InteractiveFly: GeneBrief

mad2: Biological Overview | References

Gene name - mad2

Synonyms -

Cytological map position-64E11-64E12

Function - signaling

Keywords - spindle assembly checkpoint, sequestration of Cdc20, centrosome

Symbol - mad2

FlyBase ID: FBgn0035640

Genetic map position - 3L: 5,805,232..5,806,183 [-]

Classification - HORMA (for Hop1p, Rev7p and MAD2) domain

Cellular location - cytoplasmic

NCBI links: EntrezGene

Mad2 orthologs: Biolitmine

Recent literature
Wong, H. W., Shaukat, Z., Wang, J., Saint, R. and Gregory, S. L. (2014). JNK signaling is needed to tolerate chromosomal instability. Cell Cycle 13: 622-631. PubMed ID: 24335260
Chromosomal instability (CIN), as a common feature of tumors, represents a potential therapeutic target if ways can be found to specifically cause apoptosis in unstably dividing cells. Previous studies has shown that if signaling through the JNK pathway is reduced, apoptosis is triggered in models of chromosomal instability induced by loss of the spindle checkpoint (reducing Mad2 in adult flies by RNAi). This study identified components upstream and downstream of JNK that are able to mediate this effect, and tested the involvement of p53 and DNA damage in causing apoptosis when JNK signaling is reduced in CIN cells. Cell cycle progression timing has shown to have a strong effect on the apoptosis seen when JNK signaling is reduced in genetically unstable cells: a shortened G 2 phase enhances the apoptosis, while lengthening G 2 rescues the JNK-deficient CIN cell death phenotype. These findings suggest that chromosomal instability represents a significant stress to dividing cells, and that without JNK signaling, cells undergo apoptosis because they lack a timely and effective response to DNA damage.

Stormo, B. M. and Fox, D. T. (2016). Distinct responses to reduplicated chromosomes require distinct Mad2 responses. Elife 5 [Epub ahead of print]. PubMed ID: 27159240
Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. This study, using Drosophila, uncovered two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. These results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.


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).

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal-D
Bicaudal-D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. As BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize them to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles, and the tandem spindles during female meiosis II. Furthermore, BicD is required to correctly localize clathrin and the microtubule-stabilizing factors, D-TACC and Msps, to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, right after the establishment of the female pronucleus, D-TACC and C. elegans BicD, tacc, and Chc are also needed for pronuclear fusion, pointing to the possibility that the underlying mechanism might be more widely used (Vazquez-Pianzola, 2022).

Encoded by a single gene, the Drosophila Bicaudal D (BicD) protein is part of a family of evolutionarily conserved dynein adaptors responsible for the transport of different cargoes along microtubules (MTs). The founding member of this protein family, Drosophila BicD, was identified because of its essential role during oogenesis and embryo development, in which it transports mRNAs that control polarity and cell fate. This process is mediated by its binding to the RNA-binding protein Egalitarian (Egl). Since its initial discovery, BicD and its orthologs have been shown to control a diverse group of MT transport processes through binding to different cargoes or adaptor proteins (Vazquez-Pianzola, 2022).

BicD can alternatively bind to Clathrin heavy chain (Chc) and this interaction facilitates Chc transport of recycling vesicles at the neuromuscular junctions and regulates endocytosis and the assembly of the pole plasm during oogenesis. The best-known function of Chc is in receptor-mediated endocytosis, in which it forms part of clathrin, a trimeric scaffold protein (called a triskelion), composed of three Chc and three Clathrin light chains (Clc). Aside from this, clathrin was shown to localize to mitotic spindles in mammalian and Xenopus cells and to have non-canonical activity by stabilizing the spindle MTs during mitosis. This function depends on clathrin trimerization and its interaction with Aurora A-phosphorylated Transforming Acidic Coiled-Coil protein 3 (TACC3) and the protein product of the colonic hepatic Tumor Overexpressed Gene (ch-TOG). This heterotrimer forms intermicrotubule bridges between kinetochore fibers (K-fibers), stabilizing these fibers and promoting chromosome congression. Recently, TACC3 and a mammalian homolog of Chc (CHC17) were shown to control the formation of a new liquid-like spindle domain (LISD) that promotes the assembly of acentrosomal mammalian oocyte spindles (Vazquez-Pianzola, 2022).

In order to transport its cargos along MTs, BicD interacts with the dynein/dynactin motor complex, a minus-end-directed MT motor. This complex is involved in different cellular processes, including intracellular trafficking of proteins and RNAs, organelle positioning and microtubule organization, some of which also require BicD. The dynein/dynactin complex also plays essential roles during cell division, in which it is required for centrosome separation, chromosome movements, spindle organization and positioning and mitotic checkpoint silencing (Vazquez-Pianzola, 2022).

Given that Drosophila BicD forms complexes with Chc and Dynein, both of which, as described above, perform essential activities during mitosis, this study set out to investigate possible BicD functions during cell division. Reducing BicD levels by specific protein-targeted degradation in freshly laid eggs revealed that BicD is essential for pronuclear fusion. In addition, it is required for metaphase arrest of female meiotic products after meiosis II completion. This activity appears to be mediated by the role of BicD in localizing the spindle assembly checkpoint (SAC) components. Furthermore, BicD interacts with its cargo protein, Chc, and they both localize to the mitotic spindles and centrosomes and the female tandem meiotic II spindles. In addition, BicD localizes D-TACC, clathrin, and Mini spindles (Msps; ch-TOG homolog) to the meiosis II spindles. The failure to localize these proteins accurately might also contribute to the SAC function defects observed in embryos with reduced BicD levels. D-TACC and Caenorhabditis elegans bicd-1, tac-1 and chc-1 are also needed after fertilization for pronuclear fusion, revealing an evolutionary conserved and essential role of these proteins in early zygote formation and suggesting that their mechanism of action on MTs might be widely used across species (Vazquez-Pianzola, 2022).

This study found that BicD localizes to the female tandem spindles and the central aster during MII. After fertilization, BicD also localizes to the mitotic spindles and the centrosomes. BicDnull mutants rarely survive and are sterile, but this study generated embryos with reduced levels of BicD at the beginning of embryogenesis (BicDhb-deGradFP embryos) by setting up a strategy based on the deGradFP technique. Consistent with BicD localization at the female MII spindles, it was discovered that BicDhb-deGradFP embryos arrest development, displaying aberrant meiotic products and no pronuclear fusion. Especially if combined with the CRIPSP-Cas9 strategy first to produce functional GFP-tagged proteins of interest, the construct designed in this study could be helpful for studying the role of female-sterile and lethal mutations during very early embryonic development (Vazquez-Pianzola, 2022).

In unfertilized BicDhb-deGradFP eggs, the female meiotic products were not arrested in metaphase as normally happens. Instead, they underwent additional rounds of replication. They failed to recruit or maintain the recruitment of the SAC pathway components BubR1 and Mad2, which are normally present at the kinetochores in the wild-type female meiotic polar bodies. Interestingly, in Drosophila eggs mutated for Rod, mps1 and BubR1, well-conserved orthologs of the SAC pathway, the polar bodies also cannot remain in a SAC-dependent metaphase-like state and decondense their chromatin. Furthermore, in these mutants, the polar bodies cycle in and out of M-phase, replicating their chromosomes similarly to those in BicDhb-deGradFP eggs. Thus, it appears that BicD functions to localize the SAC components to induce and/or maintain metaphase arrest of the polar bodies. Several mechanisms could explain the failure to maintain SAC activation observed in BicDhb-deGradFP embryos. BicD might be needed to recruit the SAC components to kinetochores directly. By contrast, during mitosis, the Rod-Zw10-Zwilch (RZZ) complex binds to the outer kinetochore region and recruits Mad2, Spindly and the dynactin complex. Spindly and dynactin act cooperatively to recruit dynein, which then transports the SAC components along the MTs away from kinetochores as a mechanism to trigger checkpoint silencing and anaphase onset. Given that the BicD N-terminal domain binds dynein and dynactin and promotes their interaction, it is also possible that BicD helps to move the SAC components away from the kinetochores. If this does not happen, the SAC remains persistently activated. It was also found that BicD activity in BicDhb-deGradFP embryos is insufficient to localize clathrin, TACC and Msps efficiently along the MTs of the spindle. During mitosis, impairment of MT motors, such as dynein, and treatments that prevent the TACC/clathrin complex from binding to the mitotic spindles and affecting K-fiber stability, also persistently activate the SAC. Thus, reduced levels of BicD in BicDhb-deGradFP embryos could additionally trigger SAC hyperactivation through its role in stabilizing the K-fibers. Although these data strongly suggest that the lack of BicD contributes to SAC defects through its role in localizing clathrin, D-TACC and Msps, further work is needed to elucidate whether BicD also acts more directly by binding to, and localizing, the SAC components, or indirectly by affecting the function of dynein (Vazquez-Pianzola, 2022).

Whereas persistent SAC activation leads to metaphase arrest and delayed meiosis (D-meiosis), this delay is known to be rarely permanent, at least during mitosis. Most cells that cannot satisfy the SAC ultimately escape delayed mitosis (D-mitosis) and enter G1 as tetraploid cells by a currently poorly understood mechanism. It is possible that, in BicDhb-deGradFP, the SAC pathway is constantly activated, delaying meiosis. However, at one point, the nuclei might escape metaphase II arrest, cycling in and out of M-phase, thereby replicating their chromosomes and decondensing their chromatin. The fact that female meiotic products over-replicate in BicDhb-deGradFP eggs and show no or only pericentromeric PH3 staining supports the notion that these nuclei are on an in-out metaphase arrest phase. That meiotic products in about half of the BicDhb-deGradFP embryos failed to stain for the SAC components BubR1 and Mad2 supports this hypothesis (Vazquez-Pianzola, 2022).

Chc, its partner Clc and BicD are enriched at mitotic spindles and centrosomes. Furthermore, these proteins and the clathrin-interacting partners D-TACC and Msps localize to the tandem spindles and the central aster of the female MII apparatus. The interaction of Drosophila Chc with D-TACC is conserved, and Chc interacts through the same protein domain directly with D-TACC and BicD. Moreover, BicD is needed for localizing D-TACC, Msps and clathrin throughout the MII tandem spindles. The TACC3/Chc interaction was proposed to form a domain in tandem to bind spindle MTs. It is hypothesize that BicD could help recruit Chc to the MTs by association with dynein. Given that Chc usually acts as a trimer with Clc (triskelion), each Chc might interact with either BicD or D-TACC. Thus, a mixed complex could be formed, and BicD might help to move, recruit or stabilize Chc and D-TACC along the spindles via the interaction of BicD/Chc in the same trimer. The fact that expression of D-TACC enhances the Chc/BicD interaction and that overexpression of Chc and D-TACC (tacc) arrested early development in a background in which BicD is reduced to a level that does not produce visible phenotypes on its own, supports this model. These results suggest that, with respect to BicD, the levels of D-TACC and Chc should be tightly balanced for these proteins to perform their normal function during early development, as has been shown previously for other BicD transport processes (Vazquez-Pianzola, 2022).

BicD has a role in pronuclear fusion that is conserved during evolution given that C. elegans eggs depleted for bicd-1 also failed to undergo pronuclear fusion. Moreover, Drosophila D-TACC and C. elegans chc-1 and tac-1 are also needed for pronuclear fusion. These genes might be required indirectly through their role in meiosis because preliminary data suggest that MII is also compromised in bicd-1 and chc-1 dsRNA-fed worms. Alternatively, they might play a more direct role in pronuclear migration, which depends on dynein and MTs in bovine, primate and C. elegans embryos. This would then suggest that the underlying mechanism may be used to build correctly or stabilize different types of MT. Determining their precise mechanistic involvement in pronuclear fusion is an interesting question for further studies (Vazquez-Pianzola, 2022).

Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion

The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin. Cleavage of cohesin marks anaphase onset. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC). To identify specific conditions capable of restoring defects associated with cohesion loss, a screen was carried out for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised (Silva, 2018).

In agreement with its 'safeguard' function, mitotic errors are often exacerbated by impairment of the SAC. These include defects associated with multiple centrosomes, defective microtubule assembly, or kinetochore structure. This study demonstrates that the opposite happens with cohesion defects. Absence of the SAC alleviated mitotic errors and improved mitotic fidelity after cohesion loss. Cells with a functional SAC undergo extensive chromosome shuffling and consequent genome randomization, whereas virtually no shuffling is observed in absence of the SAC. The detrimental nature of the SAC in the absence of cohesin is likely related to irreversibility of cohesion loss. Most mitotic defects can be corrected over time, but premature cohesin loss is an irreversible error and prolonging mitosis further enhances genome randomization (Silva, 2018).

Improved mitotic fidelity after cohesion loss in absence of the SAC is likely a consequence of slow kinetics of error-correction engagement coupled with a bias toward correct chromosome alignment. Several mechanisms are known to bias chromosome segregation toward the right orientation, including chromosome positioning, centromere geometry, bias on microtubule growth toward the kinetochores, and/or kinetochore-mediated microtubule nucleation. Among these, chromosome geometry is believed to facilitate bipolar attachment by facing one kinetochore to the opposite pole upon attachment. If so, what ensures geometric arrangement during the initial mitotic stages, even in the absence of cohesin? A possible mechanism enabling transient orientation of chromatids toward opposing poles is the incomplete resolution of sister chromatid intertwines. Yet residual catenation in metaphase chromosomes is unable to confer functional cohesion, as removal of cohesin is sufficient to induce immediate chromatid separation. Additional mechanisms may thus impair resolution of sister chromatids during early mitosis, in contrast to metaphase chromosomes. Spindle forces enhance decatenation, and thus resolution of DNA intertwines may only be achieved upon chromosome capture. Recent findings propose that efficient decatenation requires constant 'guiding action' from condensin I, whose maximal levels are observed on chromosomes in late metaphase or anaphase. This gradual condensin loading could limit full decatenation to later stages of mitosis, making residual catenation sufficient to allow transient pseudo-metaphase alignment that biases initial chromosome attachment toward biorientation. Although error prone, this process would be more accurate than total genome randomization resulting from chromosome shuffling (Silva, 2018).

Why cohesion loss is insufficient at triggering error correction during early mitosis remains to be addressed. This could be related to a partial tension state facilitated by pseudo-metaphase chromosomal configuration. Additionally, an intrinsic delayed action of error-correction machinery may further account for observed late shuffling onset. Indeed, slow kinetics or a lag time of Aurora-B-mediated chromosome detachment has been hypothesized in several theoretical studies with little experimental validation. Such intrinsic delay would solve the 'problem of initiation of biorientation' whereby initial low-tension interactions could survive such a tension-sensitive error correction (Silva, 2018).

Interestingly, the interplay between mitotic timing and sister chromatid cohesion has been previously reported in mammalian cells whereby extension of mitosis predisposes to sister chromatid cohesion defects. Cells arrested in mitosis for long periods were shown to display sister chromatid separation (referred as 'cohesion fatigue'). Moreover, defective sister chromatid cohesion was described to be synthetically lethal with impaired APC/C function in Warsaw breakage syndrome (WABS) patient-derived cells as well as several cancer cell lines with cohesion defects. The current observations demonstrate how reduction of mitotic timing is sufficient to rescue segregation defects associated with premature cohesin loss. Importantly, these experiments highlight the detrimental effect of the SAC upon cohesion defects. When sister chromatid cohesion is compromised and mitotic fidelity irreversibly affected, the SAC exacerbates mitotic errors in contrast to its canonical protective function (Silva, 2018).

In vivo dynamics of Drosophila nuclear envelope components

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). This study examined the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. Evidence is provided that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, the dynamics of the spindle checkpoint proteins Mad2 and Mad1 was examined. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells (Katsani, 2008).

A fraction of Drosophila Mad1 and Mad2 associates with the NE in syncytial embryos, which probably reflects their binding to the nuclear side of the NPCs as demonstrated in yeast and vertebrates. The release of GFP-Mad2 at an early stage of Drosophila NPC disassembly in prophase and the late recruitment of Mad1 and Mad2 at the NE corroborate previous observations in Xenopus and human cells. However, in addition this study shows that relocalization of Drosophila Mad1 and Mad2 to the NE occurs in two steps, with their nuclear import preceding their NE association. This shared behavior suggests that both proteins might be reimported into the nucleus as a complex (Katsani, 2008).

The coincident NPC recruitment of Mad1 and Mad2 with Mtor indicates that Drosophila Megator likely represents their NPC-anchoring determinant as are the yeast Mtor orthologues (Mlp1/2) (Scott, 2005). This seems to be an evolutionarily conserved interaction, because small interfering RNA-induced depletion of Tpr (the vertebrate orthologue of yeast Mlp1/2-Drosophila Mtor) in HeLa cells significantly reduces Mad2 labeling at the NE. In vivo study further revealed that in syncytial embryos, a fraction of GFP-Mad2 is found within the spindle area in metaphase and subsequently localizes between the two reforming nuclei during telophase, localizations similar to that of Mtor. It is thus conceivable that an interaction between Mad1-Mad2 and Mtor may also occur at this stage of mitosis (Katsani, 2008).

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. This report shows that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. It is proposed that Mtor/Tpr functions as a spatial regulator of the SAC ensuring the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible 'wait anaphase' signal to the vicinity of the spindle. It is also suggested that the regulatory role of Mtor upon Mad2 is indirect and may be catalyzed by Mps1 (Lince-Faria, 2009).

The mitotic spindle is composed of dynamic microtubules (MTs) and associated proteins that mediate chromosome segregation during mitosis. The requirement of an additional stationary or elastic structure forming a spindle matrix where molecular motors slide MTs has long been proposed to power chromosome motion and account for incompletely understood features of mitotic spindle dynamics. However, definitive evidence for its existence in living cells or on its biochemical nature and whether it plays a direct role during mitosis has been missing (Lince-Faria, 2009).

A functional spindle matrix would be expected to (a) form a fusiform structure coalescent with spindle MTs, (b) persist in the absence of MTs, (c) be resilient in response to changes of spindle shape and length, and (d) affect spindle assembly and/or function if one or more of its components are perturbed. In Drosophila, a complex of at least four nuclear proteins, Skeletor, Megator (Mtor), Chromator, and EAST (enhanced adult sensory threshold), form a putative spindle matrix that persists in the absence of MTs in fixed preparations). From this complex, Mtor is the only protein that shows clear sequence conservation with proteins in other organisms, such as the nuclear pore complex (NPC) protein translocated promoter region (Tpr) in mammals, its respective counterparts Mlp1 and Mlp2 in yeast, and nuclear pore anchor in plants. NPC proteins, including Mtor/Tpr orthologues in yeast, have been shown to functionally interact with spindle assembly checkpoint (SAC) components. The SAC ensures correct chromosome segregation by providing time for proper kinetochore (KT) attachments to spindle MTs while inhibiting the activity of the anaphase-promoting complex/cyclosome (Lince-Faria, 2009).

Assuming that any critical function by the spindle matrix is widely conserved, this study focused on understanding the mitotic role of Mtor in living Drosophila somatic cells. The results provide a new conceptual view of a spindle matrix not as a rigid structural scaffold but as a spatial determinant of key mitotic regulators (Lince-Faria, 2009).

Mtor localizes to a dynamic nuclear derived spindle matrix in living cells. To investigate the localization of Mtor in living cells, a Drosophila S2 cell line was generated stably coexpressing Mtor-mCherry and GFP-α-tubulin. Mtor-mCherry is nuclear in interphase and at nuclear envelope breakdown (NEB) reorganizes into a fusiform structure coalescent with spindle MTs. Mtor-mCherry shows a highly adaptable morphology in response to changes in spindle shape and dynamics throughout mitosis, which is inconsistent with a static structure. Similar to endogenous Mtor, Mtor-mCherry retracts and loses the fusiform shape upon MT depolymerization but is retained in a conspicuous milieu around chromosomes, suggesting that MTs exert a pushing force on the Mtor-defined matrix (Lince-Faria, 2009).

Previous electron microscopy analysis revealed the existence of a membranous network surrounding the spindle from prophase to metaphase in S2 cells. This study used immunofluorescence to show that lamin B is not fully disintegrated at this stage. Similar results have recently been reported in living Drosophila embryos and neuroblasts, where a spindle envelope was proposed to limit the diffusion of nuclear derived Nup107 before anaphase. To test whether this membranous network works as a diffusion barrier around the spindle, the dynamic behavior of Mtor-mCherry relative was compared to GFP-α-tubulin and a known MT-associated protein, Jupiter, upon colchicine addition. GFP-α-tubulin or Jupiter-GFP fluorescence is gradually lost from the spindle region with an equivalent gain in the cytoplasm. In contrast, Mtor-mCherry remains confined to the spindle region with no detectable fluorescence gain in the cytoplasm. These results argue against the existence of a diffusion barrier around the metaphase spindle in Drosophila S2 cells and suggest that Mtor is being selectively retained in this region (Lince-Faria, 2009).

FRAP was used to shed light on the dynamic properties of Mtor. In interphase nuclei, there is ~50% recovery of fluorescence in the bleached region with an equivalent loss from a similar unbleached region and undetectable cytoplasmic exchange, suggesting that Mtor in the nucleoplasm is mobile. In mitosis, FRAP of Mtor-mCherry in one half-spindle is mirrored by an equivalent loss of fluorescence from the unbleached half-spindle as if Mtor exchanges between half-spindles. However, this recovery was slower than in interphase nuclei and had a minor contribution from a cytoplasmic pool. In both interphase and mitosis, the recovery curves of Mtor-mCherry fitted a single exponential, suggesting affinity to a yet unidentified substrate, whereas GFP-α-tubulin in the spindle displayed biphasic recovery kinetics and best fit the sum of two exponentials as result of a rapid diffusion phase followed by a slower recovery phase associated with MT turnover. Finally, in S2 cells that sporadically form two spindles in the same cytoplasm, fluorescence exchange was found within the same spindle and from surrounding cytoplasm with no apparent loss from the neighboring unbleached spindle, supporting that Mtor is unable to exchange between two spindles located <10 µm apart. Collectively, these data indicate that Mtor is part of a dynamic, nuclear derived spindle matrix surrounded by a fenestrated membranous system containing lamin B and shows mobility properties that are distinct from MTs and associated proteins (Lince-Faria, 2009).

To address the mitotic role of Mtor, RNAi was used in Drosophila S2 cells stably coexpressing GFP-α-tubulin and the KT marker mCherry-centromere identifier (CID). Mtor-depleted cells show no major spindle defects but typically form a poorly defined metaphase plate as the result of progressing ~15% faster through mitosis when compared with controls. Such problems in completing chromosome congression are corrected if anaphase onset is delayed by treating cells with the proteasome inhibitor MG132. As in Mtor RNAi, S2 cells depleted of the SAC protein Mad2 undergo a faster mitosis. Moreover, Mtor-depleted cells show a lower mitotic index as well as a weakened response to MT depolymerization, suggesting that Mtor is required for proper SAC response (Lince-Faria, 2009).

Quantitative analysis of anaphase revealed a significant attenuation in the velocity of chromosome separation in Mtor-depleted cells by affecting spindle elongation. These results could be accounted for if Mtor is part of a structural scaffold where motor proteins assemble to generate force. However, an alternative hypothesis is that Mtor may function to provide the necessary time for proper maturation of a competent spindle. To test this, anaphase onset was delayed by treating Mtor-depleted cells with MG132, and half-spindle elongation velocity was measured after drug washout. No difference was found in half-spindle elongation velocity between Mtor RNAi and control cells treated with MG132. Additionally, half-spindle elongation in Mad2-depleted cells, which progresses faster through mitosis, was similar to Mtor-depleted cells, supporting the spindle maturation hypothesis (Lince-Faria, 2009).

To shed light on the role of Mtor in SAC response, the recruitment of Mad2 and BubR1 to unattached KTs after Mtor depletion was analyzed. It was found that although BubR1 was unaltered after Mtor depletion, Mad2 KT accumulation was significantly reduced. Decreased Mad2 levels at KTs explain why Mtor-depleted cells enter anaphase prematurely, presumably because it requires binding of fewer MTs to remove all Mad2 from KTs and satisfy the SAC, whereas residual Mad2 at KTs may be sufficient to produce a weakened response to colchicine (Lince-Faria, 2009).

Next, how Mtor regulates the recruitment of Mad2 to KTs in living S2 cells stably coexpressing GFP-α-tubulin and monomeric RFP (mRFP)-Mad2 was investigated. In interphase, mRFP-Mad2 is nuclear, accumulating at unattached KTs and spindles as cells transit into mitosis. The spindle accumulation of Mad2 is thought to result from dynein-dependent poleward transport as MTs attach to KTs. Interestingly, however, it was found that a distinct pool of mRFP-Mad2 localizes to a nuclear derived spindle matrix even when MTs have just started invading the nuclear space. A similar behavior has been observed in vertebrate cells, where GFP-Mad2 accumulates as an ill-defined nuclear derived matrix during early prometaphase after its initial recruitment to unattached KTs. Like Mtor, the retention of Mad2 in the spindle matrix is resistant to MT depolymerization, suggesting that spindle-associated Mad2 is not freely diffusible. mRFP-Mad2 remains associated with the spindle matrix in the absence of Mtor, but it is unable to accumulate at KTs even after MT depolymerization with colchicine. Stable expression of Mtor-mCherry (which is RNAi insensitive) rescues normal Mad2 localization at KTs after Mtor RNAi, indicating that the observed phenotype is specific and supporting that Mtor-mCherry is a functional protein. Lastly, Mtor depletion does not affect normal Mad2 expression levels and vice versa, which rules out unspecific effects of Mtor over Mad2 mRNA transport to the cytoplasm (Lince-Faria, 2009).

The colocalization of Mtor and Mad2 in the spindle matrix suggests that these proteins may interact. Indeed, Mad2 was found to coimmunoprecipitate with Mtor in lysates obtained from Drosophila embryos harvested between 0-3 h after egg laying. Given that Mtor does not specifically accumulate at KTs, this interaction might represent an important regulatory step for the subsequent recruitment of Mad2 to unattached KTs. Several proteins such as Mad1, Rod, Ndc80, or Mps1 are involved in recruiting Mad2 to unattached KTs. Although Mad1, Rod, and Ndc80 are effectively targeted to unattached KTs after Mtor depletion, Mps1 accumulation is significantly reduced. Mps1 kinase activity has been shown to be required to specifically target Mad2 but not Mad1 to unattached KTs in human cells. To investigate whether the same regulatory role upon Mad2 is true in Drosophila, an mps1 kinase-dead (mps1KD) allele was generated by homologous recombination in flies. In agreement with the results in human cells, neuroblasts from mps1KD third instar larvae show reduced or undetectable Mad2 accumulation at KTs upon colchicine treatment. Collectively, these results support that the regulatory role of Mtor upon Mad2 is indirect and may be catalyzed by Mps1 (Lince-Faria, 2009).

Like Tpr, Mad1, Mad2, and Mps1 localize at the NPC during interphase in human cells. During mitosis, Tpr remains associated with the nuclear envelope until prometaphase. Moreover, a fraction of Tpr is associated with the mitotic spindle from late prometaphase until anaphase and is recruited to the reforming nuclear envelope during telophase. This confirms the previous identification of Tpr in isolated human mitotic spindles, no enriched fraction of Tpr was dected that resists MT depolymerization with nocodazole, including KTs. To see whether Tpr has a conserved regulatory role in the recruitment of Mad2 to unattached KTs in human cells, RNAi was used to deplete Tpr in HeLa cells. Like in S2 cells, Tpr RNAi leads to reduced accumulation of Mad2 but not Mad1 to unattached KTs accompanied by a decrease in the normal mitotic index and a weakened SAC response in the presence of nocodazole. Tpr knockdown does not enrich for cells in G2 and slightly increases the number of cells in G1, supporting that the lower mitotic index is not caused by the inability of cells to enter mitosis but rather reflects a faster exit. Moreover, Tpr, Mad1, Mad2, and Mps1 coimmunoprecipitate in mitotic enriched HeLa cell extracts prepared in the presence of nocodazole, extending the results obtained in Drosophila and reinforcing that this complex forms independently of MTs and an intact nuclear envelope. While this paper was under revision, Tpr was independently found to interact with Mad1 and Mad2 in human cells. In agreement with these results, the authors propose that Tpr is important for controlling the SAC but reject the possibility that Tpr is playing a role in mitotic timing. However, quantification of the NEB to anaphase duration in Tpr-depleted cells does show a 25% acceleration of mitosis during this period. Finally, the results are not consistent with a model in which KT-associated Tpr serves as a docking place for Mad1 because Tpr (or Mtor) were not detected at KTs, including those that were positive for Mad1, and no impairment was found in Mad1 KT recruitment in Tpr- or Mtor-depleted cells (Lince-Faria, 2009).

Overall, the results support a model in which Mtor/Tpr acts as a spatial regulator of SAC, ensuring a timely and effective recruitment of Mad2 and Mps1 to unattached KTs as cells enter mitosis. In budding yeast, Mps1 phosphorylates Mad1, which is continuously recycled to KTs from Mlps at NPCs, but N-terminal deletion mutants of Mad1 lacking the Mlp-binding domain have a functional SAC. In humans and Drosophila, Mps1 regulates Mad2 but not Mad1 accumulation at KTs. Because Mad1 localization at KTs does not depend on Mlps/Mtor/Tpr and Mps1 kinase activity, the residual Mad2 at KTs after Mtor/Tpr RNAi possibly corresponds to the Mad1-bound fraction. One possibility is that Mps1 phosphorylation of Mad1 regulates the recruitment of a fast-exchanging pool of Mad2 to KTs. Parallelly, Mtor/Tpr may spatially regulate Mps1 autophosphorylation, which is important for its normal KT accumulation, together with Mad2. The presence of Mad2 in the complex may work as a positive feedback mechanism to ensure continuous Mps1 kinase activity upon SAC activation (Lince-Faria, 2009).

SAC proteins evolved from systems with a closed mitosis like budding yeast, where the spindle assembles inside an intact nuclear envelope into more complex systems like animals and plants, where the nuclear envelope is thought to fully or partially disintegrate during spindle formation, justifying the requirement of a nuclear derived spindle matrix for an effective SAC response. What retains matrix components around the spindle in systems where mitosis is thought to be open remains an intriguing question. In this regard, lamin B was proposed to tether several factors that mediate spindle assembly in Xenopus laevis egg extracts and possibly in human cells. Additionally, a continuous endoplasmic reticulum surrounding the mitotic spindle is thought to be recycled from the nuclear envelope after its disassembly and has been observed in many systems undergoing an open mitosis, including humans. Although such fenestrated membranous systems cannot work as diffusion barriers, it is possible that they indirectly help to generate local gradients or concentrate matrix-affine substrates. The enrichment of Mad2 in the spindle matrix provides an explanation for an unsolved SAC paradigm in which the 'wait anaphase' signal emanating from unattached KTs must be diffusible to prevent premature anaphase onset of already bioriented chromosomes but at the same time is known to be restricted to the vicinity of the spindle (Lince-Faria, 2009).

The proposed role of Mtor/Tpr further supports the necessity of spindle maturation for proper KT-MT attachments and anaphase spindle elongation in which the spindle matrix may help extend the duration of mitosis for the assembly of a competent chromosome segregation machinery. Mtor/Tpr-depleted cells have a weakened SAC response that, as opposed to complete checkpoint loss, may be compatible with cell viability and lead to cancer. The involvement of Tpr in the activation of several oncogenes may translate into an unfavorable combination that facilitates transformation and tumorigenesis in humans (Lince-Faria, 2009).

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/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).

Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding

Premature anaphase onset is prevented by the mitotic checkpoint through production of a 'wait anaphase' inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, this study demonstrates that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. Purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. These results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1 (Kulukian, 2009).

While unattached kinetochores have been widely inferred to be the source of a 'wait anaphase' mitotic checkpoint inhibitor, this study has now demonstrated that kinetochores can, in fact, catalyze production of an initial Mad2-Cdc20 inhibitor, significantly accelerating the initial rate of its production. Unattached kinetochores did not affect inhibition by Bub3/BubR1 in the absence of Mad2. Production of at least two inhibitors can be enhanced by unattached kinetochores: one containing diffusible Cdc20 and another in which Cdc20 is already bound in a megadalton complex to APC/C, consistent with reports that Cdc20 and checkpoint proteins are present in two complexes with differing sizes during mitosis. Both inhibitors prevent recognition by APC/C of cyclin B as an ubiquitination substrate. Disruption of cyclin B ubiquitination by a kinetochore-derived inhibitor even while Cdc20 remains bound to APC/C provides a potential explanation for the differential timing of destruction of cyclins A and B. Instead of simple sequestration of Cdc20, a kinetochore-derived mitotic checkpoint inhibitor bound to APC/CCdc20 may block recognition of cyclin B as an ubiquitination substrate, while permitting APC/CCdc20-mediated ubiquitination and destruction of cyclin A, an event that is known to initiate immediately after mitotic entry (Kulukian, 2009).

Despite amplification of Cdc20 inhibition when equal molar levels of BubR1, Mad2, and Cdc20 were added, no evidence was found for assembly of a quaternary mitotic checkpoint- (MCC-) like complex as a bona fide inhibitor produced by unattached kinetochores. Rather, almost all Cdc20 shifted to a complex comigrating with the majority of BubR1 but containing very little Mad2. Also arguing against a contribution in kinetochore-derived checkpoint signaling, it is noted that MCC-like complexes in animal cells are present outside of mitosis, and their formation in yeast continues in the absence of a functional centromere/kinetochore. All of this supports an MCC-like, premade Cdc20 inhibitor produced in a kinetochore-independent manner in interphase that restrains APC/C ubiquitination activity for cyclin B just after mitotic entry, which has been referred to as a 'timer' (Kulukian, 2009).

More importantly, at physiologically relevant concentrations of unattached kinetochores and Mad2, chromosomes catalyzed production of Cdc20 inhibition of cyclin B recognition by APC/C by at least 8-fold relative to inhibitors formed spontaneously in the absence of chromosomes. The actual in vivo effect is likely to be much greater than observed in vitro, since chromosome purification resulted in partial loss of signaling molecules from kinetochores, including a proportion of Mad1 and kinases that include Bub1, BubR1, and Aurora B (Kulukian, 2009).

Chromosome amplification of Cdc20 inhibition required Mad1 recruitment of Mad2 to kinetochores and dimerization-competent Mad2, thereby providing a direct demonstration that a Mad1:Mad2 core complex recruits and converts soluble 'inactive' Mad2 into a more potent inhibitor of Cdc20. At least part of this is from action of kinetochores on Mad2. Although it has previously been argued that the kinetochore may sensitize the APC/C for checkpoint-mediated inhibition, direct contact of chromosomes with APC/C was not required to amplify inhibition. While a kinetochore-dependent function of BubR1 can by no means be excluded from roles in microtubule attachment and chromosome alignment or from further amplification of a kinetochore derived signal, kinetochore-mediated enhancement of Cdc20 inhibition did not require BubR1 localization to or contact with kinetochores. It is concluded that immobilized, kinetochore-bound Mad1/Mad2, but not BubR1, catalyzes conversion at the kinetochore of soluble, open Mad2 into a form with its seatbelt domain poised for Cdc20 capture. Further support for this conclusion includes evidence that kinetochore-bound BubR1 is nonessential (Kulukian, 2009).

Moreover, incubation of physiologically relevant concentrations of each component ultimately produced most Cdc20 bound to BubR1, not Mad2, whether or not chromosomes were present. In fact, amplification of Cdc20 inhibition by unattached kinetochores was accompanied by a shift to a more rapidly eluting Bub3/BubR1-Cdc20 complex, without a stable pool of Mad2-Cdc20. Evidence also demonstrated that most Cdc20 is complexed with BubR1 in vivo, rather than Mad2. A model from all of this is proposed in which Mad1/Mad2 immobilized at kinetochores templates conversion of an inactive, open Mad2 to one capable of transient capture of Cdc20 followed by relay to BubR1 as sequentially produced mitotic checkpoint inhibitors that may be soluble or APC/C bound. This evidence supports Mad2-Cdc20, and perhaps an MCC-like complex, as a transient intermediate in kinetochore-mediated checkpoint signaling and one that is a precursor to BubR1-Cdc20. Further, Bub3/BubR1 binds to APC/C, but only in a Mad2-dependent manner that is stimulated by unattached kinetochores, demonstrating that kinetochores facilitate loading of Bub3/BubR1 onto APC/C. That BubR1-APC/CCdc20 is produced indirectly by unattached kinetochores as the final Cdc20 inhibitor would also support suggestions that BubR1 acts as a nonproductive pseudosubstrate of the APC/C or mediates Cdc20 proteolytic turnover (Kulukian, 2009).

Combining kinetochore-derived Bub3/BubR1-Cdc20 with evidence for two Cdc20 binding sites on BubR1 further suggests that the spontaneous and kinetochore-derived Bub3/BubR1-Cdc20 complexes may represent generation of Cdc20 bound at the two different sites, respectively, a point now testable with the appropriate BubR1 mutants (Kulukian, 2009).

Tpr directly binds to Mad1 and Mad2 and is important for the Mad1-Mad2-mediated mitotic spindle checkpoint

The mitotic arrest-deficient protein Mad1 forms a complex with Mad2, which is required for imposing mitotic arrest on cells in which the spindle assembly is perturbed. By mass spectrometry of affinity-purified Mad2-associated factors, the translocated promoter region (Tpr), a component of the nuclear pore complex (NPC), was identified as a novel Mad2-interacting protein. Tpr directly binds to Mad1 and Mad2. Depletion of Tpr in HeLa cells disrupts the NPC localization of Mad1 and Mad2 during interphase and decreases the levels of Mad1-bound Mad2. Furthermore, depletion of Tpr decreases the levels of Mad1 at kinetochores during prometaphase, correlating with the inability of Mad1 to activate Mad2, which is required for inhibiting APCCdc20. These findings reveal an important role for Tpr in which Mad1-Mad2 proteins are regulated during the cell cycle and mitotic spindle checkpoint signaling (Lee, 2008).

Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. This study shows that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitates with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1-c-Mad2 recruitment to NPCs. Interestingly, Tpr is normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1-c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restores normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, evidence is provided that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response (Schweizer, 2013).

An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. This paper describes evidence for a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, this study demonstrates that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous 'spindle envelope' confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function (Schweizer, 2015).

The cytoplasm is more crowded than the nucleoplasm, meaning that there is more “free” space in the nucleus for nano-sized particles. When proteins like Megator, soluble tubulin, or Mad2, which are restricted to the nucleus or the cytoplasm during interphase, diffusively equilibrate in all available spaces within the cell after NEB, this consequently leads to an enrichment of these proteins in the less crowded spindle region. While other roles cannot be excluded, the spindle envelope might act as a selective barrier to impede the invasion of the spindle region by large organelles, while retaining Megator complexes that might show gel-like properties. Importantly, cyclin B/Cdk1 and many other mitotic regulators, such as Polo/Plk1, Mad2, and Fizzy/Cdc20, as well as soluble tubulin and chromosomes, accumulate in the spindle region during mitosis. This might facilitate interactions between chromosomes and MTs during spindle assembly, not only by providing a physical constraint for MT growth and chromosome distribution, but also by creating a biochemical milieu that favors spindle assembly. Importantly, a MT-independent organelle-exclusion system is conserved in human cells and might therefore represent a common strategy to control mitosis in space and time (Schweizer, 2015).

Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

The mitotic checkpoint monitors kinetochore-microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, this study shows that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of this checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores (Ballister, 2014).

A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint

Recruitment of Mad1-Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1-Mad2 to kinetochores is unclear. This paper shows that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacts with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevent MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1-MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling (Moyle, 2014).

Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. This study shows that in mammalian oocytes Shugoshin-like protein 2 (Sgol2; Drosophila homolog Mei-S332) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2's ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation (Rattani, 2013).

The production of haploid gametes from diploid germ cells depends on two rounds of chromosome segregation (meiosis I and II) without an intervening round of DNA replication. Defects during the first or second meiotic division in oocytes lead to formation of aneuploid eggs, which in humans occurs with a frequency between 10 and 30% and is a major cause of fetal miscarriage. Understanding the causes of meiotic chromosome missegregation will require clarifying not only the forces and regulatory mechanisms governing meiotic chromosome segregation but also how these are coordinated (Rattani, 2013).

At the heart of this process are two opposing forces. The pulling forces produced by kinetochore-microtubules attachments (K-fibers) and resisting forces generated by sister chromatid cohesion, which counteracts K-fiber forces if and when kinetochores attach to microtubules with different polarities. During meiosis I, cohesion along chromosome arms holds bivalent chromosomes together following the creation of chiasmata produced by reciprocal recombination between homologous non-sister chromatids. This cohesion must persist during the attachment of maternal and paternal kinetochores to microtubules from opposite poles (bi-orientation) and resist the resulting spindle forces. The degree of traction exerted by meiotic spindles must create sufficient tension to facilitate bi-orientation. During the bi-orientation process, the initial attachment of kinetochores to the surface of the microtubule lattice (lateral attachment) is converted to attachments to their plus ends (end on attachment), creating K-fibers. Because this process is intrinsically error prone, inappropriate attachments, for example those that connect maternal and paternal kinetochores to the same pole, must be disrupted through phosphorylation of kinetochore proteins by Aurora B/C kinases. However, because these kinases disrupt K-fibers, they must subsequently be down-regulated once bivalents bi-orient correctly (Rattani, 2013).

The first meiotic division is eventually triggered by activation of a gigantic ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C) whose destruction of securin and cyclin B activates a thiol protease called separase that cleaves the kleisin subunit of the cohesin complex holding sister chromatids together. This process converts chromosomes from bivalents to dyads. It is delayed until all bivalents have bi-oriented by the production, at kinetochores that have not yet come under tension, of a potent inhibitor of the APC/C called the mitotic checkpoint complex (MCC) whose Mad2 subunit binds tightly to the APC/C's Cdc20 co-activator protein. This regulatory mechanism, called the spindle assembly checkpoint (SAC), must be turned off before APC/CCdc20 can direct destruction of securin and cyclin B and thereby activate separase. Another pre-condition for cleavage, at least in yeast, is phosphorylation of cohesin's kleisin subunit by a pair of protein kinases, namely CK1δ/ε and DDK. During the first meiotic division, cohesin is phosphorylated along chromosome arms but not at centromeres, which ensures that only cohesion along arms is destroyed by separase at the onset of anaphase I. The consequent persistence of cohesion at centromeres promotes bi-orientation of dyads during meiosis II (Rattani, 2013).

Centromeric cohesin avoids phosphorylation and therefore cleavage during the first meiotic division because separase activation is preceded by the recruitment to centromeres of orthologues of the Drosophila MEI-S332 protein, called shugoshins. Members of this family contain a conserved C-terminal basic region and an N-terminal homodimeric parallel coiled coil, which provides a docking site for protein phosphatase 2A's (PP2A) C and B' subunits (Xu, 2009). In budding yeast, mutant proteins defective specifically in PP2A binding fail to confer protection of centromeric cohesion during meiosis I (Rattani, 2013).

Mammals have two members of the shugoshin family: Shugoshin-like protein 1 (Sgol1), and Shugoshin-like protein 2 (Sgol2). The former prevents centromeric cohesin from a process called the 'prophase pathway' that removes cohesin from chromosomes by a non-proteolytic mechanism soon after cells enter mitosis (McGuinness, 2005; Liu, 2013). Sgol2, on the other hand, protects centromeric cohesin from separase at the first meiotic division (Lee, 2008; Llano, 2008). Sgol2's coiled coil domain binds PP2A in vitro (Xu, 2009) but whether this is vital for protecting centromeric cohesion is not known. Sgol2 also interacts with MCAK (Huang, 2007), a microtubule depolymerizing kinesin, implicated in correcting inappropriate kinetochore-microtubule interactions (error correction), and with Mad2 an essential component of the MCC (Orth, 2011). Shugoshins, though not explicitly Sgol2, have also been implicated in recruiting to kinetochores the Aurora B kinase, necessary both for the SAC and for error correction (Tsukahara, 2010; Yamagishi, 2010). Based on its interactions, Sgol2 has been linked to cohesion protection, the spindle assembly checkpoint, and error correction pathways. However, the physiological significance of these multiple interactions remain unclear (Rattani, 2013).

This study shows that Sgol2 defective in PP2A binding fails to protect centromeric cohesin, as found for Sgo1 in yeast (Xu, 2009). However, if this were the sole function of Sgol2, then chromosome behavior during meiosis I should be unaffected. Surprisingly it was found that Sgol2 deficiency caused striking changes in chromosome and microtubule dynamics. It delayed bi-orientation of bivalents on meiosis I spindles, caused increased bivalent stretching, and greatly increased Aurora B/C kinase activity at kinetochores, which was accompanied by an increase in lateral and a decrease in end on kinetochore-microtubules attachments. Lastly, Sgol2 deficiency delayed considerably APC/CCdc20 activation, suggesting that it was required to shut off the SAC. Sgol2 helps to turn off the SAC by binding directly both PP2A and the MCC protein Mad2, it moderates chromosome stretching by recruiting to kinetochores the kinesin MCAK, and likely promotes formation of K-fibers by down-regulating the activity of Aurora B/C kinase specifically at kinetochores. Meiotic chromosome segregation is a highly complex process dependent on an array of different biochemical processes. These findings imply that through its multi-domain structure, Sgol2 has an important if not unique role in coordinating many of the key processes within this array (Rattani, 2013).


Search PubMed for articles about Drosophila Mad2

Ballister, E. R., Riegman, M. and Lampson, M. A. (2014). Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint. J Cell Biol 204: 901-908. PubMed ID: 24637323

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 ID: 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. PubMed ID: 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. PubMed ID: 15886105

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

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

Chen, R. H., et al. (1996). Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 274(5285): 242-6. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 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 ID: 17576797

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

Huang, H., Feng, J., Famulski, J., Rattner, J. B., Liu, S. T., Kao, G. D., Muschel, R., Chan, G. K. and Yen, T. J. (2007). Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments. J Cell Biol 177: 413-424. PubMed ID: 17485487

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. PubMed ID: 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. PubMed ID: 14990952

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

Katsani, K. R., Karess, R. E., Dostatni, N. and Doye, V. (2008). In vivo dynamics of Drosophila nuclear envelope components. Mol. Biol. Cell 19(9): 3652-66. PubMed ID: 18562695

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

Kulukian, A., Han, J. S. and Cleveland, D. W. (2009). Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16(1): 105-17. PubMed ID: 19154722

Lee, J., Kitajima, T. S., Tanno, Y., Yoshida, K., Morita, T., Miyano, T., Miyake, M. and Watanabe, Y. (2008). Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells. Nat Cell Biol 10: 42-52. PubMed ID: 18084284

Lee, S. H., Sterling, H., Burlingame, A. and McCormick, F. (2008). Tpr directly binds to Mad1 and Mad2 and is important for the Mad1-Mad2-mediated mitotic spindle checkpoint. Genes Dev. 22(21): 2926-31. PubMed ID: 18981471

Lince-Faria, M., (2009). Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J. Cell Biol. 184(5): 647-57. PubMed ID: 19273613

Liu, H., Rankin, S. and Yu, H. (2013). Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nat Cell Biol 15: 40-49. PubMed ID: 23242214

Llano, E., Gomez, R., Gutierrez-Caballero, C., Herran, Y., Sanchez-Martin, M., Vazquez-Quinones, L., Hernandez, T., de Alava, E., Cuadrado, A., Barbero, J. L., Suja, J. A. and Pendas, A. M. (2008). Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice. Genes Dev 22: 2400-2413. PubMed ID: 18765791

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 ID: 10411903

McGuinness, B. E., Hirota, T., Kudo, N. R., Peters, J. M. and Nasmyth, K. (2005). Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells. PLoS Biol 3: e86. PubMed ID: 15737064

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Moyle, M. W., Kim, T., Hattersley, N., Espeut, J., Cheerambathur, D. K., Oegema, K. and Desai, A. (2014). A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint. J Cell Biol 204: 647-657. PubMed ID: 24567362

Musacchio, A. and Hardwick, K. G. (2002). The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell Biol. 3: 731-741. PubMed ID: 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 ID: 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. PubMed ID: 17182852

Orth, M., Mayer, B., Rehm, K., Rothweiler, U., Heidmann, D., Holak, T. A. and Stemmann, O. (2011). Shugoshin is a Mad1/Cdc20-like interactor of Mad2. EMBO J 30: 2868-2880. PubMed ID: 21666598

Rattani, A., Wolna, M., Ploquin, M., Helmhart, W., Morrone, S., Mayer, B., Godwin, J., Xu, W., Stemmann, O., Pendas, A. and Nasmyth, K. (2013). Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes. Elife 2. PubMed ID: 24192037

Schweizer, N., Ferras, C., Kern, D. M., Logarinho, E., Cheeseman, I. M. and Maiato, H. (2013). Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. J Cell Biol 203(6): 883-93. PubMed ID: 24344181

Schweizer, N., Pawar, N., Weiss, M. and Maiato, H. (2015). An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. J Cell Biol 210: 695-704. PubMed ID: 26304726

Scott, R. J., Lusk, C. P., Dilworth, D. J., Aitchison, J. D. and Wozniak, R. W. (2005). Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 16: 4362-4374. PubMed ID: 16000377

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

Silva, R. D., Mirkovic, M., Guilgur, L. G., Rathore, O. S., Martinho, R. G. and Oliveira, R. A. (2018). Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion. Curr Biol 28(17):2837-2844. PubMed ID: 30122528 Silva, 2018

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. PubMed ID: 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. PubMed ID: 12006501

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date revised: 25 August 2023

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