Spindle checkpoint protein dynamics at kinetochores in living cells

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.     

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1^wt) or a mutant version (Mps1^as) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20’s association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B–dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.     

When Mad met Bub

The faithful segregation of chromosomes into daughter cells is essential for cellular and organismal viability. Errors in this process cause aneuploidy, a hallmark of cancer and several congenital diseases. For proper separation, chromosomes attach to microtubules of the mitotic spindle via their kinetochores, large protein structures assembled on centromeric chromatin. Kinetochores are also crucial for a cell cycle feedback mechanism known as the spindle assembly checkpoint (SAC) 1 . The SAC forces cells to remain in mitosis until all chromosomes are properly attached to microtubules. At the beginning of mitosis, the SAC proteins—Mad1, Mad2, Bub1, Bub3, BubR1, Mps1, and Cdc20—are recruited to kinetochores in a hierarchical and interdependent fashion (Fig  1 A). There they monitor, in ways that are not fully clarified, the formation of kinetochore–microtubule attachments 1 . Two studies recently published in EMBO reports by the groups of Silke Hauf 2 and Jakob Nilsson 3 , and a recent study by London and Biggins in Genes & Development 4 , shed new light on the conserved SAC protein Mad1.     

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.     

Mad3/BubR1 Phosphorylation during Spindle Checkpoint Activation Depends on both Polo and Aurora Kinases in Budding Yeast

During mitosis the spindle assembly checkpoint (SAC) delays the onset of anaphase and mitotic exit until all chromosomes are bipolarly attached to spindle fibers. Both lack of attachment due to spindle/kinetochore defects and lack of tension across kinetochores generate the “wait anaphase” signal transmitted by the SAC, which involves the evolutionarily conserved Mad1, Mad2, Mad3/BubR1, Bub1, Bub3 and Mps1 proteins, and inhibits the activity of the ubiquitin ligase Cdc20/APC, that promotes both sister chromatid dissociation in anaphase and mitotic exit. In particular, Mad3/BubR1 is directly implicated, together with Mad2, in Cdc20 inactivation in both human and yeast cells, suggesting that its activity is likely finely regulated. We show that budding yeast Mad3, like its human orthologue BubR1, is a phosphoprotein that is hyperphosphorylated during mitosis and when SAC activation is triggered by microtubule depolymerizing agents, kinetochore defects or lack of kinetochore tension. In vivo Mad3 phosphorylation depends on the Polo kinase Cdc5 and, to a minor extent, the Aurora B kinase Ipl1. Accordingly, replacing with alanines five serine residues belonging to Polo kinase-dependent putative phosphorylation sites dramatically reduces Mad3 phosphorylation, suggesting that Mad3 is likely an in vivo target of Cdc5.     

Drosophila Polo regulates the spindle assembly checkpoint through Mps1‐dependent BubR1 phosphorylation

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase‐promoting complex/cyclosome‐inhibitory complexes to levels that ensure long‐term SAC activity. We propose a model in which Polo controls Mps1‐dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.     

Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.     

Spatial regulation of the spindle assembly checkpoint and anaphase‐promoting complex in Aspergillus nidulans

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ‐tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time‐lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co‐localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase‐promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ‐tubulin mutation mipAD159 causes a nuclear‐specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.     

Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores.

The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation.     

Studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living Drosophila syncytial embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators and is also a kinetochore dynamic protein. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive. Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex. Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.     

The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint

Background: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. Results: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. Conclusions: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.     

Recruitment of Cdc20 to the Kinetochore Requires BubR1 but Not Mad2 in Drosophila melanogaster

To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that negatively regulates the activation of the anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway to prevent the destruction of two key substrates, cyclin B and securin, thereby inhibiting the metaphase-to-anaphase transition until bipolar attachment of all chromosomes has been achieved (35). A number of conserved kinetochore proteins have been identified as SAC components, such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, and Rod and Aurora B kinase (reviewed by Musacchio and Salmon [35]). In vertebrates, it is believed that a diffusible inhibitory “wait anaphase” signal is generated from unattached kinetochores or lack of spindle tension (27, 45, 47) and that its primary target is Cdc20/Fzy (Fzy is the Drosophila Cdc20 homolog that we refer to as Cdc20 here), which is an essential APC/C activator (35). Mad2, BubR1 (Mad3 in Saccharomyces cerevisiae), Bub3, and Cdc20 have been found in the mitotic checkpoint complex (MCC) or its subcomplexes Bub3-BubR1-Cdc20 and Mad2-Cdc20 (42, 50, 56). Kinetochore-dependent recruitment and activation of Mad2 have been illustrated in a “template” model (12) and later a modified “two-state” model (28, 32, 35, 36, 40, 57). This model suggests that a kinetochore-bound and conformationally rearranged Mad2 is required for Cdc20 binding and that it leads to the formation of the Mad2-Cdc20 complex (8, 9, 12, 16, 48, 49). This is further supported by a more recent report that unattached kinetochores from purified HeLa cell chromosomes can catalytically generate a diffusible Cdc20 inhibitor when presented with kinetochore-bound Mad2 and that these purified chromosomes can also promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2 but not BubR1 (27). In vitro assays also suggest that Mad2 is required for Cdc20 binding to BubR1 (7, 10, 19). Fluorescence recovery after photobleaching analysis has suggested that the ∼50% of green fluorescent protein (GFP)-Cdc20 that associates with slow-phase kinetics on PtK₂ cell kinetochores is Mad2 dependent (22). However, contradictory reports also exist to suggest that Mad2 might not be required for Cdc20 kinetochore localization in Xenopus and PtK₂ cells (22) and that BubR1 might play a crucial role for this in human cell lines (33). In contrast to the above-mentioned slow-phase GFP-Cdc20, the remaining ∼50% of GFP-Cdc20 that associates with fast kinetics on prometaphase or metaphase kinetochores is Mad2 independent, and its kinetics parallel those of GFP-BubR1 in PtK₂ cells. GFP-Cdc20 is still detectable on kinetochores through anaphase, where both Mad2 and BubR1 are greatly reduced (22, 25). Moreover, the direct requirement for the kinetochore in the formation of the SAC-inhibitory complexes has been challenged by a non-kinetochore-based formation hypothesis, with MCC found to be present in HeLa cells during S phase (50) and complex formation in yeast previously shown to be independent of intact kinetochores (17, 43). Therefore, despite the importance of Cdc20 in understanding SAC mechanisms, exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear in vertebrates.Drosophila melanogaster is a well-established model used to study the spindle assembly checkpoint (2, 3, 6, 39). More recently, phenotypes of two mad2-null Drosophila mutant alleles, mad2^Δ and mad2^P, have been characterized, showing that Mad2 protein is not essential for normal mitotic progression but remains essential for SAC when microtubule attachment, chromosome alignment, and congression are abnormal (5). This contrasts with its counterpart in mouse and human (14, 34, 54) and is also different from the lethality phenotypes reported for bubR1 and cdc20 mutations in Drosophila (3, 11). It has also been reported that Mad2 is less important for SAC than BubR1 and that it is regulated differently in Drosophila S2 culture cells (39). These observations led to the tentative conclusion that Drosophila Mad2 may possess different kinetochore molecular mechanisms and function differently from its homologs in mouse and human (14, 34, 54, 58). We therefore tested Mad2 kinetochore function and further investigated the mechanisms required for Cdc20 kinetochore recruitment and localization using Drosophila transgenic and mutant lines, as well as culture cells. We have characterized a new mad2-null mutant allele, mad2^EY, and demonstrated that Drosophila possesses a highly conserved Mad2 kinetochore dimerization mechanism required for SAC function. However, Mad2 is not required for Cdc20 kinetochore recruitment and localization. Instead, there is an essential role for BubR1 in this mechanism during normal mitosis and SAC activation.     

The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that Cdc20 and C-Mad2 levels correlate at kinetochores and that depletion of Cdc20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type Cdc20 but not Cdc20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that Cdc20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-Cdc20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.     

All together now

Maintenance of genomic stability during eukaryotic cell division relies on the Spindle Assembly Checkpoint (SAC), which has evolved as a surveillance mechanism that monitors kinetochore-microtubule attachment and prevents APC/C-mediated mitotic exit until all chromosomes are properly attached to the mitotic spindle. Reversible protein phosphorylation has long been accredited as a regulatory mechanism of the SAC. Nevertheless, knowledge of how several mitotic kinases act in concert within the signaling pathway to orchestrate SAC function is still emerging. In a recent study, we undertook a comprehensive dissection of the hierarchical framework controlling SAC function in Drosophila cells. We found that Polo lies at the top of the SAC pathway promoting the efficient recruitment of Mps1 to unattached kinetochores. This renders Mps1 fully active to control BubR1 phosphorylation that generates the 3F3/2 phosphoepitope at tensionless kinetochores. We have proposed that Polo is required for SAC function and that the molecular outcome of Mps1-dependent 3F3/2 formation is to promote the association of Cdc20 with BubR1 allowing proper kinetochore recruitment of Cdc20 and efficient assembly of the Mitotic Checkpoint Complex (MCC) required for a sustained SAC response.     

The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast

The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1.     

Dynamic Autophosphorylation of Mps1 Kinase Is Required for Faithful Mitotic Progression

The spindle assembly checkpoint (SAC) is a surveillance mechanism monitoring cell cycle progression, thus ensuring accurate chromosome segregation. The conserved mitotic kinase Mps1 is a key component of the SAC. The human Mps1 exhibits comprehensive phosphorylation during mitosis. However, the related biological relevance is largely unknown. Here, we demonstrate that 8 autophosphorylation sites within the N-terminus of Mps1, outside of the catalytic domain, are involved in regulating Mps1 kinetochore localization. The phospho-mimicking mutant of the 8 autophosphorylation sites impairs Mps1 localization to kinetochore and also affects the kinetochore recruitment of BubR1 and Mad2, two key SAC effectors, subsequently leading to chromosome segregation errors. Interestingly, the non-phosphorylatable mutant of the 8 autophosphorylation sites enhances Mps1 kinetochore localization and delays anaphase onset. We further show that the Mps1 phospho-mimicking and non-phosphorylatable mutants do not affect metaphase chromosome congression. Thus, our results highlight the importance of dynamic autophosphorylation of Mps1 in regulating accurate chromosome segregation and ensuring proper mitotic progression.     

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

Loss of spindle assembly checkpoint–mediated inhibition of Cdc20 promotes tumorigenesis in mice

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20^AAA/AAA mice died at late gestation, but Cdc20^+/AAA mice were viable. Importantly, Cdc20^+/AAA mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.     

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.Massive chromosome missegregation induces cell death as observed by Theodor Boveri in the early 1900s.¹ However, the underlying mechanism remains elusive. The spindle assembly checkpoint (SAC) is a dominant machine monitoring chromosomal segregation during mitosis by delaying the onset of anaphase until all chromosomes are properly captured by microtubules. The SAC consists of kinetochore association sensors, including Mps1 (monopolar spindle 1), Bub1 (budding uninhibited by benzimidazole 1 homolog) and Aurora B; a signaling transducer termed the mitotic checkpoint complex (MCC), including CDC20 (cell division cycle 20), BubR1 (Bub1-related kinase), Bub3 (budding uninhibited by benzimidazole 3 homolog) and Mad2 (mitotic arrest deficient-like 2); and an effector APC/C (anaphase-promoting complex/cyclosome) that is inhibited by MCC in response to an active SAC.² Loss of SAC by inactivation of checkpoint sensors or signaling transducers elicits massive chromosome missegregation, induces severe gain or loss of chromosomes and eventually causes cell death.^{3, 4, 5, 6} Meanwhile, a weakened SAC due to the haploinsufficiency of the checkpoint proteins Mad1, Mad2, Bub1, BubR1 and CENP-E (centromere protein E) does not cause cell death but facilitates tumorigenesis.^{7, 8, 9, 10, 11} These studies suggest that the fate of these cells is dependent on their respective degree of SAC deficiency. Notably, in these studies SAC proteins were constitutively disturbed, raising the possibility that other signaling pathways could be affected as SAC proteins have functions beyond SAC regulation.^{12, 13, 14}Mps1 is an essential component of SAC that senses SAC signal by promoting MCC formation via kinetochore recruitment of Mad2, CENP-E and Knl1 (kinetochore-null protein 1).^{15, 16, 17, 18, 19} Recent studies show that Mps1 can discriminate between on or off SAC signaling by binding to NDC80c via the motif that associates microtubules.^{20, 21} Following SAC, Mps1 is involved in regulating chromosome alignment by phosphorylating Borealin, a component of chromosomal passenger complex (CPC).^{22, 23} In addition, Mps1 plays multiple roles beyond mitosis, including centrosome duplication, cytokinesis, ciliogenesis and DNA damage response.^{18, 24, 25, 26, 27, 28} Mps1 is indispensable for cell survival as loss of Mps1 function by specific siRNA or Mps1 kinase inhibitors causes significant cell death; it has been proposed that Mps1 regulates this process through its roles in SAC.^{29, 30, 31}Mps1 kinase is overexpressed in a variety of tumor types.^{32, 33, 34, 35} In breast cancer, high levels of Mps1 correlate with tumor grades; reducing Mps1 level induces massive apoptosis but allows a selective survival of tumor cells with less aneuploidy.³² Our recent results in colon cancer cells showed that overexpression of Mps1 facilitate the survival of tumor cells with higher aneuploidy by decreasing SAC threshold.³⁵ To further uncover the roles of high levels of Mps1 in tumorigenesis, we examined Mps1 levels in various stages of colon cancer tissues and found that Mps1 level peaks in tissues at stage II, at which stage tumor cells encounter various survival stresses, including genome instability. Aneuploid colon cancer cell lines bear higher levels of Mps1 than diploid cell lines and the amount of Mps1 required for cell survival is far more than that of maintaining SAC, suggesting that other functions of Mps1 are also employed to maintain cell viability. Short-term inhibition of Mps1 activity in mitosis with inhibitors at a dose of more than SAC depletion is sufficient to cause dividing cell death and increase mitochondrial fragmentation simultaneously. Finally, we found that Mps1 can regulate the release of cytochrome c by associating with mitochondrial protein VDAC1 (voltage-dependent anion channel 1). Based on these findings, we postulated that high levels of Mps1 contribute to survival of aneuploid cancer cells via its roles in SAC and mitochondria.     

Requirement for proteolysis in spindle assembly checkpoint silencing

Anaphase initiation requires ubiquitin-dependent proteolysis of crucial substrates through activation of the ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) in association with its coactivator Cdc20. To prevent chromosome segregation errors, effector proteins of a safeguard mechanism called spindle assembly checkpoint (SAC), Mad2 and BubR1, bind Cdc20 and restrain APC/C^Cdc20 activation until spindle assembly. Coordinated chromosome segregation also requires timely SAC inactivation. Spindle assembly appears necessary to silence SAC, however, how resolution of the SAC effector branch is achieved is still largely unknown. We show here that the complex between Mad2 and Cdc20 peaked at prometaphase in mammalian cells, while its dissociation proceeded along with spindle assembly and required proteolysis. Proteolysis did not appear required for assembly of metaphase spindles but rather needed for Mad2-Cdc20 complex resolution by promoting reversal of phosphorylations that maintain the complex. Indeed, in the absence of proteolysis, Mad2-Cdc20 complex dissociation was reversed by treatment with cyclin-dependent kinase or Aurora kinase inhibitors. Mad2-Cdc20 disassembly was, however, resistant to the potent PP1 and PP2A phosphatases inhibitor okadaic acid. We propose that SAC silencing in mammalian cells requires proteolysis-dependent activation of okadaic acid-resistant phosphatase(s) to reverse phosphorylations that lock the Mad2-Cdc20 complex.