mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos

Drosophila double park (dup) encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. This study shows that mitotic checkpoint genes mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos. A study of double parked mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41 (Drosophila ATR); (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. double park mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16th S phase (S16). Cells carrying dupa1 allele forgo S16 altogether but enter mitosis 16 (M16). The timing of entry into M16 is similar in dupa1 and heterozygous or wild-type (wt) controls. In contrast, mutant cells carrying another allele, dupa3, undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dupa3 and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dupa3 and dupa1 mutant cells that enter M16 become arrested in M16. Mitotic cyclins are stabilized and a spindle checkpoint protein, Bub1 (Basu, 1999), localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dupa3 bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. This is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication (Garner, 2001).

Yeast mutants that cannot complete DNA replication arrest before mitosis, but yeast mutants that fail to initiate DNA replication fail to arrest; thus, initiation of DNA replication appears necessary to activate a checkpoint that couples mitosis to the completion of S phase. This can account for Drosophila dup mutants that fail to undergo S16 but enter M16. It was, however, surprising to find that cells of dupa1 and dupa3 mutant embryos, previously thought to behave similarly, enter M16 at different times. dupa1 mutant cells entered M16 approximately concurrent with heterozygous controls, whereas dupa3mutant cells entered M16 after heterozygotes. While the dupa1 allele results from a stop codon at 171 (out of a total of 743 amino acids), the dupa3allele results from a stop codon at position 592. Thus, dupa3 mutants may retain partial Dup activity that allows a partial S16 and consequently activates a checkpoint to delay M16. This idea is supported by two pieces of data: (1) a partial S16 is detected in dupa3 mutants, while confirming the observation that S16 is absent in dupa1 mutants; (2) the delay of M16 seen in dupa3 mutants is found to be absent in mei-41 dupa3double mutants. mei-41 encodes a homolog of the checkpoint kinase ATR and is required to delay mitosis upon inhibition of DNA synthesis in Drosophila syncytial embryos (cycles 11-13) (Sibon, 1999). It is concluded that partial DNA synthesis in dupa3mutants delays the entry into M16 via a Mei-41-dependent checkpoint (Garner, 2001).

The effect of dup mutations on entry into mitosis is in agreement with findings in yeast and Xenopus; the effect on exit from M16, however, is contrary to previous results. Budding yeast cells that lack CDC6 skip S phase and enter mitosis (similar to dupa1mutants) but are reported to subsequently exit mitosis with wild-type kinetics to undergo a 'reductional' anaphase. In contrast, epidermal cells of both dupa3and dupa1 mutants that enter M16, with and without a prior delay, become arrested. dupa1 cells are in M16 in stage 11 and mitotic cells are still seen in stage 13; dupa3cells are in M16 in stage 13 and mitotic cells are still visible at stage 15, if not later. Because the stages are at least 2 hr apart in each case, the arrest in M16 is likely to average at least 2 hr, significantly longer than normal embryonic mitosis of about 10 min (Garner, 2001 and references therein).

The apparent difference in the behavior of yeast and Drosophila cells that harbor unreplicated chromosomes led to an effort to determine the basis for mitotic arrest in dup mutants. To this end, mitotic spindles were examined by staining for alpha- and gamma-tubulin, and chromosomes were visualized by staining for a mitotic phosphoepitope on histone H3 (PH3). While the spindles are bipolar and appear to contain functional centrosomes, i.e., they contain gamma-tubulin and nucleate aster microtubules, chromosomes fail to align normally. Most chromosomes lie within the bipolar spindle but are scattered and not compacted into a metaphase plate. Severe alignment defects are readily visible in 84% (±11%) of mitotic cells in dupa1mutants and 80% (±7%) of mitotic cells in dupa3mutants; it is probable that higher resolution analyses may reveal higher incidences of defective alignment. Because chromosome configuration in dup mutants deviates from normal configurations, other markers of mitotic progression were examined to determine at which stage in mitosis dup mutant cells arrest (Garner, 2001).

In normal mitosis, Cyclin A degradation concludes in metaphase while Cyclin B degradation concludes in early anaphase. Spindle checkpoint proteins such as Bub1 that bind kinetochores upon spindle damage localize to kinetochores during unperturbed mitosis in metazoa, indicating that the spindle checkpoint is active through earlier parts of mitosis. Drosophila Bub1 localizes on kinetochores during prometaphase and dissociates during metaphase (Basu, 1999). In dup mutants, both cyclins persist during mitotic arrest, and Bub1 is present on discrete sites on chromosomes, presumably at kinetochores. These data suggest that dup mutant cells arrest prior to metaphase-anaphase transition (Garner, 2001).

The persistence of Bub1 on chromosomes and stabilization of Cyclin B occurs when the spindle checkpoint is active; therefore, mitotic arrest in dup mutants may be mediated by the spindle checkpoint. To test this directly, double mutants of dupa3and bub1 were examined. dupa3bub1 double mutants have fewer mitotic cells when compared to dupa3 single mutant embryos of similar stage. Two types of additional evidence indicate that this difference is due to dupa3bub1 double mutants exiting M16 (rather than reverting to previous interphase). (1) Approximately 10 times more cells are seen in the act of exiting mitosis (i.e., in telophase) in the double mutants. Most telophase cells show chromosome bridges, consistent with the failure to complete DNA replication in these mutants. (2) Nuclear density is higher in dupa3bub1 double mutants than in dupa3 single mutants, and it approaches that of heterozygotes or wt controls that complete M16. Collectively, these data indicate that a significant number of dupa3bub1 mutant cells exited M16 and suggest that bub1 is required for mitotic arrest in dup mutants (Garner, 2001).

During mitotic arrest by the spindle checkpoint, Cyclin A is degraded while Cyclin B remains stable. Therefore, persistence of Cyclin A during mitotic arrest in dup mutants suggests that additional control(s), in addition to the spindle checkpoint, operate to stabilize Cyclin A. DNA damage leads to stabilization of Cyclin A in Drosophila. Possibly, the presence of incompletely replicated DNA during mitosis also leads to stabilization of Cyclin A. bub1-mediated controls, however, appear more consequential because dupa3bub1 double mutants exited mitosis. This would be consistent with the finding that Cyclin A at endogenous levels cannot block mitotic exit in Drosophila (Garner, 2001).

CDC6-deficient cells are reported to exit mitosis with normal kinetics. An examination of these cells, however, reveals a mitotic arrest that requires MAD2. Thus, incomplete DNA replication in both yeast and Drosophila results in mitotic arrest mediated by members of the spindle checkpoint. Why might this be? A complete or partial absence of sister chromosomes would lead to a complete or partial absence of sister chromatid cohesion. The failure to duplicate centromeres, likely in dupa3mutants and certainly in dupa1mutants, would preclude the formation of kinetochore pairs. Either deficiency would preclude stable bipolar attachment of chromosomes to the spindle and thereby activate the spindle checkpoint (Garner, 2001).

In summary, dup mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41, Drosophila ATR; (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. In Drosophila syncytial cycles, nuclei delay the entry into mitosis upon inhibition of DNA synthesis, but exit from mitosis is not blocked. Instead, chromosome separation fails during the exit from mitosis, resulting in polyploid nuclei that are subsequently eliminated. In other systems, either the entry into mitosis (in fission yeast and vertebrate cells) or the exit from mitosis (in budding yeast) is blocked in response to incomplete DNA synthesis. Therefore, this is the first report of mitosis in a single cell cycle being regulated at two points via two different mechanisms in response to incomplete DNA replication. Identification of these responses in Drosophila, a genetically tractable organism with superb cytology, should enable testing of candidate checkpoint genes and searching for new genes that function at each regulatory point (Garner, 2001).

The behaviour of spindle checkpoint proteins during microtubule-kinetochore interaction

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. This study analyses how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, S2 cells were treated with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, it was found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension the kinetochore localization of spindle checkpoint proteins was analysed in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, it was observed that BubR1 (see below: The gene described in this section of the Interactive Fly is more properly termed BudR1) and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, these results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores (Logarinho, 2004).

In addition to the Drosophila Bub1 homologue referred to above, a genomic sequence (AE003565) was identified encoding a protein with homology to human Mad2. To study the localization patterns of Bub1 and Mad2 during mitosis, rabbit polyclonal antibodies were identified, and new antibodies were also produced against Bub3 and BubR1. Western blot analyses of total protein extracts from S2 cultured cells show that anti-Bub1 (Rb1112), anti-Mad2 (Rb1224), anti-Bub3 (Rb730) and anti-BubR1 (Rb666) sera are specific: (1) their corresponding pre-immune sera do not react with the predicted antigens; (2) affinity-purified Rb1112, Rb1224 and Rb730 sera and crude serum Rb666 recognise only one band of the expected molecular mass in S2 cell extracts; (3) anti-Bub1 antibodies do not cross-react with BubR1 since a single band of the predicted molecular mass is detected in extracts from bubR11 null mutant neuroblasts (Logarinho, 2004).

Immunofluorescence analysis of S2 cells was performed with the specific antibodies against Mad2, Bub1, Bub3 and BubR1 to determine their distribution during mitosis. The results show that Mad2 is mostly nuclear during prophase. At prometaphase, strong Mad2 labelling can be observed at kinetochores and spindle poles. At metaphase, Mad2 signal decreases dramatically and specifically in chromosomes that have congressed, but is still detected at the spindle poles and associated with microtubules in a punctuated pattern. In anaphase, Mad2 is no longer detected at kinetochores and staining of the spindle poles and microtubules decreases until telophase. The distribution of Mad2 in Drosophila cells is thus generally similar to that previously described for other organisms (Logarinho, 2004).

Bub1 immunolocalization shows that the polyclonal antibodies label the centrosomes strongly, as well as some spindle microtubules, from prophase until late anaphase. Kinetochore staining is mostly weak and only detectable during prophase and prometaphase. This immunolocalization pattern observed for the Bub1 protein is specific since it is not observed with pre-immune serum and is completely abolished if antibodies are pre-incubated with the recombinant protein (Logarinho, 2004).

Bub3 and BubR1 staining patterns are in agreement with reports using chicken polyclonal antibodies (Basu, 1998; Basu, 1999). During prometaphase both proteins show strong kinetochore accumulation, which decreases significantly at metaphase and becomes undetectable at later mitotic stages. However, it is consistently observed that whereas Bub3 and Bub1 localize to kinetochores during early prophase, as shown by anti-tubulin staining, BubR1 is never detected. This supports the classification adopted for the Drosophila Bub1-like proteins, since previous observations have shown that human Bub1 localizes to kinetochores at prophase before BubR1 (Logarinho, 2004).

In order to study the role of Bub1, BubR1, Bub3 and Mad2 in the spindle checkpoint, the behaviour of Drosophila S2 cells was examined when exposed to microtubule poisons. Incubation of these cells with colchicine (25 µM) appears to inhibit cell proliferation without an effect upon cell viability. This is consistent with a significant increase in the mitotic index during the first 12 hours and a strong kinetochore localization of all spindle checkpoint proteins tested. However, after this time the mitotic index starts to decrease and the culture shows an accumulation of polyploid cells suggesting that cells adapt and exit mitosis. The response of S2 cells to low doses of taxol (10 nM) is much less severe since the culture doubling time is only slightly delayed and the mitotic index shows a moderate increase. Overall these results indicate that S2 cells respond to microtubule poisons by delaying mitotic progression, and depending on the drug, the concentration used, and the incubation time, they display either a weak or a strong spindle checkpoint (Logarinho, 2004).

In mammalian cells, the distance between sister kinetochores at metaphase is considered to reflect the amount of tension exerted on them, since the microtubule poleward pulling forces cause the centromeric heterochromatin to stretch. Treatment of these cells with 10 µM taxol reduces centromeric stretch to nearly the 'rest length' (distance between sister kinetochores at prophase) when microtubules are unable to interact with kinetochores because of the nuclear envelope. To determine if S2 cells behave similarly, cells were fixed and co-immunostained for tubulin and the centromeric protein CID. Z-series optical sections of prophase and metaphase control cells were collected and interkinetochore distances were measured. The average interkinetochore distance in prophase cells was 0.73±0.12 µm. However, during metaphase the average interkinetochore distance increased significantly to 1.22±0.23 µm. To reduce the amount of tension across kinetochores, S2 cells were incubated for a brief period with low concentrations of taxol. Incubation with 10 nM taxol was sufficient to reduce tension without affecting microtubule attachment to kinetochores, as determined by measurement of interkinetochore distances in metaphase cells and tubulin immunostaining. The average interkinetochore distance in taxol-treated metaphase cells decreased to 0.82±0.13 µm, a value closer to the rest length. In order to confirm these results, control and taxol-treated cells were immunostained with the 3F3/2 monoclonal antibody. Tension is thought to induce a conformational change on the 3F3/2 kinetochore epitopes that promotes their dephosphorylation. In Drosophila, 3F3/2 was shown to label kinetochores during early stages of mitosis becoming strongly reduced or absent at metaphase. Strong 3F3/2 kinetochore labelling was found in taxol-treated metaphase cells but not in control metaphase cells. These results suggest that, in S2 cells, tension can be reduced by incubation with nanomolar concentrations of taxol and monitored by staining with the 3F3/2 antibody (Logarinho, 2004).

Recent studies suggest that Mad2, Bub1 and BubR1 might be monitoring different aspects of kinetochore-microtubule interactions, namely attachment and tension. Therefore, the kinetochore localization of Drosophila proteins was examined under conditions of reduced tension induced by taxol treatment. In taxol-treated cells, all metaphase kinetochores exhibit strong BubR1 staining in striking contrast to metaphase kinetochores from untreated cells, which only stain weakly. However, Mad2 and Bub1 do not accumulate significantly at kinetochores after taxol treatment. Interestingly, the subcellular distribution of Mad2 appears to be affected by loss of microtubule dynamics since it was never found in centrosomes or spindle microtubules after incubation with taxol. The kinetochore localization of these proteins was also characterized in the mono-oriented chromosomes occasionally seen in the taxol-treated cells. While Mad2 and Bub1 stainings are mostly detected only at the unattached kinetochore, BubR1 labelling is usually detected at both kinetochores. Staining only at the attached kinetochore was never observed for any of the antibodies. Quantification of the mono-oriented chromosome staining patterns showed that 75% and 65% exhibit Mad2 and Bub1 staining, respectively, only at the unattached kinetochore. However, most (78%) of the mono-oriented chromosomes were stained for BubR1 at both kinetochores. Taken together, these results show that BubR1 localizes to kinetochores whenever tension is absent and independent of their microtubule occupancy, whereas Mad2 and Bub1 localize preferentially to unattached kinetochores. These results are mostly consistent with recent data suggesting that localization of Bub1 is regulated by microtubule attachment, while that of BubR1 responds to tension, supporting the classification adopted for the two Drosophila Bub1-like proteins (Logarinho, 2004).

The role of tension in spindle assembly checkpoint signalling is not easy to distinguish from that of attachment, as application of tension on kinetochores can enhance both the stability of individual microtubule attachments and the overall occupancy of microtubules. Therefore, a protocol using S2 lysed cells was developed as an in vitro system to study 'tension in the absence of microtubules'. In this system, mechanical tension is analysed indirectly through the observation of 3F3/2 kinetochore phosphorylation, known to correlate with tension in vivo. Previously, it was shown that washed chromosomes from lysed cells, as well as isolated chromosomes, have dephosphorylated kinetochores that do not stain with 3F3/2 antibody. However, these kinetochores can be phosphorylated simply by incubation with ATP and a phosphatase inhibitor, showing that they contain a complete phosphorylation/dephosphorylation system, consisting of the kinase, the substrate and the phosphatase. In this system, kinetochore phosphorylation can be controlled experimentally rather than by tension, which is the situation in vivo. S2 cells lysed with detergent in the absence of phosphatase inhibitors rapidly lose 3F3/2 phosphoepitopes at their kinetochores. However, if cells are lysed in the presence of the phosphatase inhibitor microcystin, 3F3/2 staining is clearly detectable, in particular before metaphase. When dephosphorylated S2 cells (lysed in the absence of microcystin) are incubated for 20 minutes with ATP and microcystin, all kinetochores label positively with the 3F3/2 antibody. Interestingly, even metaphase chromosomes exhibit strong labelling at their kinetochores, while anaphase kinetochores are no longer rephosphorylated. In control preparations incubated with ATP in the absence of microcystin, none of the kinetochores becomes labelled with the 3F3/2 antibody. Therefore, S2 lysed cells provide an in vitro phosphorylation system that can be manipulated in order to study the behaviour of checkpoint proteins with respect to tension-sensitive 3F3/2 kinetochore phosphorylation in the absence of microtubules (Logarinho, 2004).

In order to study the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive phosphorylation, S2 lysed cells were double immunostained to detect 3F3/2 and Mad2, Bub1, BubR1 or Bub3. S2 cells lysed in the presence of microcystin showed strong kinetochore labelling for 3F3/2 and all spindle checkpoint proteins prior to anaphase. In S2 cells lysed in the absence of microcystin, 3F3/2 labelling was, as expected, undetectable at all kinetochores, but spindle checkpoint proteins showed different staining patterns. Mad2 and Bub1 were easily detected in the absence of 3F3/2 kinetochore phosphoepitopes. However, BubR1 and Bub3 kinetochore accumulation was not detected at any mitotic stage after detergent extraction in the absence of microcystin. As a control, the centromeric protein CID was observed to behave independently of the kinetochore phosphorylation status, suggesting that BubR1 and Bub3 depletion from kinetochores is not caused by disruption of kinetochore structure resulting from the procedure. These results show that, unlike Mad2 or Bub1, BubR1 and Bub3 retention at kinetochores depends specifically on the presence of kinetochore phosphoepitopes (Logarinho, 2004).

To unequivocally demonstrate that BubR1 and Bub3 depletion from dephosphorylated kinetochores is independent of microtubules, the phosphorylation assays were performed on isolated chromosomes. These chromosomes were purified from S2 cells incubated with colchicine to depolymerise microtubules. Moreover, since microcystin was included during all purification steps, and since these chromosomes were isolated from cells with an activated spindle checkpoint, the kinetochores stained very brightly with the 3F3/2 and anti-BubR1 antibodies. Incubation of these chromosomes with lambda phosphatase removed completely the 3F3/2 phosphoepitopes from the kinetochores, as well as BubR1. Control incubations with lambda phosphatase buffer or alternatively, with lambda phosphatase plus microcystin, showed that BubR1 loss is caused specifically by dephosphorylation. The same experiments were performed for Bub3 with similar results. To further determine whether the loss of BubR1 and Bub3 proteins from kinetochores is caused by dephosphorylation of 3F3/2 epitopes and not by dephosphorylation of some other epitopes, 3F3/2 phosphoepitopes were blocked with the antibody before lambda phosphatase treatment. Under these conditions, it was found that BubR1 and Bub3 accumulation at the kinetochores is preserved. The results indicate that, even when other phosphoproteins are dephosphorylated, as long as 3F3/2 phosphoepitopes are present, BubR1/Bub3 proteins are retained at the kinetochores (Logarinho, 2004).

Thus BubR1 and Bub3 retention or depletion from kinetochores correlates with the presence of 3F3/2 phosphoepitopes. To determine whether their accumulation at kinetochores also depends on 3F3/2 phosphorylated proteins, relocalization studies were carried out. Incubation of dephosphorylated lysed cells with ATP buffer without microcystin does not regenerate the 3F3/2 phosphoepitopes and BubR1 staining is also absent. However, if lysed cells are incubated with ATP plus microcystin, 3F3/2 phosphoepitopes are rephosphorylated and a very weak, mostly inconsistent, BubR1 signal can be detected. This result suggested that residual levels of BubR1 that had escaped detergent extraction could be recruited to kinetochores. Therefore, in order to test whether BubR1 reaccumulation at kinetochores, like its release, could be dependent on kinetochore phosphorylation, the ability of exogenously added BubR1 to bind to dephosphorylated or phosphorylated kinetochores was tested. Exogenously added BubR1 does not accumulate at dephosphorylated kinetochores, but it accumulates strongly at phosphorylated kinetochores. This recruitment is only observed before anaphase. Accordingly, rephosphorylated kinetochores stain brightly for 3F3/2 in metaphase but, at anaphase, 3F3/2 rephosphorylation does not occur. Incubation of rephosphorylated cells with a BubR1-depleted mitotic extract confirmed that BubR1 accumulation is specific. Similar experiments were performed for Bub3, with identical results. These results indicate very clearly that BubR1 and Bub3 accumulate only at kinetochores containing 3F3/2 tension-sensitive phosphoepitopes (Logarinho, 2004).

Thus, Drosophila, similar to higher eukaryotes, contains two genes that encode Bub1-like proteins. Both proteins share homology at the N terminus with Mad3 and additionally have a putative kinase domain in the C terminus typical of other known Bub1 and BubR1 proteins. However, whereas phylogenetic analysis in vertebrates places Bub1 and BubR1 proteins into defined clusters, in Drosophila the two Bub1-like proteins are more closely related to each other than to either Bub1 or BubR1 from other species. Therefore, their classification on the basis of sequence analysis turns out to be rather difficult. This is mainly due to the fact that both proteins have highly conserved Ser/Thr kinase domains, while in vertebrates BubR1 proteins are easily distinguishable from Bub1 proteins because they are less conserved at their C termini. Nevertheless, the data suggests that the previously reported Bub1 protein is BubR1 instead, and that the newly identified protein is Bub1, and this classification was adopted in this study. (1) Protein sequence analysis indicates that the previously described Bub1 protein contains a KEN-box motif at the N terminus while the new Bub1-like protein does not. The KEN box is an APC/CCdh1 recognition signal and was identified on the N terminus of yeast Mad3 and Mad3/BubR1 homologues, but not in any Bub1 homologue. It is therefore thought to be the distinguishable feature between Bub1 and Mad3/BubR1 proteins. Since there are no species with both BubR1 and Mad3, the functions fulfilled by BubR1 in mammalian cells and by Mad3 in yeast cells may be partially analogous, even though Mad3 does not have a C-terminal kinase domain. (2) Analysis of their intracellular pattern of localization shows that, during early prophase, only the newly identified Bub1 localizes to kinetochores, in agreement with previous observations showing that Bub1 localizes to kinetochores before BubR1 at prophase. (3) The Drosophila Bub1-like proteins were found to behave differently with respect to tension and microtubule attachment. BubR1 proteins accumulate at kinetochores in the absence of tension and are not as sensitive to microtubule attachment as Bub1. In this study, the protein that contains the KEN-box is the one whose localization responds to changes in tension but not microtubule attachment, once again suggesting that is the BubR1 homologue (Logarinho, 2004).

It has remained unclear whether tension and attachment are separable events in terms of checkpoint function. Therefore, two different approaches to study tension and attachment separately were used to determine whether the spindle checkpoint monitors those events independently. To study attachment in the absence of tension, Drosophila S2 culture cells were treated with nanomolar concentrations of taxol. At these low doses, microtubules can still attach to the kinetochores, but tension is severely reduced because of loss of microtubule dynamics. Measurement of interkinetochore distances and tubulin staining confirmed that, in S2 cells treated with taxol, tension is lost without disturbing microtubule attachment. Furthermore, 3F3/2 kinetochore staining correlates with the presence or absence of tension as previously shown in other cell types. In control cells, bioriented chromosomes showed dephosphorylated kinetochores as a result of tension while in taxol-treated cells, metaphase kinetochores were strongly phosphorylated (Logarinho, 2004).

To study 'tension in the absence of attachment', detergent-extracted S2 cells were used. In these cells, microtubules are depolymerised and tension can be analysed indirectly through the observation of 3F3/2 kinetochore phosphoepitopes. Mechanically applied tension diminishes kinetochore phosphorylation in lysed cells just as it does in living cells. Therefore, lysed cells can be used as an in vitro phosphorylation system to simulate the in vivo tension effect in the absence of microtubules. In vitro phosphorylated chromosomes mimic the in vivo improperly attached chromosomes, while in vitro dephosphorylated chromosomes mimic the in vivo bi-oriented chromosomes under tension (Logarinho, 2004).

How the kinetochore localization of spindle checkpoint proteins is affected by disrupting tension but not microtubule attachment was tested. Interestingly, different behaviours were found. When tension was reduced by low doses of taxol, BubR1 accumulated at kinetochores. This is in agreement with recent reports showing that reduced tension at kinetochores containing a full complement of microtubules induces a checkpoint-dependent metaphase delay associated with elevated levels of BubR1 at kinetochores. However, Mad2 and Bub1 proteins do not localize at kinetochores of aligned bioriented chromosomes after taxol treatment. Nevertheless, in mono-oriented chromosomes, Mad2 and Bub1 staining was consistently detected at the unattached kinetochore. These results are fully consistent with previous observations showing that Mad2 depletion from kinetochores is governed by microtubule attachment. In the case of Bub1, there are contradictory results regarding its behaviour. Whereas some reports show kinetochore accumulation of Bub1 under conditions of reduced tension, others show that its release from kinetochores is regulated by microtubule attachment. Thus, either Bub1 responds to both attachment and tension, or the results reflect differences between cell types. Nevertheless, the current results suggest that the behaviour of Bub1 is globally more similar to that of Mad2, suggesting that it is mainly sensitive to microtubule binding (Logarinho, 2004).

How the spindle checkpoint proteins behave with respect to tension-sensitive kinetochore phosphorylation in the absence of microtubule attachment was examined. These results were fully consistent with those obtained using taxol treatment. Whereas BubR1 and Bub3 are lost from kinetochores after dephosphorylation, Mad2 and Bub1 remain localized at kinetochores independent of their phosphorylation status, in agreement with the fact that these proteins are displaced by microtubule attachment. Previously, an inverse correlation between the amount of tubulin staining and the amount of Bub3 was shown for the two sister kinetochores of lagging chromosomes (Martinez-Exposito, 1999). However, this asymmetric labelling at lagging chromosomes might not necessarily reflect sensitivity to microtubule-attachment since 3F3/3 staining has been also reported to be asymmetric in lagging chromosomes. The phosphoepitopes are more strongly expressed on the leading kinetochore than in the trailing one. Indeed, BubR1 and Bub3 depletion from kinetochores occurs specifically because of 3F3/2 epitope dephosphorylation. Dephosphorylation of 3F3/2 epitopes might induce a conformational change in kinetochore proteins rendering them unable to interact with BubR1 and Bub3. BubR1 and Bub3 are unlikely to bind directly to the phosphoepitopes since pre-blocking isolated chromosomes with the anti-BubR1 antibody, does not inhibit 3F3/2 dephosphorylation by the lambda phosphatase treatment. Finally, from the experiments with lysed S2 cells it was found that, similar to their release, the binding of BubR1 and Bub3 to kinetochores is dependent on tension-sensitive kinetochore phosphorylation. Previous observations have demonstrated that Mad2 binding is also promoted by kinetochore phosphorylation. On the basis of all these results, a model is suggested for the behaviour of spindle checkpoint proteins during microtubule-kinetochore interaction (Logarinho, 2004).

Phosphorylation establishes a biochemical difference between kinetochores that are under tension and those that are not. Since the disruption of normal microtubule dynamics is sufficient to cause rephosphorylation of the 3F3/2 epitopes at metaphase kinetochores, even though they still contain many microtubules, this biochemical signal must act independently of microtubule attachment. Therefore, checkpoint proteins whose kinetochore localization is regulated exclusively by tension-sensitive phosphorylation are necessary to activate the checkpoint when microtubule dynamics is affected or when sister kinetochores are attached by microtubules from the same pole (syntelic attachment). Mad2 does not fit in this group of checkpoint proteins. Although Mad2 binding to kinetochores is initially governed by phosphorylation, it is inhibited later as kinetochores start being occupied by microtubules. However, the results show that BubR1 and Bub3 proteins behave differently from Mad2 because their removal from kinetochores is governed by dephosphorylation and is insensitive to microtubule attachment. Compelling data suggests that BubR1 and Mad2 operate independently, with the first sensing tension and the second monitoring attachment. The strength of the checkpoint response induced by these two events also appears to be different since S2 cells show only a short mitotic delay when exposed to low taxol doses, while complete microtubule depolimerization by colchicine, strongly compromises mitotic progression (Logarinho, 2004).

However, even though BubR1 and Mad2 may sense different spindle assembly signals, these must be integrated at some point. Evidence for the convergence of the two sensing mechanisms comes from the observation that the metaphase delay induced by reduced tension and increased levels of BubR1 at the kinetochores, is Mad2 dependent. This indicates that BubR1 and Mad2 cannot suppress the Cdc20-APC/C activity independently of each other. Indeed, Mad2 and BubR1 were found as components of the same APC/C-inhibiting complex (Logarinho, 2004).

The effect of tension on the spindle checkpoint might be direct. In the absence of tension, the 3F3/2 kinase(s) might directly activate spindle checkpoint proteins at kinetochores. Generation of tension, which pulls sister kinetochores apart, could then impair phosphorylation of 3F3/2 substrates by separating them from the kinase(s) or by changing their conformational structure. Only a few 3F3/2 epitopes have been identified so far. Interestingly, among these are the APC/C components Apc1 (Tsg24) and Cdc27, which concentrate at kinetochores during mitosis. Considering the results, which strongly suggest that BubR1 interacts closely with 3F3/2 phosphoproteins at kinetochores, and the recent evidence showing BubR1 interaction with the APC/C, it is possible that at unattached kinetochores, active kinases might catalyse phosphorylations that indirectly inhibit APC/C activity by enhancing the binding of BubR1 to the APC/C. Furthermore, it is believed that BubR1 is not a 3F3/2 kinase, since Drosophila bubR1 null mutants exhibit 3F3/2 staining. Curiously, bubR1 mutant cells enter anaphase precociously and with strong 3F3/2 labelling at the kinetochores. This supports BubR1 as being a component of the spindle checkpoint pathway that monitors tension-sensitive kinetochore phosphorylation. When BubR1 is absent, cells can override an arrest that would be otherwise induced by the presence of phosphorylated kinetochores (Logarinho, 2004).

Alternatively, tension might inactivate the spindle checkpoint indirectly. Recent genetic work in budding yeast suggests that the Aurora kinase Ipl1 plays an important role in tension-dependent spindle assembly checkpoint signalling. Aurora/Ipl1 is required for the spindle checkpoint activity induced by the absence of tension but not for the one induced by microtubule depolymerization. In addition, Aurora/Ipl1 was demonstrated to be critical for reorienting monopolar-attached sister chromatids whose kinetochores are not under tension so that they become attached to microtubules from opposite poles. The signal generated by lack of tension might therefore induce the release of microtubules from the syntelically attached sister kinetochores to allow the amphitelic reattachment. Accordingly, loss of tension indirectly maintains spindle checkpoint signalling by generating loss of microtubule occupancy, which is then sensed by Mad2. Recent results have shown that, in higher eukaryotes, Aurora B activity is also required to correct syntelic attachments and to activate the spindle checkpoint in the absence of tension. Furthermore, inhibition of Aurora B function in nocodazole-treated cells was shown to compromise kinetochore localization of the spindle checkpoint protein BubR1 but not Mad2. These results would be fully consistent with the observations if Aurora B/Ipl1 were the kinase that phosphorylates the 3F3/2 epitopes at kinetochores lacking tension (Logarinho, 2004).

Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. It is proposed that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization (Blower, 2006; full text of article)

Drosophila CENP-C is essential for centromere identity

Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. This study used RNA interference depletion in S2 cells to address this question, and it was found that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore-microtubule interactions, and spindle size are severely misregulated. Importantly, CENP-C was shown to be required for centromere identity, since CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. It is suggested that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila (Orr, 2011).

Kinetochores are assembled at the centromere of each replicated sister chromatid and provide an essential protein interface to allow binding of spindle microtubules and consequent chromosome segregation during mitosis. This study found that in Drosophila, CENP-C plays a major role not only in kinetochore organization but also in the proper assembly/maintenance of important centromere components suggesting that communication between the inner kinetochore and the centromere is an essential step in determining centromere identity (Orr, 2011).

CENP-C inactivation in vertebrate cells has been performed by antibody microinjection in HeLa cells, using CENP-C knockout mice or by tetracyclin-inducible knockouts in DT40 cells, and all studies concluded that CENP-C is essential for cell viability and mitotic progression. Detailed immunofluorescence analysis in CENP-C-deficient DT40 cells revealed a partial disruption of the inner kinetochore accompanied by a BubR1-dependent mitotic delay. While CENP-C inactivation in vertebrate cells causes partial disruption of the inner kinetochore, in Drosophila, CENP-C appears to perform more important roles. Consistently, bioinformatic approaches directed at evaluating CENP-C conservation between species reveals that while CENP-C is highly conserved among other Drosophila species, it bears very limited homology with its counterparts in higher eukaryotes. These differences may reflect different functions for the Drosophila CENP-C homolog and argue in favor of a different centromere-kinetochore interface specific to Drosophila chromosomes (Orr, 2011).

This study shows that CENP-C is required for the loading/maintenance of all kinetochore proteins tested including the SAC proteins (Mad2, Bub1, BubR1, and Bub3), mitotic regulator Polo kinase, microtubule motor protein CENP-meta, and KMN proteins (Ndc80, Nuf2, and Mitch). Interestingly, the kinetochore null phenotype observed after CENP-C depletion appears to be specific to Drosophila and C. elegans chromosomes, since CENP-C has been shown not to be required for full kinetochore organization in higher eukaryotes. Similar to Drosophila, no Constitutive Centromere-Associated Network (CCAN) homologs have yet been identified in C. elegans, which suggests that in systems lacking CCAN, centromere function relies uniquely on the structural role of CENP-C. Different to what has been reported in vertebrate cells, the current results are consistent with a model in which CENP-C is required to lay the foundation for all components essential for kinetochore assembly (Orr, 2011).

Previous reports have shown that loss of CENP-C in mammalian cells causes a mitotic delay. In chicken cells, this mitotic delay is BubR1 dependent and associated to a 3-fold increase in the overall duration of mitosis. This study demonstrated that in the absence of CENP-C, Drosophila kinetochores are unable to recruit essential SAC proteins Mad2, Bub1, BubR1, and Bub3, even if mitotic exit is prevented and microtubules removed. Nevertheless, consistent with the observed loss of SAC proteins, these cells are insensitive to microtubule poisons and rapidly exit mitosis in the presence of spindle damage. As expected when analyzing SAC-deficient phenotypes, these cells undergo fast mitotic exit accompanied by premature sister chromatid separation. Cells exit mitosis with a mitotic timing similar to what has been observed after Mad2 depletion in the same cell line, which suggests that this 12-min period is the minimum time these cells require to complete all processes required for mitotic exit. Two possible hypotheses could explain why CENP-C inactivation in other systems causes cells to block in mitosis. Either CENP-C inactivation was not as efficient in other species as it is in Drosophila S2 cells or these discrepancies could reflect structural differences in kinetochore organization specific to Drosophila chromosomes. Interestingly, Drosophila CID mutants display mislocalization of several kinetochore components accompanied by a BubR1-dependent mitotic delay, which suggests that CID inactivation cannot account for the loss of SAC maintenance observed when disrupting Drosophila CENP-C. However, in the case of CID mutants, maternally contributed CID might have occluded phenotypes that may explain the SAC-dependent mitotic delay in these cells. This study shows that kinetochore null cells fail to maintain SAC activity even in the presence of microtubule poisons, which suggests that kinetochore inactivation is not compatible with a functional SAC. Taken together, these data demonstrate that CENP-C is essential for full kinetochore assembly, a pre-requisite for efficient SAC maintenance (Orr, 2011).

In Drosophila, the localization of all outer kinetochore proteins appears to be dependent on CENP-C. Moreover, CENP-C is an essential factor for CID assembly at Drosophila centromeres. In accordance, it was recently proposed that CCAN copy number at kinetochores varies between vertebrates and yeast, suggesting that although specific centromere/kinetochore assembly models appear to be conserved, differences in protein copy number may reflect structural discrepancies between phenotypic analyses. The current data confirm that efficient CENP-C depletion causes CID mislocalization at centromeres, and this appears to be specific to Drosophila centromeres as it has never been observed in other systems. Collectively, these studies highlight potential differences in kinetochore organization between Drosophila and vertebrate cells (Orr, 2011).

The data also demonstrate that in Drosophila, CENP-C is essential for the proper localization of other centromere-specific proteins including the cohesion protector MEI-S332 and the CPC components INCENP and Aurora B. Taken together, these results are consistent with the proposal that Drosophila CENP-C is essential for maintaining normal centromeric architecture and identity, which appears to be species specific. In vertebrates, however, a large cluster of constitutive centromere-associated proteins (CENP-C, CENP-H, CENP-I, and CENP-K to CENP-U, and CENP-X) was identified as the CCAN which associates with CENP-A throughout the cell cycle, although a recent report also identified CENP-W that forms a DNA-binding complex together with CENP-T, all of which have no identified Drosophila orthologs. However, similarly to CENP-C, many of the CCAN proteins may have failed to be detected in the Drosophila genome because they lack significant conservation. At this point, it is not possible to rule out this possibility, although it is clear that in Drosophila, CENP-C plays an essential role in overall centromere and kinetochore organization, a role that might be shared with the CCAN protein complexes in other systems (Orr, 2011).

Together with the cumulated published evidence on the functional analyses of CID and CENP-C, the data suggest that the Drosophila centromere/kinetochore interface is simpler than that of higher eukaryotes. It is proposed that CENP-C plays a direct role in maintaining centromere identity and may fulfill many of the structural roles of CCAN complex proteins present in other organisms. Importantly, it was shown that there is functional communication between the inner kinetochores and the centromere, and at this point, it would be essential to understand which proteins are responsible for performing analogous functions at centromeres of higher eukaryote cells (Orr, 2011).

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

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