progression of control and S2 cells treated with RNAi. Mitotic cells were identified by immunostaining with PH3 specific antibodies. (H) Quantification of cells in anaphase and prometaphase-like cells in which sister chromatids are clearly separated over time. (I) Treatment of S2 cells with Bub3 RNAi reduces the number of cells in prometaphase and metaphase over time. (J) Depletion of Bub3 causes a marked increase in the number of cells in prophase by 72 hours after treatment. Bar, 5 µm.

View larger version (46K): [in a new window]   Fig. 3. In vivo analysis of S2 cells shows that Bub3-depleted cells transit faster through mitosis, despite a delay in prophase. (A) Selected images from time-lapse movies (see supplementary material Movie 1) of either control or Bub3 RNAi-treated cells expressing GFP-tubulin recorded every 2 minutes from the time asters first appeared to the re-formation of the daughter nuclei. The two movies have been aligned setting nuclear envelope breakdown (NEBD) as 0. In control cells, NEBD occurred approximately 17 minutes after asters were clearly visualized, whereas anaphase started 32 minutes after NEBD. In Bub3 RNAi-treated cells, NEBD occurred 32 minutes after aster formation, showing a significant delay in prophase. Anaphase started approximately 20 minutes after NEBD, indicating a rapid transition from prometaphase to anaphase. (B) Quantitative analysis of the different cell cycle stages, in both control (n=14) and Bub3 RNAi-treated cells (n=29). **P<0.005 when compared to corresponding stages in control cells. (C) Analysis of prophase cells by PH3 labelling reveals that the majority of the Bub3-depleted cells (53%) exhibits strong PH3 labelling and DNA condensation but no aster formation, in contrast to the control population where PH3 staining correlated with aster formation in more than 98% of cells (n=40).

View larger version (55K): [in a new window]   Fig. 4. Bub3-depleted cells exhibit lower levels of cyclin B during G2 and mitosis. (A) Analysis of cyclin B at different stages of the cell cycle in control and Bub3 RNAi-treated cells for 96 hours, followed by incubation for 6 hours in colchicine. -tubulin staining (red) was used to discriminate between G1/S, G2 and mitotic cells. DNA is shown in white and cyclin B in green. Images show that Bub3-depleted cells do not accumulate cyclin B to control levels in G2 or during mitosis. (B) Quantification of cyclin B levels. The mean pixel intensity per cell in control and RNAi-treated cells was measured at different cell cycle stages (see also supplementary material Table S2). ***P<0.0005 when compared to intensity in control cells. (C) Cyclin B accumulation during G1/S and G2 in control and Bub3-depleted cells after 96 hours in culture without colchicine treatment. -tubulin staining (red) was used to classify cells as before, cyclin B is shown in green and DNA in white. Bub3-depleted cells fail to accumulate cyclin B during G2, unlike control cells. (D) Quantification of cyclin B levels (see also supplementary material Table S3). ***P<0.0005 when compared to intensity in control cells. (E) Analysis of cyclin B levels by western blotting of total protein extracts (10 µg) isolated from control or RNAi-treated cells before or after 3 and 6 hour incubations with colchicine. -tubulin was detected as a loading control. (F) Quantitative analysis of the western blot shown in E. Bar, 5 µm.

View larger version (58K): [in a new window]   Fig. 5. Mutation of the APC/C subunit cdc27 in bub31 mutant cells allows cyclin B accumulation during G2 and restores normal progression through early mitotic stages. (A) Neuroblasts of different genotypes were isolated, incubated in colchicine (1 hour), fixed and stained for cyclin B (green). DNA was counterstained with DAPI (white). Images show high levels of cyclin B in cdc27 mutant cells whereas cdc27;bub31 double mutant cells show normal cyclin B levels during G2. (B) Quantification of cyclin B levels. The mean pixel intensity per cell in the wild type and mutant strains at different cell cycle stages was measured (see also supplementary material Table S4). *P<0.05; **P<0.005; ***P<0.0005 when compared to intensity in corresponding control cells. (C) Mitotic index of wild-type (control), double mutant (cdc27;bub31) and single mutant (cdc27 or bub31) cells. Mutation of cdc27 in a bub31 background leads to an increase in the mitotic index relative to bub31 alone, but still reduced when compared to the wild-type index. (D) Quantification of cells in prophase for the different genetic backgrounds showing that double mutant cells (cdc27; bub31) transit normally through prophase. (E) A non-degradable form of cyclin B (CyCBND) was expressed in bub31 mutant neuroblasts (Mz1061;CycBND-GFP;bub31). Mz1061 was used to drive neuroblast expression. Mz1061;CycBND-GFP neuroblasts were used as a control. Expression of a non-degradable form of cyclin B in bub31 neuroblasts restores the mitotic index to wild-type values. Bar, 5 µm.

View larger version (36K): [in a new window]   Fig. 6. Bub3 is required to promote accumulation of cyclin A during G2. (A) Cyclin A accumulation during G1/S and G2 in control and Bub3-depleted cells. -tubulin staining (red) was used to classify G1/S and G2 cells. Cyclin A is shown in green and DNA in white. Bub3-depleted cells fail to accumulate cyclin A in G2. (B) Quantification of cyclin A levels for control and RNAi-treated cells. Image analysis was performed as described for cyclin B and the mean pixel intensity per cell at the different stages of cell cycle was determined (see also supplementary material Table S6); ***P<0.0005 when compared to intensity in control cells. Bar, 5 µm.

View larger version (54K): [in a new window]   Fig. 7. Mutations in bubR11 do not affect cyclin B accumulation during G2. (A) bubr11 mutant neuroblasts were treated with colchicine as previously described, fixed and stained for DNA (white) and cyclin B (green). The results show that unlike bub31, bubR11 mutant cells do not compromise accumulation of cyclin B during G2. Nevertheless, as expected, cyclin B levels during mitosis decrease significantly because of loss of spindle checkpoint activity. (B) Quantification of cyclin B levels at different cell cycle stages in Oregon R (wild-type strain) and bubR11, was performed as described for bub31 (see also supplementary material Table S7). **P<0.005; ***P<0.0005 when compared to intensity in the relevant control cells. Bar, 5 µm.

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