Bud1 expression and demonstration of a spindle checkpoint accompanying spermatogenesis

In order to examine Drosophila Bub1 distribution during the cell cycle, affinity-purified antibodies were generated against a LacZ/Bub1 fusion protein. Affinity-purified IgY identified two bands of ~165 kD (the predicted molecular mass for Drosophila Bub1) on Western blots of larval brain extracts. These bands disappear almost completely in brain extracts made from bub1 mutants to levels <2%-3% of wild-type, and are completely absent in identical blots probed with preimmune IgY made from the same chickens. The antibody preparations also recognize a 100-kD band unaffected by bub1 mutations; this band is also seen when blots are probed with preimmune IgY. The two Bub1-specific bands probably represent alternatively spliced or phosphorylated forms of Bub1. The near complete absence of these bands in extracts from l(2)K06109 and l(2)K03113 homozygotes indicate that these mutations represent strongly hypomorphic, near null alleles of Drosophila bub1. It is possible that the residual low levels (seen at longer exposures) represent the perdurance of maternally supplied product contributed by the heterozygous mothers of these mutant animals (Basu, 1999).

In other organisms, Bub1 and other components of the spindle checkpoint associate with the kinetochore during early prophase and remain until late metaphase, but when mitotic arrest is induced by microtubule depolymerizing agents such as nocodazole or colchicine, they do not leave the kinetochore. Treatment of Drosophila cells with colchicine leads to prolonged arrest in a prometaphase-like configuration, demonstrating that the checkpoint responds to this drug in flies as well. Bub1 is recruited to kinetochores in chromosomes isolated from colchicine-treated Drosophila S2 tissue culture cells. No Bub1 signal is observed at the kinetochores of larval neuroblasts from prometaphase-arrested bub1 mutants. Thus, affinity-purified anti-Bub1 antibodies recognize epitopes recruited to the kinetochore when the spindle is perturbed, as would be expected for a Bub1 protein. These results additionally confirm the observations gained from the Western blots that the Bub1 protein recognized by this antibody is nearly completely absent from the larval brains of bub1 mutants (Basu, 1999).

Affinity-purified anti-Bub1 antibodies were used to examine in detail the distribution of Bub1 during mitosis in cycling Drosophila S2 cells. Interphase cells show a generalized, diffuse nucleoplasmic staining pattern. At prophase, Bub1 associates strongly with the kinetochore regions of the condensed chromosomes; Bub1 indeed substantially colocalizes with the kinetochore marker ZW10. Kinetochore staining becomes weaker at prometaphase. At metaphase, the Bub1 signal weakens, specifically for those chromosomes that have migrated to the metaphase plate. Chromosomes in the same cells that have not yet reached the metaphase plate continue to show strong Bub1 staining at their kinetochores. Depending on the orientation of the chromosome with respect to the spindle, one kinetochore may stain more strongly for Bub1 than the other. Very weak kinetochore signals continue to be visible into anaphase, but are not observed during late anaphase or telophase. Some staining of the spindle midzone is detectable at late anaphase (Basu, 1999).

Similar intracellular protein distributions have already been documented for the Drosophila mitotic checkpoint control component Bub3 (Basu, 1998), and have also been observed for human Bub1 and BubR1. A previous report for mouse Bub1 failed to detect its association with kinetochores during metaphase or subsequent stages of mitosis (Taylor, 1997) -- it is not clear whether this represents a true difference between the mouse and the human or Drosophila patterns of Bub1 distribution, or is instead the result of lower signal intensities obtained with the monoclonal anti-mouse Bub1 antibody employed in that study (Basu, 1999).

The existence of a spindle checkpoint in meiotically dividing Drosophila spermatocytes is currently uncertain. The presence of univalents (chromosomes without pairing partners) does not prevent primary spermatocytes from entering anaphase. Furthermore, although mei-S332 or ord mutations cause sister chromatids to separate during the first meiotic division, chromosomes in mutant secondary spermatocytes still undergo obvious anaphase pole-ward movements. If a spindle checkpoint is active, it should prevent anaphase onset under either of these conditions because chromosomes could not be subject to the bipolar tension needed to deactivate the checkpoint. Finally, testes treated with colchicine contain many spermatids with polyploid nuclei, showing that spermatocytes with aberrant spindles do not arrest in metaphase and instead progress through meiosis and differentiate into spermatids (Basu, 1999).

To explore the apparent absence or weakness of the spindle checkpoint in meiotic Drosophila spermatocytes, the distribution of Bub1 was examined during spermatogenesis. Bub1 localizes to the kinetochores of bivalents in primary spermatocytes during prometaphase I. The kinetochore association of Bub1 decreases significantly as the bivalents align at the metaphase plate and become undetectable at anaphase, although some nuclear and spindle staining above background is visible during these cell cycle stages. This dynamic localization pattern is repeated during the second meiotic division. Thus, the pattern of Bub1 distribution during both meiotic divisions parallels that seen during mitosis (Basu, 1999).

Is the association of Bub1 with the kinetochores during male meiosis in Drosophila responsive to tension? To answer this question, the distribution of Bub1 was analyzed in a situation in which primary spermatocytes contain univalents: the attached XY (X^Y) and the compound 4th [C(4)RM], which are never subject to bipolar tension as they can attach only to a single pole. The intensity of Bub1 staining in this genotype is comparable between univalents and bivalents at prometaphase I. However, once the bivalents align at the metaphase plate, the intensity of Bub1 staining on their kinetochores decreases drastically, while the univalents in the same cell retain strong Bub1 signals. Thus, the spindle checkpoint component Bub1 is not only properly localized during male meiosis, but it is also capable of discriminating between the presence or absence of bipolar tension at kinetochores (Basu, 1999).

Larval testes from bub1 mutants were analyzed for evidence of mitotic and meiotic defects. These testes are significantly smaller than wild-type testes, suggesting that mitotic proliferation of the germline has been substantially suppressed or that many mutant germline cells are directed into an apoptotic fate as seen in neuroblasts. As a result, although it is difficult to find meiotic or post-meiotic figures in mutant larval testes, the limited observations that were made indicate that bub1 mutations strongly affect meiosis as well. Living testes from bub1 mutants examined by phase contrast optics show meiotic figures with severe spindle abnormalities at metaphase and anaphase, and multiple nuclei of variable volume at telophase. Onion stage spermatids from bub1 mutant testes contain abnormal numbers of nuclei of variable size (including micronuclei) associated with a single Nebenkern of normal size. This phenotype results from chromosomal missegregation not accompanied by cytokinesis defects (Basu, 1999).

Effects of Mutation or Deletion

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).

A search of the BDGP database of genomic sequences flanking P-element insertion sites with the complete sequence of the Bub1 cDNA identified the lethal P-element insertions l(2)K06109 and l(2)K03113 as mutations that could potentially affect the expression of the bub1 gene. The P-elements in the two separate mutants are inserted in exactly the same position within sequences transcribed into the 5'-untranslated leader of the bub1 mRNA, 48 bp upstream of the initiator ATG (Basu, 1999).

The lethality and associated mitotic phenotypes of l(2)K06109 and l(2)K03113 homozygotes is due to the P-element insertions into the bub1 gene. In brief, these two independently isolated mutations are allelic to one another, and they do not complement either of two deletions [Df(2R)nap1 and Df(2R)nap2] that remove polytene chromosome region 42A1-3, the location to which the bub1 gene and the l(2)K06109 and l(2)K03113 P-element insertions map by in situ hybridization. In addition, precise excision of the P-element in both mutant stocks by remobilization with a source of P-element transposase results in complete rescue of the lethality and associated mitotic defects seen in l(2)K06109 or l(2)K03113 homozygotes, showing that the P-element alone is responsible for the phenotype of these mutants. Importantly, many of the mitotic phenotypes visible in the larval neuroblasts of mutant animals are precisely those that would be expected from mutations affecting the expression of a component of the spindle checkpoint in Drosophila. These observations, taken together with the Western blot and immunofluorescence data, argue strongly that these mutant stocks contain P-element-induced hypomorphic mutations specifically affecting the Drosophila bub1 gene (Basu, 1999).

To determine the developmental stage at which bub1 mutant homozygotes arrest their development, P-element l(2)K06109- or l(2)K03113-bearing chromosomes were rebalanced over a balancer chromosome bearing the dominant marker Tubby, whose effects are visible in larvae and pupae. In these rebalanced stocks, ~30% of the third instar larvae were non-Tubby, in line with Mendelian expectations that bub1 homozygotes would constitute one-third of the animals that hatch from embryos. Many pupae were also non-Tubby, but these constituted a slightly smaller percentage (approximately 22%) of the total pupae. These results indicate that the lethality caused by the two bub1 mutations occurs mainly during the pupal stages, with most mutant homozygotes surviving through the third larval instar. It has been argued that animals homozygous for mutations in genes controlling essential cell cycle functions in Drosophila should survive to third instar larval stages or to the larval-pupal transition, because cell divisions prior to these stages could be supported by maternally supplied components contributed by their heterozygous mothers. This expectation has been borne out by many subsequent investigations of cell cycle mutants in flies. Indeed, no mitotic abnormalities have been detected in any of >1,000 post-cellularization divisions from a total of 22 homozygous mutant embryos observed at high resolution. Thus, squashed preparations of neuroblasts taken from the brains of homozygous bub1 mutant third instar larvae were analyzed to define the functional role of Bub1 in Drosophila cell divisions (Basu, 1999).

It was initially observed that bub1 mutants possess the hallmark of a defect in the spindle checkpoint: that is, a failure to maintain sister chromatid cohesion when the spindle is disrupted. In wild-type brains incubated with colchicine for 1 h, sister chromatids remain attached in 98% of all mitotic figures, revealing activity of the spindle checkpoint. Under identical conditions, sister chromatids remain attached in only 32% of mitotic figures in bub1 brains, indicating that the spindle checkpoint has often been bypassed. In fact, the frequency of neuroblasts with separated sister chromatids in bub1 mutant brains is essentially unaffected by colchicine treatment, in stark contrast with wild-type (Basu, 1999).

In brains untreated with colchicine, the percentage of bub1 mutant mitotic cells with separated sister chromatids is much higher, and the percentage of mitotic cells in prophase or prometaphase is much lower, than in wild-type. Relative to wild-type controls, the brains of bub1 homozygotes show a threefold reduction in the mitotic index, operationally defined as the number of mitotic figures per optic field, with every optic field in the brain being scored. More limited data sets obtained through observations of l(2)K06109 homozygotes or of l(2)K06109/l(2)K03113 trans-heterozygotes yielded almost identical results (Basu, 1999).

In all particulars, the brains of larvae heterozygous for either bub1 mutation with either of two deletions removing the bub1 locus display phenotypes qualitatively identical to those seen in bub1 homozygotes. However, there is some slight quantitative variation in mitotic parameters between these deletion heterozygotes and the mutation homozygotes; it is not known whether these effects are due to the activity of the bub1 gene or due to background effects. Based on these genetic criteria, the two bub1 mutations behave as very strong hypomorphs that are probably nearly but not completely null alleles (Basu, 1999).

A significant proportion of the anaphase figures in bub1 mutant brains is aberrant (61%-84%, depending upon genotype). Three major kinds of abnormalities are seen at high frequency. (1) In many neuroblast anaphases, chromatin bridges extend between the two separating groups of chromosomes. (2) In other anaphase figures, lagging chromatids remain at the position of the metaphase plate while the other chromosomes have migrated to positions near the poles. (3) Extensive chromosome fragmentation is observed in many mutant anaphases. It is believed that these anaphase aberrations explain the observation that many of the cells in colchicine-treated mutant brains appear to be aneuploid. These aneuploid cells could be produced by the maldistribution of intact chromosomes during anaphase of a previous cell generation. However, it is suspected that many of the chromatids seen in mitotic cells may actually be chromosome fragments, resulting in an overestimate of the degree of aneuploidy (Basu, 1999).

A striking feature of bub1 mutant brains examined with DNA staining is the occurrence of extremely high frequencies of pycnotic nuclei with highly condensed chromatin. These nuclei are strongly positive when labeled by TUNEL-based techniques. Because the TUNEL procedure detects chromosome damage (normally induced in the pathway for apoptosis), the TUNEL signals could reflect either the occurrence of bona fide programmed cell death, or alternatively simply the chromosome fragmentation that occurs during anaphase in bub1 mutant cells. To discriminate between these possibilities, it was asked whether mutant nuclei showed elevated expression of two apoptotic events independent of chromosome breakage. The first of these markers was the redistribution of phosphatidylserine, which early in apoptosis rapidly moves from the internal face of the plasma membrane to the outside of the membrane; this redistribution was detected by use of FITC-conjugated Annexin V, a protein with very strong affinity for the serine in phosphatidylserine. The second marker was a ß-galactosidase reporter for reaper, a gene whose expression is needed to activate programmed cell death in Drosophila. Use of both markers verifies that mitotic cells in bub1 mutants undergo vastly elevated levels of apoptosis. Levels of apoptotic nuclei are similar in l(2)K06109 or l(2)K03113 homozygotes as well as in trans-heterozygotes for either of the two alleles with deletions of the region (Basu, 1999).

Dephosphorylation of 3F3/2 epitopes (detected by anti 3F3/2 antibody) is associated with the metaphase-anaphase transition. Microinjection of anti-3F3/2 antibodies into cultured cells blocks 3F3/2 dephosphorylation and delays anaphase onset, implying that dephosphorylation of 3F3/2 epitopes may be a prerequisite for entry into anaphase. The Bub1 kinase has been suggested as a candidate 3F3/2 kinase, both because of its function in the spindle checkpoint and because its intracellular distribution shows similarities with that of 3F3/2 epitopes. In order to examine these questions in more detail, it was asked whether bub1 mutations would affect the distribution of 3F3/2 epitopes. F3/2 signals are present at the kinetochores in bub1 prophase/prometaphase and metaphase figures at levels comparable to those of wild-type brains (see Bousbaa, 1997 for a description of 3F3/2 staining in wild-type Drosophila neuroblasts). This result demonstrates that Bub1 kinase does not contribute significantly to 3F3/2 kinase activity in vivo (Basu, 1999).

Interestingly, 3F3/2 staining continues to be detectable at the kinetochore at significant levels in many anaphase figures from bub1 mutant brains. In wild-type Drosophila neuroblasts, 3F3/2 phosphoepitopes at the kinetochore are completely lost by the start of anaphase (Bousbaa, 1997). This observation indicates that dephosphorylation of kinetochore-associated 3F3/2 phosphoepitopes is not essential for entry into anaphase, at least in a bub1 mutant background (Basu, 1999).

To establish the possible relationship between bub1 and other genes known to influence the fidelity of cell division in Drosophila, the effects of mutations in these genes on the intracellular distribution of Bub1 were explored. Focus was placed on genes encoding other proteins that localize to the kinetochore, since the results of this analysis would further an understanding of kinetochore assembly. Mutations in zw10 and rough deal disrupt the segregation of chromosomes during anaphase of mitosis and meiosis. Intriguingly, mutations in both genes cause precocious sister chromatid separation in colchicine treated larval neuroblasts, indicating a bypass of the spindle checkpoint. Both the ZW10 and Rod proteins are associated with the kinetochore during prophase/prometaphase of mitosis and both meiotic divisions. Mutations in zw10 or rod do not affect the localization of Bub1 to the kinetochore. Interestingly, in these mutant cells Bub1 continues to be associated with the kinetochores of precociously separated sister chromatids, indicating that sister chromatid separation does not require the loss of Bub1 from the kinetochore. Similar results were observed when precocious sister chromatid separation was induced in wild-type colchicine-arrested neuroblasts subjected to prolonged hypotonic shock. Conversely, bub1 mutations do not block the association of ZW10 with the kinetochore (Basu, 1999).

The mitotic mutation polo also affects mitotic fidelity and leads to chromosome missegregation and spindle abnormalities. The polo gene product is a protein kinase that shows a dynamic, cell cycle-dependent localization with several components of the mitotic apparatus, including the kinetochores. However, mutations in polo do not affect the distribution of Bub1, and the Polo protein kinase is localized normally to the kinetochores in a bub1 mutant background (Basu, 1999).

Mutations in the Drosophila gene fizzy lead to metaphase arrest, and Fizzy/Cdc20/Slp1/p55CDC has been shown to be required to mediate the Bub/Mad-dependent inactivation of the APC. p55CDC, the mammalian homolog of Fizzy, is concentrated at kinetochores from late prophase to telophase. Because the action of the Fizzy protein is thought to be downstream of Bub1, it was predicted that mutations in fizzy would not affect the ability of Bub1 to localize to the kinetochores; this is indeed the case (Basu, 1999).

Progression through mitosis requires the ubiquitin-mediated proteolysis of several regulatory proteins. A large multisubunit complex known as the anaphase-promoting complex or cyclosome (APC/C) plays a key role as an E3 ubiquitin-protein ligase in this process. The APC/C adds chains of ubiquitin to substrate proteins, targeting them for proteolysis by the 26S proteasome. The gene makos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens gamma-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product (Deak, 2003).

During mitosis, a checkpoint mechanism delays metaphase-anaphase transition in the presence of unattached and/or unaligned chromosomes. This delay is achieved through inhibition of the anaphase promoting complex/cyclosome (APC/C) preventing sister chromatid separation and cyclin degradation. Bub3 is an essential protein required during normal mitotic progression to prevent premature sister chromatid separation, missegreation and aneuploidy. Bub3 is required during G2 and early stages of mitosis to promote normal mitotic entry. Loss of Bub3 function by mutation or RNAi depletion causes cells to progress slowly through prophase, a delay that appears to result from a failure to accumulate mitotic cyclins A and B. Defective accumulation of mitotic cyclins results from inappropriate APC/C activity, since mutations in the gene encoding the APC/C subunit Cdc27 (see Drosophila Cdc27) partially rescue this phenotype. Furthermore, analysis of mitotic progression in cells carrying mutations for cdc27 and bub3 suggests the existence of differentially activated APC/C complexes. Altogether, these data support the hypothesis that the mitotic checkpoint protein Bub3 is also required to regulate entry and progression through early stages of 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).

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. The conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions in a manner similar to somatic cells. To determine BubR1 function during embryogenesis, a new hypomorphic semi-viable female sterile allele was generated. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, it has been found that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. These results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis (Perez-Mongiovi, 2005).

The localisation of BubR1 was examined at 0-180 minutes after egg laying (AEL) in wild-type embryos. BubR1 is first detected at kinetochores of mitotic chromosomes during prophase and becomes stronger in prometaphase. During metaphase, BubR1 kinetochore staining decreases significantly and during anaphase it is undetectable. BubR1 localisation follows the same pattern during early or late cycles. Co-localisation with anti-CID antibody shows that BubR1 accumulates at kinetochores of condensed chromosomes. In addition, BubR1 was detected on spindle microtubules at metaphase. Overall, these results indicate that BubR1 protein has a dynamic pattern of kinetochore localisation, consistent with its role in monitoring microtubule kinetochore interaction during the syncytial nuclear cycles of Drosophila embryos (Perez-Mongiovi, 2005).

Western blot analysis and immunodetection on stage 10 egg chambers revealed a maternal contribution for BubR1 to ensure normal progression through early stages of embryo development prior to the onset of zygotic gene expression. During syncytial divisions, BubR1 localisation corresponds to previous description in Drosophila larval neuroblast and S2 cells, except for BubR1 localisation on spindle microtubules at metaphase. Dynein-dependent redistribution of BubR1 and other checkpoint proteins was previously observed in mammalian cells and Drosophila embryos. Accordingly, the observations of BubR1 localisation along spindle microtubules at metaphase suggests that during syncytial division, it could be partially removed from kinetochores via microtubule transport. Because BubR1 spindle localisation is observed in only one third of the metaphases, it is suspected that the removal and redistribution of BubR1 is likely to occur within a very short period of time at this stage. Further studies using live imaging in different mitotic cell populations in Drosophila will elucidate if this observation reflects different mechanism of BubR1 checkpoint regulation between the syncytial and somatic divisions (Perez-Mongiovi, 2005).

In order to study BubR1 checkpoint activity during the syncytial nuclear divisions, bubR1Rev1, a new hypomorphic female sterile allele, was characterised. Analysis of nuclear proliferation in bubR1Rev1 embryos shows that starting at cycle 4-5 all embryos present abnormal mitotic progression as described for bubR11 mutant allele. BubR1 has been shown to be involved in chromosome alignment/congression (Ditchfield, 2003; Lampson, 2005) and in the inhibition of APC/C activity by signalling lack of tension at the kinetochore (Logarinho, 2004). The lagging chromatids, DNA bridges and aneuploidy observed in bubR1Rev1 embryos indicate precocious anaphase onset without stable microtubule-kinetochore attachment and an increase local degradation of APC/C targets. Therefore, the observations of mitotic progression on fixed and live embryos, in addition to the failure to delay mitosis exit in the presence of colchicine, support the notion that BubR1 spindle checkpoint activity is required during the rapid nuclear proliferation of Drosophila embryo. In the absence of BubR1, nuclear mitotic exit is initiated too rapidly, resulting in nuclear cycle asynchrony and developmental failure (Perez-Mongiovi, 2005).

In the wild-type Drosophila embryo, three out of the four haploid meiotic products form the polar body, which is formed by Phospho-H3-positive condensed chromosomes containing radiating microtubules without centrosomes. Although it has been hypothesised that F-actin and myosin II are involved in pulling and anchoring the polar body to the embryonic cortex with the plus ends of microtubules attached to the kinetochores and the minus ends facing outwards, the establishment and maintenance of the polar body mitotic arrest imply that specific factors are required to exclude the polar body from the mitotic oscillation, since arrest occurs in the same cytoplasm where neighbouring nuclei undergo synchronous divisions. Surprisingly, the immunodetection study revealed that BubR1 accumulates at polar body kinetochores in unfertilised and fertilised wild-type embryo. In bubR1Rev1 embryos; the polar body fails to establish and maintain its structure. Instead, polar bodies undergo cycle of DNA condensation-decondensation in phase with the mitotic cycle of the neighbouring nuclei. Thus, these results strongly suggest that at least the maintenance of the mitotic arrest and the exclusion of the polar body from the mitotic oscillator activity in the cytoplasm are dependent on normal BubR1 levels. Accordingly, BubR1 localisation at the polar body kinetochores appears to have a dominant effect by allowing maintenance of the arrest (Perez-Mongiovi, 2005).

Similarly, it has been shown in Drosophila that the conserved mitotic checkpoint protein Mps1 is require for polar body mitotic arrest (Fischer, 2004) and while BubR1 polar body localisation is affected by Mps1 mutation, its nuclear syncytial localisation remains unaffected. Moreover, gnu, png and plu gene products, which form a multi-protein complex, have been shown to be required for entry and exit into mitosis during early syncytial cycles in Drosophila embryos through Cyclin B stabilisation. Mutant alleles of these genes induce nuclei to undergo DNA replication in the absence of chromosome segregation and polar body de-condensation. Furthermore, it has been shown that increase Cyclin B levels in a png mutant genetic background can restore polar body chromosome condensation. These observations have led to speculation of a potential genetic interaction between BubR1, Mps1 and gnu-png-plu during the syncytial stages. However, it remains to be determined whether Mps1 and BubR1 are involved in the same pathway during early syncytial cycles and polar body structure. Moreover, while polar body structure differs between bubR1 and gnu mutant embryos, it is possible that they interact genetically to establish and maintain polar body structure through local cyclin B stabilisation (Perez-Mongiovi, 2005).

It has been proposed that syncytial nuclear proliferation should be divided in three phases: a first phase of synchronous proliferation (cycle 1 to 6), a second phase being Cdk1/cyclinB dependent with local variation in metaphase-anaphase duration (cycle 7 to 10), and a third phase of meta-synchronous divisions that is DNA damage checkpoint dependent and shows an increase in the duration of interphase and M phases (Ji, 2004). Within each embryo, the total nuclear cycle time between cycles 7 until 10 remains equal, but those located at the centre undergo prolonged metaphase, which is compensated by a shorter anaphase/telophase. These variations are regulated by local Cdk1/cyclin B activity and the total cycle length can be modified and influenced by variation in cyclin B maternal gene dose. It has been proposed that the meta-synchronisation observed between cycle 7-10 is solely induced by cytoplasmic flux and local oscillation in Cdk1/cyclin B activity (Ji, 2004) and that early cycles are driven by the dynamics of the mitotic apparatus. However, the observations that in bubR1Rev1 embryos almost half of the embryos show extensive loss of nuclear synchrony as early as cycle 4-5, suggest that BubR1 checkpoint activity could be directly involved in the process of nuclear synchrony (Perez-Mongiovi, 2005).

Since all nuclei share a common cytoplasm, a key factor in the regulation of mitotic progression is the transduction of a global state to the local nuclear level so as to ensure proper synchrony. Although the abnormal mitotic progression in bubR1Rev1 embryos can be easily explained in terms of spindle checkpoint activity, the nuclear cycle asynchrony suggests that BubR1 regulates the mitotic apparatus at a local level by timing the proper progression of chromosome congression and anaphase onset. It has been proposed that higher Cdk1/cyclin B activity decreases microtubule stability and increases sister chromatid velocity at anaphase to maintain constant nuclear cycle length within the embryo. The observations of this study suggest that local variation in BubR1 checkpoint activity can provide a feedback mechanism to ensure proper chromosome segregation and local variation in the timing of metaphase/anaphase transition through regulation of microtubule attachment/tension at kinetochore pairs and APC/C inhibition. However, analysis of local cyclin B levels by immunodetection did not provide conclusive results, probably owing to the severe abnormalities observed in bubR1Rev1 embryos. Furthermore, since an increase in cyclin B copy number induces local changes in nuclear progression and only a global embryonic phenotype at six extra copies, the observations led to the speculation that variation in BubR1/cyclin B copy number should induce local variation in timing mitotic progression without affecting the total embryonic phenotype. Accordingly, a decrease in BubR1 protein level should result in a decrease in metaphase delay during cycle 7, as well as a reduced metaphase delay induced by higher cyclin B levels (Perez-Mongiovi, 2005).

In summary, this analysis of the requirement of BubR1 indicates that during syncytial development the spindle checkpoint appears to operate at various levels. It ensures that nuclei respond to global perturbations by imposing a mitotic arrest especially during later cycles. BubR1 also appears to work at a local level during early and late cycles to ensure proper synchrony of nuclear divisions. In parallel, BubR1 is required to sustain the mitotic arrest of the polar body so as to exclude it from undergoing further rounds of DNA replication during embryonic development (Perez-Mongiovi, 2005).

Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex

The partially conserved Mad3/BubR1 protein is required during mitosis for the spindle assembly checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast, whereas in mice, reduced dosage leads to severe chromosome missegregation. These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. A viable bubR1 allele was identified in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, a dose-sensitive requirement was demonstrated for BubR1 in maintaining sister-chromatid cohesion at anaphase I, whereas the mutant BubR1 protein localizes correctly. In bubR1 mutant females, both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister-chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency, and although the synaptonemal complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. These results demonstrate that BubR1 is essential to maintain sister-chromatid cohesion during meiotic progression in both sexes and for normal maintenance of SC in females (Malmache, 2007).

Observations of increased levels of pericentric exchange and defects in SC maintenance may be consistent with a role of BubR1 in regulating prophase progression, as observed for Mad3 in yeast. This activity in yeast, however, is particularly important for the segregation of achiasmate chromosomes. In contrast, the majority of nondisjunction events in bubR1 mutant females involve chromosomes that have undergone exchange, and achiasmate chromosomes are not particularly susceptible to nondisjunction. Furthermore, that the nondisjunction is largely equational suggests a function in late MI or MII rather than prophase. Although the possibility that these outcomes obtain from an earlier prophase defect cannot be ruled out, the simplest explanation is that relative to Mad3, BubR1 has a different, or additional, activity in maintaining sister-chromatid cohesion. Indeed, all the data are more consistent with BubR1 playing a direct role in sister-chromatid cohesion, similar to that of MeiS332. One possibility is that BubR1 affects the centromeric loading and/or maintenance of MeiS332, an essential protein required to prevent PSCS during MI, as has been observed for Bub1 in S. pombe. This could occur by a direct regulation of MeiS332 or by an indirect alteration of centromeric heterochromatin that in turn affects its loading at centromeres. There is precedent for a SAC-independent role of BubR1 at centromeres. Null mutations in CID, the fly homolog of human centromeric CENP-A protein, trigger a BubR1-dependent early mitotic delay, indicating that BubR1 is somehow involved in monitoring centromere assembly and/or behavior prior to metaphase. In summary, these results suggest that BubR1 plays an essential role in maintaining sister-centromere organization and function during meiotic progression (Malmache, 2007).

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).

In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).

To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav+ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).

These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype. cav homozygotes and cav1/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).

The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).

It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).

SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).

Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).

To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).

Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).

It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).

Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).

Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).

It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).

Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).


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Bub1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 12 January 2018

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