The isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3 is reported. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. Localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways (Basu, 1998).
During a search for Drosophila genes involved in the regulation of mitotic progression, a recessive mutant line was isolated in which the lethality mapped to the bub3 locus. Genomic sequencing of the bub3 gene in this line showed that it contained a single point mutation in the coding region, leading to the substitution of a conserved glycine at position 193 by aspartic acid. This mutation is responsible for the lethality of the line, since expression of wild-type bub3 cDNA during larval development using a specific GAL4 driver rescues the mitotic phenotype and gives rise to viable adults. Thus, in Drosophila, bub3 is an essential gene and this mutant allele has been designated bub31. Western blot analysis of total protein extracts from bub31 homozygous or bub31/Df(3R)Dr-rv1 hemizygous individuals indicates that the mutant protein is expressed. To evaluate whether the mutant protein is able to localize properly, antibodies were used to determine its intracellular localization in bub31 third instar larvae neuroblasts. Although in wild-type cells Bub3 always localizes to kinetochores during prometaphase, in approximately half bub31 cells the mutant protein is not detected at kinetochores. However, some bub31 cells (45%) show localization of the mutant protein at one or two kinetochore pairs, whereas only very few (9%) show localization to all kinetochores, suggesting that this allele might behave as a hypomorph (Lopez, 2005).
To determine whether bub31 cells have an abnormal mitotic checkpoint response, the mitotic index of both bub31 homozygous and hemizygous neuroblasts was measured in the presence and absence of spindle damage. The results show that in the absence of spindle damage, the mitotic index of mutant larvae is not significantly different from wild-type controls. However, if the spindle is disrupted by colchicine, neither bub31 homozygous nor hemizygous mutant cells are able to arrest in mitosis and undergo precocious sister chromatid separation (PSCS) indicating loss of mitotic checkpoint response (Lopez, 2005).
Mitotic progression was examined in both homozygous and hemizygous mutant individuals. The phenotype associated with both genotypes is nearly identical. bub31 mutant cells undergo cytologically normal prophase but, a significant proportion of cells with a prometaphase-like configuration (19%) display PSCS. Furthermore, most of the anaphases (70%) observed are disorganized and contain lagging chromatids, and of the total mitotic figures a high proportion (38%-42%) are aneuploid, probably reflecting the occurrence of PSCS in prior rounds of cell division (Lopez, 2005).
Although observations of late mitotic stages indicate that bub31 homozygous and hemizygous mutant cells have nearly identical phenotypes, the two genotypes behave differently during the early stages of mitosis. Although the frequency of prophase and prometaphase cells in bub31 homozygous mutants does not differ significantly from the wild type, hemizygous individuals have a higher frequency of cells in prophase and a correspondingly lower frequency of cells in prometaphase. This classical genetic test verifies the conclusion from cytological analysis that the bub31 allele behaves as a hypomorph. Importantly, these results further suggest that a more severe depletion of Bub3 function causes a slow progression through early stages of mitosis, a phenotype that has not been described before for any checkpoint protein (Lopez, 2005).
These results suggest an unexpected role for Bub3 during early mitotic stages that only became apparent in bub31 hemizygous individuals. The consequences of depleting Bub3 in Drosophila S2 cells by double-stranded RNA interference (RNAi) was investigated. Treatment of S2 cells with bub3 RNAi causes a significant reduction of the Bub3 protein levels within 48 hours and by 96 hours the protein is barely detectable. Immunofluorescence analysis on RNAi-treated cells failed to detect Bub3 at kinetochores in all mitotic cells examined. Accordingly, Bub3-depleted cells are unable to arrest in mitosis when the spindle is damaged, and show elevated levels of PSCS. Similar to the results presented for bub31 mutant neuroblasts, depletion of Bub3 by RNAi demonstrates that in Drosophila S2 cells, Bub3 is required for an efficient mitotic checkpoint response (Lopez, 2005).
The role of Bub3 in mitotic progression was addressed. Depletion of Bub3 causes a reduction in cell proliferation that is not due to loss of cell viability, but instead to an increase in the culture doubling time. Quantification of the mitotic index over the course of the experiment shows that in comparison with control cells, the number of cells in mitosis increases after depletion of Bub3. These results contradict a role for Bub3 as a negative regulator of mitotic exit and suggest that Bub3-depleted cells spend more time in mitosis, leading to a slower proliferation rate. To better understand this unexpected behaviour, mitotic progression was carefully analysed. The results show that depletion of Bub3 leads to an increase in the number of cells with PSCS. Compared to control cells, there is also a twofold increase in the total number of cells in anaphase and most anaphases (68%) are disorganized or have lagging chromatids. Notably, after depletion of Bub3 there is a significant reduction in the number of cells in prometaphase and metaphase, whereas approximately 57% of the prometaphase cells exhibit PSCS. These data are all consistent with a premature mitotic exit. If these cells exit mitosis earlier than normal, then the higher mitotic index observed after depletion of Bub3 must result from a delay at an earlier stage of mitosis. Indeed, there is a twofold increase in the percentage of cells in prophase 72 hours after treatment with RNAi. Therefore, a severe depletion of Bub3 in S2 cells results in mitotic alterations similar to those observed in bub31 hemizygous neuroblasts. Both sets of observations indicate that besides its function in the mitotic checkpoint response, Bub3 has a role in promoting normal transit through early stages of mitosis, particularly through prophase (Lopez, 2005).
To clarify further a possible role of Bub3 in mitotic progression, in vivo time-lapse analysis of S2 cells stably expressing GFP-tubulin after depletion of Bub3 by RNAi was performed. Control and Bub3-depleted cells were recorded every 2 minutes from the time asters first appeared to the reformation of the daughter nuclei. GFP-tubulin is an excellent marker to follow mitotic progression, since various events can be timed. The visualization of well-defined asters was taken to indicate initiation of prophase, nuclear envelope breakdown (NEBD) can be seen by rapid entry of GFP-tubulin into the nuclear area, retraction of kinetochore bundles shows the beginning of anaphase A, spindle elongation marks anaphase B and nuclear re-formation indicates telophase. Thus, the duration can be determined of all major mitotic phases including prophase (from appearance of the asters until NEBD), prometaphase-metaphase (from NEBD until anaphase initiation), anaphase A (from microtubule bundle retraction to spindle elongation) and anaphase B (from spindle elongation to nuclear reformation) allowing a direct correlation between the in vivo data and results from fixed material. The results show that depletion of Bub3 causes significant changes in mitotic progression during both prophase and prometaphase. The appearance of the asters was set as time zero, and the duration of each mitotic stage was plotted. In vivo analysis of untreated S2 cells shows that prophase takes on average 16 minutes, however, after Bub3 depletion, prophase is significantly delayed lasting on average 21 minutes. Furthermore, although in control cells the timing between NEBD and anaphase onset takes on average 34 minutes, after Bub3 depletion it is significantly reduced to 21 minutes on average, indicating an accelerated progression through prometaphase-metaphase. These data are in agreement with the proposed function of Bub3 in the mitotic checkpoint response (Lopez, 2005).
Thus, depletion of Bub3 causes a significant delay in prophase, strongly supporting observations in fixed cells. However, it was surprising not to find a more significant prophase delay since depletion of Bub3 causes a twofold increase in the percentage of cells in prophase. To clarify this discrepancy, the number of cells in prophase (in S2 cells) expressing GFP-tubulin after Bub3 RNAi was quantified using PH3 as a mitotic marker. Two hallmark events of prophase were analysed: the appearance of Phosphohistone H3 (PH3) at Ser10, which in Drosophila correlates with the beginning of prophase and separation of the centrosomal asters. This analysis also showed a twofold increase in the number of cells in prophase after Bub3 depletion. However, unlike control cells, in Bub3 depleted cells, two types of PH3 positive cells were clearly observed: cells in which formation of the asters was markedly visible and cells with an interphase arrangement of microtubules without visible asters. The latter phenotype accounted for 53% of the prophase cells observed after Bub3 depletion and was never observed in control cells. In the control population, the appearance of the asters always correlated with PH3 staining. These observations suggest that the in vivo analysis is likely to underestimate the prophase delay observed after depletion of Bub3 (Lopez, 2005).
These results suggest that after Bub3 depletion, nuclear and cytoplasmic events, namely chromosome condensation and spindle formation, appear to uncouple. It is well established that accumulation of mitotic cyclins and the activation of the cdk/cyclin complexes is essential for entry and progression through mitosis. Besides being important for chromosome condensation, cdk activity is responsible for the dramatic changes in microtubule dynamics that occur at the onset of mitosis, resulting in microtubule nucleation from centrosomes and assembly of the mitotic spindle. Accordingly, tests were performed to see whether the slower progression through early mitotic stages observed after Bub3 depletion could result from abnormal accumulation of mitotic cyclins. Cyclin B levels were measured by immunofluorescence and Western blot analysis in cells that had been depleted of Bub3 by RNAi. Control and RNAi-treated cells were incubated in colchicine, to promote high levels of cyclin accumulation and increase the number of cells in mitosis, and cyclin B levels were measured by immunofluorescence. The centrosomal marker gamma-tubulin was used to distinguish between different cell cycle stages. Interphase cells without gamma-tubulin staining were classified as G1/S, whereas those with centrosomal staining but without centrosome separation were classified as G2. Cells in mitosis (prophase and prometaphase) had gamma-tubulin staining, well-separated centrosomes and clear chromosome condensation. The fluorescence intensity values obtained for G1/S cells did not differ between control and RNAi-treated cells, allowing a direct comparison between the two samples. In agreement with the expected pattern for cyclin B accumulation, S2 control cells start to accumulate cyclin B during G2 and attain their highest levels of cyclin B during mitosis. However, cyclin B accumulation in Bub3-depleted cells is surprisingly different. These cells already show significantly lower levels of cyclin B in G2 and also during mitosis. The failure to maintain high levels of cyclin B during mitosis is in agreement with the predicted function of Bub3 in the mitotic checkpoint response (similar results were also observed in bub31 mutant neuroblasts). However, these results also indicate that Bub3 might be required during G2 to ensure accumulation of cyclin B. To eliminate the possibility that colchicine treatment could indirectly affect cyclin B accumulation during G2, cyclin B levels were measured in control and Bub3-depleted cells in the absence of the drug. Quantification of cyclin B levels shows that in contrast to control cells, Bub3-depleted cells fail to accumulate cyclin B during G2. In agreement with the immunofluorescence data, western blot analysis of total protein extracts shows that after Bub3 depletion, accumulation of cyclin B is significantly reduced reaching only 50% of the levels observed in untreated cells. In addition, although treatment with colchicine leads to accumulation of cyclin B in control cells, the same is not observed after Bub3 depletion, where accumulation of cyclin B is severely compromized. These results show that Bub3 does indeed appear to be required during G2 to promote accumulation of cyclin B, strongly suggesting that Bub3 might have a function in G2 before its well-established role in the mitotic checkpoint during prometaphase (Lopez, 2005).
Accumulation of cyclins during G2 in somatic cells depends primarily upon transcriptional activation and the fact that the anaphase promoting complex/cyclosome (APC/C) is inhibited at this stage by early mitotic regulators. To test whether APC/C activity might be required to mediate the reduction in cyclin B levels after depletion of Bub3, the effect of removing the APC/C subunit cdc27 in bub3 mutant cells was analysed. It has been shown that in Drosophila, the APC/C subunit cdc27 is required for the degradation of cyclin B but not of securin (another APC/C substrate), since mutations in cdc27 mutant neuroblasts or depletion of cdc27 by RNAi in S2 cells, results in cells with high levels of cyclin B and separated sister chromatids. Accordingly, mutations in the bub3 and cdc27 genes would be predicted to have opposite effects on the accumulation of cyclin B and the combination of the two mutations might even lead to normal mitotic entry. Therefore cyclin B levels were measured in single (bub31 or cdc27) and double (bub31;cdc27) mutant cells after incubation in colchicine. The results show that cdc27 mutant cells have significantly higher levels of cyclin B both in G2 and mitosis than do control wild-type cells, in agreement with the phenotype previously described for this mutant. As expected, accumulation of cyclin B in double mutant cells (bub31; cdc27) during G2 is not significantly different from wild-type controls and it is higher than in bub31 cells. These results support the hypothesis that the inability to accumulate normal levels of cyclin B after depletion of Bub3 is mediated by the APC/C (Lopez, 2005).
Next, several mitotic parameters in single (bub31 or cdc27) and double (bub31; cdc27) mutant cells were analysed after spindle damage induced by incubating the cells in colchicine. Under these conditions, mutant cdc27 neuroblasts arrest in mitosis with well-condensed chromosomes and unseparated sister chromatids resulting in an increased mitotic index when compared to the wild-type control. This behaviour is due to the additive effect of the mutation in the APC/C subunit and the checkpoint-dependent arrest induced by spindle damage. In neuroblasts mutant for both bub31 and cdc27, the percentage of cells in mitosis is increased relative to bub31 alone. This result shows that the premature mitotic exit characteristic of bub31 mutant cells is dependent on APC/C activity. Nevertheless, the mitotic index of double mutant cells (bub31 and cdc27) is significantly lower than that seen in wild-type cells. This difference relative to control cells is most likely explained by premature mitotic exit due to the absence of an effective checkpoint response when Bub3 activity is absent, coupled with the fact that cdc27 does not seem to be required for APC/C-mediated securin degradation. Finally, the frequency of cells in prophase in double mutant cells was observed to be indistinguishable from wild-type controls, suggesting that the double mutant transits normally through this early stage of mitosis. To rule out the possibility that cyclin B levels could be regulated by a mechanism other than APC/C-driven proteolysis, a non-degradable form of cyclin B was overexpressed in bub31 mutant neuroblasts using the GAL4/UAS system. The results show that stabilization of cyclin B in bub31 mutant cells results in an increase in the mitotic index to values similar to those observed in control cells. Taken together, these results indicate that Bub3 appears to negatively regulate APC/C activity during the G2-M transition, allowing the accumulation of sufficient cyclin B for timely progression through the early stages of mitosis (Lopez, 2005).
These results suggest that Bub3 is required for normal accumulation of cyclin B before and during mitosis. However, cyclin A is also required to promote mitotic entry in Drosophila. Cyclin A accumulates during S phase and G2 and it is degraded by the APC/C prior to the degradation of cyclin B as cells progress through early stages of mitosis. However, in contrast to cyclin B, cyclin A levels are not stabilized by the spindle damage-associated checkpoint response. In order to determine if Bub3 is also required to promote accumulation of cyclin A, its levels were analysed during cell cycle progression in Bub3-depleted cells. The centrosomal marker gamma-tubulin was used to distinguish cells in G1/S or G2. The results show that although cyclin A accumulates from G1 to G2 in control cells and can still be detected in some early mitotic cells, cyclin A fails to accumulate and can hardly be detected in cells depleted for Bub3. Analysis of cyclin A accumulation in bub31 homozygous mutant neuroblasts gave very similar results. These observations further support the role of Bub3 as a negative regulator of the APC/C during G2 and mitosis (Lopez, 2005).
Unlike other checkpoint proteins like Mad2 or BubR1, Bub3 has never been found to interact directly with the APC/C or with its activator Cdc20. Thus, it was of interest to determine if other proteins that interact simultaneously with Bub3 and Cdc20 or APC/C subunits could mediate Bub3-dependent APC/C inhibition. BubR1 is, at first glance, a good candidate to perform this function as it has been found in an interphase high molecular weight complex. Accordingly, tests were performed to see whether mutations in bubR1 could affect cyclin B accumulation in G2 or mitosis. Wild-type or bubR11 third larval neuroblasts were incubated in colchicine and immunostained to reveal the level of chromatin condensation and cyclin B. Analysis of control cells shows that cells with no cyclin B and no chromosome condensation (classified as G1/S) could be identified; cells with cyclin B levels but no visible chromatin condensation (classified as G2), and cells with high levels of cyclin B and well-condensed chromosomes (classified as mitotic), were identified as expected for normal cyclin B accumulation. Similarly, bubR11 mutant neuroblasts were detected in G1 and also in G2 with normal levels of cyclin B. However, mitotic cells showed significantly lower levels of cyclin B consistent with its known function in the mitotic checkpoint response. These results show that the pattern of cyclin B accumulation in the absence of bubR11 is significantly different from that of bub31 mutant cells. Therefore, the data suggest that accumulation of cyclin B during G2 and early mitosis requires Bub3, independent of its interaction with BubR1 (Lopez, 2005).
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date revised: 20 January 2018
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