InteractiveFly: GeneBrief

mad2: Biological Overview | References

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. The role of the putative Drosophila Mad2 homologue was examined in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2 has been shown to block mitotic exit by sequestering Cdc20 (Fizzy in Drosophila). Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together these results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation (Orr, 2007).

The spindle assembly checkpoint (SAC) is a carefully orchestrated quality control mechanism required to ensure accurate chromosome segregation during cell division. The SAC is responsible for preventing anaphase onset in cells whose chromosomes have not yet reached a stable bipolar attachment. SAC activation/maintenance is thought to be mediated by a signal continuously generated at unattached or improperly attached kinetochores during prometaphase. Studies in primary spermatocytes demonstrated that not only microtubule occupancy but also tension across kinetochore pairs is required in order to satisfy the SAC. The delayed metaphase-anaphase transition imposed by the SAC is ultimately controlled by the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase that targets several mitotic substrates (including mitotic cyclins and securin) for destruction by the 26S proteasome to allow sister chromatid separation and mitotic exit (Orr, 2007).

The main components of the SAC molecular machinery were originally identified by genetic screens in budding yeast. MAD1-3 and BUB1 and BUB3 were shown to be required for a mitotic arrest in the presence of spindle damage. These genes have been found to be conserved from yeast to man with the exception of Mad3, which in higher eukaryotes is called Bub1-related kinase (BubR1; see Drosophila Bub1) because it is highly similar to Bub1 and unlike Mad3 contains a protein kinase domain within the C-terminal half (Orr, 2007).

Significant progress has been made in unraveling the molecular mechanism by which SAC proteins like Mad2 impose the mitotic arrest in response to inappropriately attached kinetochores. Mad2 is required for the establishment of a checkpoint-mediated arrest in response to spindle damage in Xenopus egg extracts (Chen, 1996) and in mammalian cells in culture (Gorbsky, 1998). Studies in Xenopus and human cells have also shown that Mad2 blocks mitotic exit by sequestering Cdc20, an APC/C activator (Musacchio, 2002; Bharadwaj, 2004). Mad2 localizes to kinetochores early in mitosis after binding Mad1 (Chung, 2003) where it undergoes rapid turnover (Howell, 2004). This rapid turn over at kinetochores is thought to underlay the formation of Mad2-Cdc20 inhibitory complexes that signal abnormal microtubule-kinetochore attachment (Sironi, 2001). Extensive work using fixed cells in a variety of organisms has supported this model by showing that indeed Mad2 accumulates strongly at kinetochores in the absence of microtubules (Chen, 1998, Chen, 1999; Logarinho, 2004). However, recent studies in Drosophila using a green fluorescent protein (GFP)-Mad2 transgene and time-lapse microscopy have suggested an alternative view. The data indicates that even after microtubule kinetochore attachment takes place, a low level of Mad2 continues to enter the kinetochore and is removed mostly along spindle microtubules in a poleward direction (Howell, 2004; Buffin, 2005). These results suggest that perhaps the inhibitory signal provided by unattached kinetochores results not from the absence of kinetochore microtubule attachment per se but from the inability of Mad2 (and maybe other checkpoint proteins) to exit the kinetochore through microtubules, causing the accumulation of Mad2 at the kinetochore and the consequent formation of complexes that can now freely diffuse throughout the cytoplasm and inhibit the APC/C (Orr, 2007).

Although the role of kinetochores in the generation of a soluble inhibitory signal that delays metaphase-anaphase transition is consistent with most published data, recent experiments have suggested that cytoplasmic Mad2 is also required for the proper timing of early prometaphase independently of kinetochores (Meraldi, 2004). Studies in human tissue culture cells show that when Mad2 is depleted in cells with disrupted kinetochores, sister chromatid separation follows very shortly after nuclear envelope breakdown (NEBD). However, if kinetochore-deficient cells now contain cytosolic Mad2, prometaphase is extended significantly, even though these cells still show a defective SAC response (Meraldi, 2004). These results suggest that Mad2 has a kinetochore-associated function in maintaining SAC activity and a kinetochore-independent function in timing mitotic progression (for discussion see Kops, 2005). Mad2 might therefore perform additional, SAC-unrelated functions during progression through mitosis. Interestingly, recent studies have also shown that besides their role in maintaining SAC activity, other checkpoint proteins also perform additional roles during mitosis progression. Bub3 has been shown to be required for the accumulation of cyclins during G2 and early mitosis. Furthermore, BubR1 and Bub1 were shown to be required for maintaining proper microtubule kinetochore interaction and chromosome congression (Orr, 2007).

Therefore, to gain insight into the primary role of Mad2 during mitosis, Drosophila S2 tissue culture cells were treated with double-stranded RNA against Mad2, and mitotic progression was analyzed in detail. Consistent with previous studies in other organisms, it was found that depletion of Mad2 causes loss of the SAC response in Drosophila S2 cells. Moreover, Mad2-depleted cells fail to reach metaphase, exit mitosis very soon after NEBD, and show highly abnormal chromosome segregation that is characterized by the formation of extensive chromatin bridges and severe aneuploidy. However, the results indicate that Mad2 is unlikely to have any specific role in either chromosome condensation or microtubule kinetochore interaction because a checkpoint-independent arrest in mitosis allows normal chromosome condensation and congression. Also, release from the mitotic arrest allows cells to exit mitosis without chromatin bridges and with chromatid segregation profiles that are indistinguishable from controls. More significantly, if Mad2-depleted cells are released from the mitotic arrest into media containing the microtubule-depolymerizing agent colchicine, cells arrest in mitosis with intact sister chromatid cohesion and strong kinetochore accumulation of SAC proteins, suggesting an active SAC response. Taken together these results suggest that Mad2 has a major role in delaying mitotic progression during early stages of prometaphase so that the SAC can be activated and chromosome segregation can be properly conducted (Orr, 2007).

This study found that all Mad2-associated phenotypes can be reverted and the checkpoint is effectively activated if cells are subjected to a transient mitotic arrest. Thus, contrary to current models which view Mad2 at the center of the inhibition of the APC/C by the SAC, it is hypothesize that Mad2 is required for proper timing of mitotic progression only during prometaphase, allowing cells to fully engage the SAC through kinetochore accumulation of other checkpoint proteins so that complete chromosome condensation and congression can be achieved before the controlled metaphase-to-anaphase transition takes place (Orr, 2007).

Checkpoint proteins have been shown to be essential for the fidelity of mitosis because they are responsible for sensing errors in microtubule kinetochore interaction. Loss of the Mad2 homologue causes inactivation of the SAC in Drosophila S2 cells. To find out whether Mad2 is required for the kinetochore localization of other checkpoint components, immunolocalization studies of other checkpoint proteins was performed. All three proteins tested (Bub1, Bub3, and BubR1) show strong accumulation at kinetochores, demonstrating that they do not require Mad2 for their localization and also that proper kinetochore localization of these checkpoint components does not per se prevent premature mitotic exit. Previous studies in Xenopus and HeLa cells were performed in the presence of microtubule poisons, and therefore it was unclear whether the absence of protein localization reflected a hierarchical relationship or the inability to analyze a large number of mitotic cells because of fast mitotic exit (Chen, 2002; Johnson, 2004). Because individual depletion of Bub3 in Drosophila (Lopes, 2005) and analysis of the hypomorphic allele of BubR1 (Basu, 1999) resulted in a nonfunctional SAC response, it is very likely that these proteins work through parallel signaling pathways that are mutually required at some stage to sustain checkpoint activity. Consistent with previous work, it seems probable that removing Mad2 may abrogate the spindle checkpoint not only because a sensor is being removed, but because the MCC, a multisubunit complex containing BubR1-Mad2-Bub3-Cdc20 (a far more potent APC/C inhibitor; Sudakin, 2001) cannot form in its absence (Orr, 2007).

Previous results have shown that inactivation of Mad2 by antibody microinjection during either prophase or prometaphase induced abnormal sister chromatid segregation in PtK1 cells (Gorbsky, 1998). This phenotypic analysis revealed that Drosophila S2 cells lacking Mad2 also display severe abnormalities during mitotic progression. Mad2-depleted cells fail to reach metaphase and exit mitosis with extensive chromatin bridges. The anaphase bridges formed by Mad2-depleted cells appear to be exclusively due to a premature exit from mitosis because extending the time spent in mitosis is enough to revert this phenotype. This is fully consistent with recent data suggesting that proper chromosome condensation and sister chromatid resolution is only fully completed during early prometaphase. Furthermore, the results suggest that in S2 cells, full chromosome condensation is achieved only late in prometaphase, after the minimal time cells spend in prometaphase when the SAC is inactivated. This is in full agreement with a previous analysis of mitotic progression in Bub3-depleted cells (Lopes, 2005), where it was shown that in the absence of Bub3 the SAC is inactive but that cells do not exit mitosis with inappropriately condensed chromosome because of an extended period in prophase (Orr, 2007).

Several studies have shown that loss of SAC proteins causes premature sister chromatid separation (PSCS) and significant aneuploidy. This study found that loss of Mad2 causes premature degradation of cohesins during prometaphase resulting in high levels of PSCS. In addition, quantification of kinetochore segregation at anaphase shows that loss of Mad2 results in a high frequency of cells showing unequal kinetochore segregation. Furthermore, FACS analysis shows that the DNA content of the mitotic population changes significantly over time in the absence of Mad2. These results suggest that unlike in yeast where MAD2 is not essential for chromosome segregation (Cohen-Fix, 1997), Drosophila Mad2 is required to maintain the long viability of cells (Orr, 2007).

Early studies on the role of Mad2 in the SAC response using cultured animal cells revealed premature anaphase onset (Gorbsky, 1998). More recently, it was found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision in which ~80% of cells complete mitosis in 32 ± 6 min and that Mad2 inactivation in these cells consistently shortened mitosis (Jones, 2004). Furthermore, depletion of Mad2 by RNAi showed that HeLa cells exit mitosis prematurely (Meraldi, 2004). The current results are fully consistent with this data because depletion of Mad2 in S2 cells causes a threefold reduction in the time from NEBD to anaphase onset. Interestingly, these same studies proposed a role of Mad2 in timing mitotic progression that is more complicated that previously expected. It was shown in HeLa cells that inactivation of kinetochore-bound Mad2 disrupts the SAC without significantly affecting the timing of mitotic progression. However, when the cytosolic pool of Mad2 present in these cells is depleted, then both the SAC response is abnormal and the timing of NEBD to anaphase onset is severely reduced, suggesting that Mad2 is also required to time mitotic progression in a kinetochore-independent manner. This contrasts with current models which propose that Mad2 plays an essential role in SAC activation and maintenance by providing a kinetochore-based signal that inhibits the APC/C (Musacchio, 2002). The observations with Drosophila suggest a much more subtle role for Mad2 in ensuring a SAC response. Surprisingly, it was found that after a transient mitotic arrest, Mad2-depleted cells were able to respond to spindle damage and arrest in mitosis with cohesin still located at the centromere of chromosomes and high kinetochore levels of BubR1, suggesting that the SAC is fully functional. Thus, providing time in a checkpoint-independent and transient manner appears to be sufficient for Mad2-depleted cells to either reactivate or maintain SAC activity and respond correctly to microtubule depolymerization. Given that this sustained SAC activity cannot be observed after depletion of other SAC proteins such as BubR1, it is hypothesized that the APC/C inhibitory signal provided by Mad2 is specifically required during early stages of prometaphase to ensure maintenance of SAC activity. Subsequently, SAC activity could be ensured by other checkpoint proteins such as BubR1 that could then accumulate strongly at kinetochores. These observations are in full accordance with previous biochemical studies that identified at the G2-M transition the MCC, a multisubunit complex containing BubR1-Mad2-Bub3-Cdc20 (Sudakin, 2001). The MCC was shown to be the most powerful APC/C inhibitor (Sudakin, 2001; Tang, 2001; Sudakin, 2004). Interestingly, formation of the MCC does not require unattached kinetochores given that it is present well before the NEBD. Taken together, these observations have suggested a 'two-step' model for the activation and maintenance of SAC activity (Chan, 2005). This model proposes a first step involving the formation of the MCC as cells reach the G2/M transition, allowing cyclin accumulation and mitotic entry. Subsequently, in a second step after NEBD, SAC proteins can bind unattached kinetochores and produce additional inhibitory complexes that sustain SAC activity until all kinetochore pairs are properly attached and congression is achieved. Subsequent studies both in yeast (Fraschini, 2001) and Drosophila (Lopes, 2005) provide strong support for this model. The results reported in this study, provide a further refinement of this model in that the second step can be separated into two events: one at NEBD when cytoplasmic Mad2 is required to extend prometaphase and provide enough time so that in a second event, SAC proteins such as BubR1 and Bub3 can fully engage checkpoint activity. Further studies on the role of Mad2 and other SAC proteins in the inhibitory activity of the MCC before and during early stages of mitosis will be required to unravel how the different levels of regulation are organized. Nevertheless, the current observations provide new insights into how the signals provided by different SAC proteins might contribute to a fully integrated checkpoint response (Orr, 2007).

Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores. Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, a new protein (Spindly) has been identified that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. A human homologue of Spindly serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. These findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores (Griffis, 2007).

The spindle assembly checkpoint (SAC) is critical for preventing the onset of anaphase until all chromosomes are aligned on the metaphase plate. A single misaligned kinetochore is sufficient to generate a wait anaphase signal, thereby ensuring that all sister chromatids segregate to opposite ends of the spindle and are equally distributed to the daughter cells. Failure of the SAC can lead to premature anaphase onset and aneuploidy. Such defects can have consequences for a whole organism; mice that lack a full complement of SAC genes have more frequent DNA segregation errors and are more susceptible to tumor development (Griffis, 2007).

The presence of the SAC was initially inferred from observations that cells delay in metaphase when meiotic sex chromosomes fail to pair and align or after the spindle is perturbed by either microtubule poisons or microsurgery. Molecules responsible for the SAC were later identified in yeast genetic screens and named Mad1, -2, and -3 (Mad for mitotic arrest deficient) and Bub1, -2, and -3 (Bub for budding unperturbed by benzimidazole). Subsequent work showed that these proteins together with the MPS1 kinase form distinct complexes that target to the kinetochore. Two additional metazoan checkpoint proteins, Zw10 and Rough Deal (Rod), were later isolated as cell cycle mutants in Drosophila melanogaster. These two proteins, together with a third protein called Zwilch (for review see Karess, 2005), form a complex (Rod-Zw10-Zwilch complex [RZZ]) that regulates the levels of Mad1 and Mad2 on the kinetochore (Griffis, 2007).

Ultimately, the SAC pathway must lead to inhibition of the anaphase-promoting complex (APC), a multisubunit ubiquitin E3 ligase that targets multiple mitotic regulators (e.g., mitotic cyclins as well as the securin protein that inhibits the cleavage of cohesin molecules) for proteosome degradation to allow mitotic exit. Several studies have shown that localization of the checkpoint proteins to misaligned kinetochores is essential for establishing the SAC and keeping the APC inhibited, most likely by generating a diffusible signal that inhibits the APC. The nature of the diffusible signal is still subject to debate. However, a current model (for review see Musacchio, 2007) suggests that the kinetochore-bound Mad1-Mad2 complex acts as a template that coverts the free, inactive Mad2 to an active form that can diffuse away from the kinetochore and bind to and sequester Cdc20, a regulatory component of the APC (Griffis, 2007).

The capture of microtubules by the kinetochore and the downstream activity of two different microtubule motors are required for silencing the SAC in metazoans. One of these motors is the kinesin centromere protein (CENP) E, which may act as a tension sensor that, when stretched, inactivates the BubR1-dependent inhibition of Cdc20 (Chan, 1999; Mao, 2005). The second motor is dynein, which transports Mad1, Mad2, and RZZ from the kinetochore to the spindle pole. Dynein-based removal of Mad1 and Mad2 from the kinetochore may disrupt the template mechanism that generates the active Mad2 that inhibits the APC (for review see Musacchio, 2007). After inhibition or depletion of dynein or its cofactors, metazoan cells arrest in metaphase with correctly aligned chromosomes and high levels of kinetochore-bound Mad1, Mad2, and RZZ (Griffis, 2007).

Resolving the mechanism of dynein recruitment to kinetochores is important for understanding how kinetochore-microtubule binding ultimately leads to inactivation of the SAC. Currently, it is thought that dynein is brought to the kinetochore by binding directly to dynactin (a multisubunit complex required for multiple dynein functions), which, in turn, binds to the Zw10 subunit of the RZZ complex. Lis1, another dynein cofactor, also has been proposed to play a role in targeting dynein to kinetochores. Dynactin, Lis1, and Zw10 are not kinetochore-specific factors, as they are involved in targeting dynein to multiple other locations in the cell. It has not been clearly established whether dynactin and Lis1 are sufficient for targeting dynein to kinetochores or whether other proteins might be involved (Griffis, 2007).

To find new proteins that might participate in the SAC, an automated 7,200 gene mitotic index RNAi screen was undertaken in S2 cells. This screen uncovered a novel gene, which was also identified in an independent screen of genes involved in S2 cell spreading and morphology. This protein (termed Spindly) localizes to microtubule plus ends in interphase and to kinetochores during mitosis. Cells depleted of Spindly arrest in metaphase with high levels of Mad2 and Rod on aligned kinetochores, a defect caused by a failure to recruit dynein to the kinetochore. However, Spindly is not required for other dynein functions during interphase and mitosis. A human homologue of Spindly, which is similarly involved in recruiting dynein to kinetochores, was identifed. Thus, these results have uncovered a novel conserved dynein regulator that is involved specifically in dynein's function in silencing the SAC (Griffis, 2007).

An RNAi screen has identified Spindly as an essential factor for docking dynein to the kinetochore. Spindly is recruited to the kinetochore in an RZZ-dependent manner, and there, together with dynactin, Spindly recruits dynein to the outermost region of the kinetochore. The dynein motor complex then transports Spindly along with Mad2 and the RZZ complex to the spindle poles to inactivate the SAC. A Spindly homologue plays a similar role in human cells, revealing a conserved dynein kinetochore targeting mechanism in invertebrates and vertebrates. These data provide new insight into the mechanism and importance of recruiting dynein to the kinetochore to inactivate the SAC. Spindly also plays a role in maintaining S2 cell morphology during interphase and localizes to the growing ends of microtubules (Griffis, 2007).

The depletion of Spindly creates several mitotic defects that appear to reflect a loss of dynein activity exclusively at the kinetochore. Metaphase arrest is the most evident defect observed after the RNAi-mediated depletion of Spindly in Drosophila or human cells. This metaphase arrest phenotype is most likely explained by the absence of kinetochore-bound dynein in Spindly-depleted cells, and, indeed, the data support a model proposes that kinetochore-bound dynein is required for transporting Mad2 from the kinetochore to inactivate the SAC. Nevertheless, the possibility that the mitotic delay seen after dynein or Spindly depletion is caused by another kinetochore aberration that keeps the checkpoint activated. However, Spindly-depleted cells ultimately overcome metaphase arrest, as seen in live cell imaging experiments and by the modest increases in the mitotic indices of Spindly-depleted S2 and HeLa cells (three- to seven-fold and two-fold, respectively). The mechanism of slippage from this metaphase arrest is not clear, but it might involve proteins (e.g., p31 comet) that silence the SAC by disrupting the interaction between Mad2 and Cdc20 (Griffis, 2007).

In addition to mitotic arrest, chromosomes in Spindly- and dynein-depleted S2 cells require a longer time to align on the metaphase plate. This result may be attributable either to the displacement of CLIP-190 (a microtubule tip-binding protein) from kinetochores after Spindly or dynein depletion or the loss of dynein-mediated lateral attachments to microtubules in early prometaphase. In HeLa cells, a defect in chromosome alignment was noticed after Hs Spindly depletion, which also has been observed after the depletion of dynein (perhaps mediated through a loss of kinetochore-bound CLIP-170) (Griffis, 2007).

Thus, the spectrum of mitotic defects observed in Spindly-depleted cells is consistent with a loss of dynein function specifically at the kinetochore. Spindly depletion does not produce any other defects seen after dynein depletion, such as centrosome detachment and spindle defocusing. Dynactin is another protein that is required for recruiting dynein to kinetochores, but it is important for other mitotic and interphase dynein functions. Depletion of the RZZ complex inhibits the kinetochore recruitment of dynein, but this also prevents Mad1 and Mad2 recruitment and reduces kinetochore tension to a greater degree than Spindly or dynein depletion alone. Thus, Spindly depletion appears to be the most specific means identified to date for interfering with dynein function only at the kinetochore (Griffis, 2007).

These findings provide new insight into how dynein localizes to kinetochores. Previous studies have led to a model in which dynactin binds to the RZZ complex and then, either alone or in collaboration with Lis1, recruits dynein to the kinetochore. Because it was found that both dynactin and Spindly are required for dynein localization to kinetochores, an updated model is proposed in which Spindly and dynactin target to the kinetochore independently and work together to recruit dynein (Griffis, 2007).

Thus, dynein recruitment to the kinetochore may involve multiple weak interactions. Consistent with the possibility of weak interactions, endogenous dynein, dynactin, and Rod did not coprecipitate with GFP in pull-down experiments, and Spindly did not coenrich with these proteins in sucrose gradient fractions. Lis1 is not included in the dynein localization model, since it was found that Lis1 RNAi does not block dynein recruitment to the kinetochore (using a colchicine treatment localization assay), although Lis1 depletion does cause a mitotic delay and substantial increase in GFP-Spindly on aligned kinetochores. Thus, a role is favored for Lis1 in dynein activity but not in recruiting dynein to the kinetochore (Griffis, 2007).

Spindly's role in the spreading morphology of S2 cells makes it unusual among proteins involved in silencing the SAC (including dynein and dynactin), which did not produce phenotypes in the interphase morphology screen. The Spindly RNAi interphase phenotype of defective actin morphology and the formation of extensive microtubule projections is still not understood. However, a clue may be Spindly's dynamic localization to the growing microtubule plus end. Other plus end-binding proteins (+TIPs) interact with signaling molecules that regulate cell shape, one example being the binding and recruitment of RhoGEF2 to the microtubule plus end by EB1. Spindly may similarly interact with and carry an actin regulatory molecule to the cortex, but this hypothesis will require identifying proteins that interact with Spindly during interphase (Griffis, 2007).

The mechanism of Spindly recruitment to the microtubule plus end also warrants further investigation. This interaction must be regulated by the cell cycle because GFP-Spindly no longer tracks along microtubule tips in prometaphase. Seven consensus CDK1 phosphorylation sites are present in the positively charged C-terminal repeats of Spindly, and phosphorylation of these sites could reverse the charge of these repeats and regulate the transition from microtubule tip binding to kinetochore binding at the onset of mitosis (Griffis, 2007).

Motor proteins must be guided to the correct subcellular site to execute their biological function. To carry out the multitude of transport activities required in eukaryotic cells, metazoans have evolved numerous kinesin motors (25 genes in Drosophila) with distinct domains that dictate their localization and regulation. In contrast, a single cytoplasmic DHC performs numerous roles in interphase and mitosis, suggesting that additional regulatory factors guide dynein to specific cargoes (e.g., organelles, mRNAs, and vesicles). The main dynein-associated proteins (the dynactin complex, Lis1, and NudEL) are involved in dynein function at many sites and, thus, do not appear to be cargo specific. Zw10 was initially thought to specifically regulate the recruitment of dynein-dynactin to the kinetochore, but it now also appears to play an essential role in targeting dynein to membrane-bound organelles. Bicaudal D is another multifunctional adaptor molecule that has a role in the dynein-based transport of multiple cargoes such as RNA, vesicles, and nuclei. Perhaps the most site-specific dynein recruitment factor is the Saccharomyces cerevisiae Num1 protein that binds to the DIC Pac11p to target the motor to the cortex of daughter cells, where it pulls the nucleus into the bud neck. However, dynein only serves this one function in yeast compared with its plethora of activities in metazoans, and Num1p homologues have yet to be identified in higher eukaryotes (Griffis, 2007 and references therein).

Spindly appears to be a highly selective dynein-recruiting factor, and, unlike other dynein cofactors, it does not appear to be involved in the motor's nonkinetochore functions in mitosis (e.g., pole focusing) or in interphase (e.g., endosome transport). However, the mechanism by which Spindly recruits dynein to the kinetochore remains to be elucidated. Observations that Spindly moves from kinetochores to the spindle poles as discrete punctae strongly suggests that it may incorporate into a large and somewhat stable particle that contains the RZZ complex, Mad1-Mad2, dynein, and likely additional proteins. Therefore, Spindly not only serves to recruit dynein to the kinetochore but also is part of a cargo that dynein transports. Future studies will be needed to better understand the protein composition of these transport particles and the contacts that Spindly makes within them (Griffis, 2007).

Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. This study simultaneously followed GFP-Mad2 and RFP-Rod and found they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores (Buffin, 2005).

To gain insight into the role of Rod/Zw10 relative to other checkpoint proteins, a study of fluorescently tagged (GFP and mRFP1 Rod (CG1569), Mad2 (CG17498), and BubR1 (CG7838) in a single cell type, the Drosophila larval neuroblast. All three fusion proteins are controlled by their natural promotors, and all three retain their biological activity (Buffin, 2005).

Consistent with earlier reports, Rod and BubR1 are cytoplasmic in interphase, whereas Mad2 is associated with the nucleoplasm and nuclear envelope. In fly neuroblasts, as in Hela cells but unlike in the marsupial cell line PtK, BubR1 is the first to accumulate on kinetochores during prophase. Mad2 and Rod begin to label kinetochores only during nuclear-envelope breakdown (NEB), easily recognized by the invasion of Rod into the nucleoplasm. The first kinetochore-associated Mad2 signals above the nucleoplasmic background are seen simultaneously with the first Rod signal (Buffin, 2005).

In prometaphase, the kinetochores brightly label with all three proteins. Because cytoplasmic Mad2 signal is consistently higher than either BubR1 or Rod, Mad2 kinetochore labeling appears relatively less prominent. As the kinetochores capture MTs, Mad2 and Rod both are transported poleward. This process, called 'shedding', requires dynein/dynactin and may be important for shutting off the checkpoint once MTs are properly attached (Buffin, 2005).

These live images reveal a robustness that was not evident for Mad2 transport in earlier studies in PtK cells and Drosophila cells , although it can be seen sometimes even by immunostaining. It is difficult to quantify these signals, but the films clearly show that new cytosolic Mad2 is continuously recruited to kinetochores even after MT capture and replaces that lost to shedding; the total Mad2 signal on kinetochore microtubules (KMTs) over the duration of prometaphase and metaphase is far greater than the original kinetochore-associated signal. This is particularly evident where metaphase is prolonged. Thus Mad2, like Rod, establishes a flux of recruitment to and shedding from attached kinetochores (Buffin, 2005).

GFP-Rod and RFP-Mad2 show a near-perfect coincidence of signal in prometaphase and early metaphase, not only on kinetochores but also along the KMTs. The overall patterns of the two proteins are superimposable. Where discrete particles of GFP-Mad2 could be followed, they always contained RFP-Rod. These results suggest that Mad2 and Rod/Zw10 remain associated as they leave the kinetochore along the KMTs (Buffin, 2005).

By late metaphase, Mad2 signal has essentially disappeared from kinetochores and is only faintly visible on the spindle above the cytoplasmic Mad2 background, whereas Rod shedding continues robustly up to anaphase onset. In larval neuroblasts, the timing of NEB to anaphase onset is typically 7-12 min, of which metaphase lasts 2-8 min. There does not appear to be much delay between Mad2 disappearance from the spindle and anaphase onset. On average, Mad2 is gone less than 1 min prior to anaphase, and sometimes just seconds before. This contrasts with the situation in PtK cells, where anaphase occurs on average 10 min after the disappearance of the last detectable Mad2 signal. The significance of this difference is for now unclear. It may reflect simply an adaptation to the very rapid mitosis in flies (7-12 min NEB-anaphase, compared to 25 min after alignment of the last chromosome for Ptk cells). Alternatively, it may reflect a more fundamental difference in the way the spindle checkpoint is turned off (Buffin, 2005).

The behavior of Mad2 and Rod was distinguishable from that of BubR1 in several ways. BubR1 remained tightly associated with kinetochores and was not detectable along the spindle after MT capture. Although in PtK cells BubR1 may be transported from kinetochores to poles after energy depletion, in normal fly neuroblasts shedding does not appear to be a major route by which BubR1 levels are reduced on attached kinetochores. Moreover, close inspection of in vivo double-labeled cells revealed that, as the metaphase plate develops, BubR1 becomes enriched in a kinetochore domain slightly internal to that of Rod and Mad2 (Buffin, 2005).

Rod/Zw10, dynein/dynactin, Mad2 and BubR1, and all the transient kinetochore proteins are normally classified as outer-domain kinetochore components, and indeed they all form enlarged crescents around the MT-free kinetochores. The outer domain can be further subdivided into a more interior 'outer plate' which appears to be the MT attachment site as well as the location of BubR1, and an outer fibrous corona that is believed to contain Rod/Zw10, dynein/dynactin, and CenpE. The relative locations of the various checkpoint proteins have not been compared in attached kinetochores of living cells. The observation that Mad2 colocalizes with Rod but not with BubR1 is the first demonstration that Mad2 is part of the corona (Buffin, 2005).

The different locations of Mad2 and BubR1 are consistent with certain distinct features of their behavior. For example, Mad2 accumulation is highly sensitive to MT attachment and is depleted from kinetochores by shedding along KMTs. BubR1 by contrast is not depleted significantly by shedding and responds more to changes in tension. If this correlation holds, perhaps other proteins with robust shedding (for example, CenpF will prove to colocalize in the corona with Mad2, Rod/Zw10, and dynein (Buffin, 2005).

In summary, Mad2 and Rod/Zw10 behavior on kinetochores and spindles are qualitatively closely linked. They are simultaneously recruited and are shed together during prometaphase and early metaphase. BubR1, by contrast, is independently recruited to a different kinetochore domain and does not undergo detectable shedding (Buffin, 2005).

To further probe the relationship of Mad2 and Rod/Zw10, the behavior of GFP-Mad2 was examined in rod and zw10 null-mutant cells. Given the importance of dynein-dynactin for shedding and the role of Rod/Zw10 in dynein recruitment, it was anticipated that rod or zw10 mutants would show abnormal retention of Mad2 on kinetochores. In fact, however, in these cells kinetochore-associated GFP-Mad2 was significantly reduced, although Mad2 was still prominent on interphase rod nuclei. The reduction of kinetochore-associated Mad2 was evident in every rod or zw10 mutant cell examined, although the extent of reduction was somewhat variable. In three of 15 rod cells (20%) filmed from NEB to anaphase onset, no kinetochore-associated Mad2 was detectable above the cytoplasmic background at any stage. In the rest, a weak signal was briefly detectable on some kinetochores during prometaphase. Quantitation of these signals revealed that the kinetochore intensity in rod cells was only about 20% above the cytoplasmic level, at their maximum, whereas in wild-type cells kinetochore Mad2 signals averaged 4.4-fold higher than cytoplasmic signals. Depolymerizing microtubules with colchicine, which normally elevates kinetochore levels of checkpoint proteins, including Mad2, did not increase Mad2 kinetochore signals in rod cells. These observations indicated that Mad2 requires the Rod/Zw10 complex to achieve its normal levels on kinetochores. An earlier report did not find that inactivating Rod by antibody injection of Hela cells had any effect on Mad2 recruitment, although the antibody did block Rod recruitment at the kinetochore and did lead to premature mitotic exit. The discrepancy with the current results may be due to the different methodologies employed (Buffin, 2005).

Several other checkpoint proteins were examined in rod and zw10 mutants. BubR1 and Bub3 were still present. Mps1 and Bub1 were also unaffected by rod mutants. Thus, the requirement for Rod/Zw10 seems to be specific to Mad2. By contrast, treatments that remove Mad2 from kinetochores in vertebrate cells have no effect on Rod/Zw10 (Buffin, 2005).

It was possible that the failure of rod and zw10 mutant cells to recruit Mad2 was caused by the premature degradation of cyclin B in these checkpoint-defective cells; perhaps Mad2 cannot bind kinetochores when cyclinB/cdc2 kinase activity is low. To test this possibility, Mad2 behavior was examined in cells doubly mutant for rod and imaginal discs arrested (ida), the gene encoding APC5, a component of the APC/C. ida cells arrest in M phase with consistently elevated cyclin B. The ida phenotype is epistatic to rod: i.e., ida rod double mutants do not exit mitosis, and they retain elevated cyclin B (Buffin, 2005).

In ida cells, chromosomes are frequently found unattached to spindles, and Mad2 accumulation on kinetochores is therefore prominent even without colchicine. Significantly, in ida rod or ida zw10 double mutants, Mad2 signal on kinetochores was greatly reduced, just as in rod or zw10 mutants alone. This result argues that the Rod/Zw10 complex is physically required, directly or indirectly, for normal Mad2 accumulation on kinetochores (Buffin, 2005).

This study has shown that many aspects of Mad2 behavior are intimately associated with the Rod/Zw10 complex. Rod/Zw10 accompanies Mad2 as it accumulates on unattached kinetochores and as it leaves kinetochores after MT attachment, and in the absence of Rod/Zw10, little or no Mad2 accumulates on kinetochores. Given that Rod/Zw10 is also required for dynein/dynactin recruitment, which removes Mad2 from attached kinetochores, one can say that the entire kinetochore cycle of Mad2 depends, directly or indirectly, on Rod/Zw10. The checkpoint defect of rod and zw10 mutants is now presumably explained by this failure to recruit Mad2. These results suggest that Rod/Zw10 is physically interacting with a complex containing Mad2 (or Mad1, see below) throughout mitosis. However, two-hybrid screening, immunoaffinity columns, and coimmunoprecipitation experiments have not revealed any interaction between Rod/Zw10 and Mad1 or Mad2. Thus, unlike dynein/dynactin, Mad1/Mad2 may be binding only indirectly to Rod/Zw10, perhaps via an unknown protein. Alternatively, there may be direct interactions between Rod/Zw10 and Mad1/Mad2, but only under native conditions on intact kinetochores (Buffin, 2005).

Kinetochore recruitment of Mad2 initially occurs as part of a complex with Mad1, to which it is tightly bound even in interphase (Chen, 1998; Chung, 2002). The Mad1/Mad2 complex is relatively stable at unattached kinetochores (Howell, 2004; Shah, 2004), but a second Mad2 population, which depends on the first, turns over rapidly and presumably becomes an activated form, the 'wait anaphase' signal (Shah, 2004; Sironi, 2002). Once MTs have attached, however, the Mad1/Mad2 complex is rapidly depleted, at least partially by dynein-mediated shedding along KMTs, and this is believed to be part of the mechanism that extinguishes the checkpoint signal. It is therefore likely that the Rod/Zw10 complex is exerting its effect on the Mad1/Mad2 complex and not on Mad2 alone. Recent work in Hela cells supports this contention by showing that depletion of Zw10 by RNAi (Kops, 2005) reduces both Mad1 and Mad2 recruitment to unattached kinetochores (Buffin, 2005).

It is unclear what kinetochore components constitute the Mad1/Mad2 'binding site'. The hierarchy of kinetochore assembly has been studied in several model systems, not always with consistent results. However, it appears that the Ndc80 complex, Bub1, and Mps1 kinase activity are required for the subsequent assembly of Mad1/Mad2 on kinetochores. Conversely, interfering with Mps1 or the Ndc80 complex in Hela cells has no effect on Rod or dynein recruitment, and rod and zw10 mutants have no effect on BubR1, Bub3, CenpMeta (the fly homolog of CenpE), Bub1, or Mps1 (this study), nor in all likelihood on the Ndc80 complex (in rod mutants, chromosomes are efficiently captured by MTs and congress). Thus Rod/Zw10, with Ndc80 complex, Bub1, and Mps1, all contribute to Mad2 kinetochore recruitment. The role of Rod/Zw10 may be to enhance the affinity of Mad1/Mad2 for its binding site (because some Mad2 binds even in rod mutants), increasing its stability on kinetochores prior to MT capture, perhaps by interacting with Ndc80 complex (Buffin, 2005).

The current results also demonstrate that, just like Rod/Zw10, Mad1/Mad2 is continuously recruited to and then released from kinetochores, even following MT capture, and only disappears from spindles just prior to anaphase onset. This differs significantly from the behavior reported in vertebrate cells, in which MT capture appears to shut off new Mad2 recruitment. The difference need not conflict with the basic model in which kinetochore Mad2 generates the anaphase inhibitor. In both cases, there is a rapid decline, perhaps below a critical threshold, in the net steady-state abundance of Mad2 on attached kinetochores. Alternatively, MT capture may render the remaining kinetochore-associated Mad2 inactive. Perhaps the difference is in the rate of Mad2 recruitment in the two cell types. Even in PtK cells there is some evidence that Mad2 is capable of recruitment to attached kinetochores: If dynein activity (and therefore shedding) is blocked after chromosome alignment, Mad2 eventually reaccumulates at attached kinetochores, suggesting that prior to dynein inhibition, Mad2 was being recruited and immediately shed from these kinetochores. This continuous recruitment of Mad1/Mad2 to attached kinetochores may ensure that it will always be available to begin generating anaphase inhibitor should one or more MTs inadvertently detach. At the same time, the continued presence of Rod/Zw10 ensures the dynein levels required both to remove unneeded Mad1/Mad2 and, later, to power anaphase movement (Buffin, 2005).

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 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/C^Cdh1 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).

Search PubMed for articles about Drosophila Mad2

Basu, J., et al. (1998). Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod. Chromosoma 107(6-7): 376-85. PubMed citation: 9914369

Basu, J., et al. (1999). Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J. Cell Biol. 146: 13-28. 10402457

Bharadwaj, R. and Yu, H. (2004). The spindle checkpoint, aneuploidy, and cancer. Oncogene 23: 2016-2027. Medline abstract: 15021889

Buffin, E., Lefebvre C., Huang J., Gagou M. E. and Karess R. E. (2005). Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex. Curr. Biol. 15: 856-861. Medline abstract: 15886105

Buffin, E., Emre, D. and Karess, R. E. (2007). Flies without a spindle checkpoint. Nat. Cell Biol. 9(5): 565-72. Medline abstract: 17417628

Chan, G. K., Liu, S. T. and Yen, T. J. (2005). Kinetochore structure and function. Trends Cell Biol. 15(11): 589-98. PubMed citation: 16214339

Chen, R. H., et al. (1996). Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 274(5285): 242-6. PubMed citation: 8824188

Chen, R. H., Shevchenko, A., Mann, M. and Murray, A. W. (1998). Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J. Cell Biol. 143: 283-295. Medline abstract: 9786942

Chen, R. H., Brady, D. M., Smith, D., Murray, A. W. and Hardwick, K. G. (1999). The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins. Mol. Biol. Cell. 10: 2607-2618. Medline abstract: 10436016

Chen, R. H. (2002). BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J. Cell Biol. 158(3): 487-96. 12163471

Chung, E., and Chen, R. H. (2002). Spindle checkpoint requires Mad1-bound and Mad1-free Mad2. Mol. Biol. Cell 13: 1501-1511. Medline abstract: 12006648

Chung, E. and Chen, R. H. (2003). Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint. Nat. Cell Biol. 5: 748-753. Medline abstract: 12855955

Cohen-Fix O. and Koshland, D. (1997). The metaphase-to-anaphase transition: avoiding a mid-life crisis. Curr. Opin. Cell Biol. 9: 800-806. Medline abstract: 9425344

Fraschini, R., Beretta, A., Sironi, L., Musacchio, A., Lucchini, G. and Piatti, S. (2001). Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. EMBO J. 20: 6648-6659. Medline abstract: 11726501

Gorbsky, G. J., Chen, R. H. and Murray A. W. (1998). Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase. J. Cell Biol. 141: 1193-1205. Medline abstract: 11726501

Griffis, E. R., Stuurman, N. and Vale, R. D. (2007). Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. J. Cell Biol. 177(6): 1005-15. PubMed citation: 17576797

Howell, B. J., et al. (2004). Spindle checkpoint protein dynamics at kinetochores in living cells. Curr. Biol. 14: 953-964. Medline abstract: 15182668

Johnson, V. L., Scott, M. I., Holt, S. V., Hussein, D. and Taylor, S. S. (2004). Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression. J. Cell Sci. 117: 1577-1589. Medline abstract: 15020684

Jones J. T., Myers J. W., Ferrell J. E. and Meyer T. (2004). Probing the precision of the mitotic clock with a live-cell fluorescent biosensor. Nat. Biotechnol. 22: 306-312. Medline abstract: 14990952

Karess, R. (2005). Rod-Zw10-Zwilch: a key player in the spindle checkpoint. Trends Cell Biol. 15: 386-392. PubMed citation: 15922598

Kops G. J., et al. (2005). ZW10 links mitotic checkpoint signaling to the structural kinetochore. J. Cell Biol. 169: 49-60. Medline abstract: 15824131

Logarinho, E., Bousbaa, H., Dias, J. M., Lopes, C., Amorim, I., Antunes-Martins, A. and Sunkel, C. E. (2004). Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells. J. Cell Sci. 117: 1757-1771. 15075237

Lopes, C. S., et al. (2005). The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis. J. Cell Sci. 118: 187-198. 15615783

Martinez-Exposito, M. J., Kaplan, K. B., Copeland, J. and Sorger, P. K. (1999). Retention of the BUB3 checkpoint protein on lagging chromosomes. Proc. Natl. Acad. Sci. 96(15): 8493-8. PubMed citation: 10411903

Meraldi, P., Draviam, V. M. and Sorger P. K. (2004). Timing and checkpoints in the regulation of mitotic progression. Dev. Cell. 7: 45-60. Medline abstract: 15239953

Musacchio, A. and Hardwick, K. G. (2002). The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell Biol. 3: 731-741. Medline abstract: 12360190

Musacchio, A. and Salmon, E. D. (2007). The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8: 379-393. PubMed citation: 17426725

Orr, B., Bousbaa, H. and Sunkel, C. E. (2007). Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells. Mol. Biol. Cell 18(3): 850-63. Medline abstract: 17182852

Shah, J. V., et al. (2004). Dynamics of centromere and kinetochore proteins: Implications for checkpoint signaling and silencing, Curr. Biol. 14: 942-952. Medline abstract: 15182667

Sironi, L., et al. (2001). Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint. EMBO J. 20: 6371-6382. Medline abstract: 11707408

Sironi, L., et al. (2002). Crystal structure of the tetrameric Mad1-Mad2 core complex: Implications of a 'safety belt' binding mechanism for the spindle checkpoint, EMBO J. 21: 2496-2506. Medline abstract: 12006501

Starr, D. A., et al. (1998). ZW10 helps recruit dynactin and dynein to the kinetochore, J. Cell Biol. 142: 763-774. Medline abstract: 9700164

Sudakin, V., Chan, G. K. T. and Yen, T. J. (2001). Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, MAD2. J. Cell Biol. 154: 925-936. 11535616

Sudakin, V. and Yen, T. J. (2004). Purification of the mitotic checkpoint complex, an inhibitor of the APC/C from HeLa cells. Methods Mol. Biol. 281: 199-212. Medline abstract: 15220531

Tang, Z., Bharadwaj, R., Li, B. and Yu, H. (2001). Mad2-independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1: 227-237. 11702782

date revised: 30 March 2008

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