Signalling dynamics in the spindle checkpoint response.

Nature Reviews Molecular Cell Biology | AOP, published online 10 October 2014; doi:10.1038/nrm3888

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Signalling dynamics in the spindle checkpoint response Nitobe London1,2 and Sue Biggins1

Abstract | The spindle checkpoint ensures proper chromosome segregation during cell division. Unravelling checkpoint signalling has been a long-standing challenge owing to the complexity of the structures and forces that regulate chromosome segregation. New reports have now substantially advanced our understanding of checkpoint signalling mechanisms at the kinetochore, the structure that connects microtubules and chromatin. In contrast to the traditional view of a binary checkpoint response — either completely on or off — new findings indicate that the checkpoint response strength is variable. This revised perspective provides insight into how checkpoint bypass can lead to aneuploidy and informs strategies to exploit these errors for cancer treatments. Bi-oriented The kinetochore–microtubule attachment state where sister kinetochores are attached exclusively to opposite spindle poles by similar numbers of microtubules.

Cohesin complex A protein complex that physically links DNA on sister or homologous chromosomes following S‑phase and that must be cleaved for mitotic progression.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., PO Box 19024, Seattle, Washington 98109, USA. 2 Molecular and Cellular Biology Program, University of Washington/Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. Correspondence to S.B. e-mail: [email protected] doi:10.1038/nrm3888 published online 10 October 2014 1

Accurate chromosome segregation is essential for genome inheritance and cellular fitness. Chromosome mis-segregation results in lethality or aneuploidy, the state in which cells have an aberrant number of chromosomes. Aneuploidy leads to abnormal gene dosage and exposes detrimental recessive mutations, potentially causing birth defects and promoting cancer cell proliferation (for reviews, see REFS 1,2). Accurate segregation is achieved by linking sister chromatids after replication and then segregating them to opposite spindle poles prior to cytokinesis. Segregation is mediated by spindle microtubules that attach to chromosomes through kinetochores, large protein complexes that assemble on centromeric DNA. Microtubule disassembly provides the force to segregate chromosomes at anaphase3. Several different attachment states are possible within the mitotic spindle because sister kinetochores are capable of binding to microtubules from either pole (FIG. 1). Sister kinetochores become bi-oriented by attaching to microtubules from opposite poles (that is, forming amphitelic attachments), but they can also make mono-oriented attachments. Monooriented attachments occur when microtubules from the same pole attach to both sister kinetochores (forming syntelic attachments) or when only one of the two sister kinetochores attaches (forming monotelic attachments). Individual kinetochores typically bind multiple microtubules (from approximately three in fission yeast to approximately 30 in mammalian cells), whereas the unusual budding yeast kinetochore binds to a single microtubule. Therefore, in most organisms,

some microtubules from the same spindle pole can bind to both sister kinetochores (forming merotelic attachments). However, only bi-oriented attachments will reliably lead to correct segregation, and cells must therefore attain bi-orientation before anaphase onset. To monitor bi-orientation, cells sense forces at the kinetochore. Before anaphase, sister chromatids are linked by the cohesin complex, which resists microtubule pulling forces (FIG. 1). Evidence suggests that the tension generated on sister kinetochores by the pulling forces of spindle microtubules signals proper bi-orientation4. Two central mechanisms ensure the formation of bi-oriented attachments. First, error correction mechanisms detect and correct mono-oriented attachments. These mechanisms destabilize incorrect microtubule attachments, thus allowing cells another chance to achieve bi-orientation. The second major mechanism is the spindle checkpoint signalling cascade (also known as the spindle assembly checkpoint (SAC) or mitotic checkpoint), which senses the attachment state of kinetochores and prevents anaphase onset until biorientation is achieved. Kinetochores that lack tension or attachment induce a spindle checkpoint arrest, giving cells time to resolve incorrect attachments. Because tension defects generate unattached kinetochores through error-correction mechanisms, it has been unclear whether tension and attachment use different upstream pathways to trigger the checkpoint. In addition, a precise tension signal has not been conclusively identified4. Here, we review recent results that elucidate the molecular basis for checkpoint activation at kineto chores as well as novel findings that suggest that

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Figure 1 | Kinetochore–microtubule attachment states on the mitotic spindle. Kinetochores can bind to Nature Reviews | Molecular Cell Biology microtubules attached to either spindle pole, making several different configurations possible. An amphitelic attachment is when the correct number of spindle microtubules attach to each sister kinetochore from each spindle pole. A monotelic attachment is when one kinetochore is unattached and the other kinetochore is attached to microtubules from a single spindle pole. A syntelic attachment is when both kinetochores are attached to microtubules from a single spindle pole. Finally, a merotelic attachment is when a single kinetochore is attached to microtubules from both spindle poles. Spindle poles nucleate spindle microtubules, which are shown attaching to kinetochores, as well as astral microtubules, which position the spindle within the cell. Sister chromosomes are linked by the protein complex cohesin until anaphase onset. Microtubules tend to pull chromosomes towards spindle poles, resulting in tension across bi-oriented sister chromosomes. The spindle checkpoint is activated by syntelic attachments and monotelic attachments but not necessarily by merotelic attachments, because in these cases some tension is applied across sister kinetochores134.

nuclear pores can also generate a checkpoint signal. We then relate these mechanistic findings to current reports on the dynamics and kinetics of checkpoint signalling. Overall, these findings resolve long-standing questions about checkpoint activation and redefine our view of the checkpoint response.

The spindle checkpoint response The spindle checkpoint was discovered in budding yeast in two landmark studies, which screened for mutants that failed to arrest in response to microtubuledestabilization5,6. These screens identified the budding uninhibited by benzimidazole genes (BUB1, BUB2 and BUB3)5 and the mitotic arrest-deficient genes (MAD1, MAD2 and MAD3)6. Most of the proteins encoded by these genes are recruited to kinetochores in a hierarchical manner (FIG. 2), indicating that the kinetochore serves as the hub for checkpoint activation. However, because kinetochores have a complex composition, it has been difficult to elucidate the molecular details underlying the recruitment of these proteins. Seminal observations established that phosphorylation is essential for the checkpoint 7,8 (for a review, see REF. 9), and at least one kinetochore phospho-epitope persists on both unattached and low-tension kinetochores but is extinguished on bi-orientation10–13. Although numerous kinases have direct or indirect roles in the checkpoint (BOX 1), the conserved kinetochore kinase monopolar spindle 1 (MPS1) has emerged as a dominant effector of the checkpoint. The Aurora B kinase has a central role in both error correction and the checkpoint (BOX 1), and is reviewed elsewhere9,14,15. New studies have now succeeded in reconstituting kinetochore signalling events in vitro and in vivo, showing that a specific set of kinetochore proteins termed the KMN (kinetochore null protein 1 (KNL1)–mis-segregation 12 (MIS12)– nuclear division cycle 80 (NDC80)) network16 (FIG. 2a) are the scaffold for checkpoint signalling. The KMN network is also a major microtubule-binding interface of the kinetochore and is therefore well suited for linking microtubule attachment states to checkpoint signalling 14,16,17. Pioneering laser ablation and cell biology studies7,18–20 determined that kinetochores activate the checkpoint by responding to missing or tensionless microtubule attachments. Because even a single unattached kineto chore is sufficient to arrest the cell cycle20, and because this is a minimal defect in the mitotic spindle, it led to the view that the checkpoint is switch-like: either completely on or off. However, cells seemed to arrest for different lengths of time depending on the severity of the attachment state defect (that is, depending on the number of unattached or incorrectly attached kinetochores21, or the concentration of microtubule poison 22). Consistent with this, new reports now show that the checkpoint is graded, with more severe defects leading to a stronger response23,24. Furthermore, complementary findings about the kinetics of checkpoint signalling help to explain why the checkpoint is not reactivated during anaphase when tension on kinetochores is lost 25–28.

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Figure 2 | Kinetochore activation of the checkpoint through hierarchical checkpoint protein recruitment. NatureKinetochore Reviews | Molecular Cell Biology a | Assembly of a functional checkpoint-signalling complex on kinetochores is stepwise. null protein 1 (KNL1) and nuclear division cycle 80 (NDC80) are core kinetochore proteins that form a scaffold for checkpoint protein recruitment. Budding uninhibited by benzimidazole 3 (BUB3) forms separate complexes with BUB1 and BUB1‑related protein kinase (BUBR1; mitotic arrest-deficient 3 (Mad3) in yeast). Monopolar spindle 1 (MPS1) kinase activity recruits BUB1–BUB3, and BUB1 permits localization of BUBR1. Next, a MAD1–MAD2 heterotetramer binds to the kinetochore through a MAD1–BUB1 interaction and possibly through an additional receptor. Kinetochore-bound MAD1–MAD2 facilitates checkpoint activation through catalytic conversion of soluble ‘open’ (O)-MAD2 to ‘closed’ (C)-MAD2. b | Kinetochores catalyse the assembly of the mitotic checkpoint complex (MCC). C-MAD2 generated through kinetochore MAD1–C-MAD2 binds to cell division control protein 20 (CDC20), possibly through an intermediate conformation, I-MAD2 (not shown), and then to BUBR1–BUB3. Conserved motifs in BUB1 and MAD1 are necessary for checkpoint signalling (see main text). CDC20 and BUBR1 localize to kinetochores in most organisms, although it is unresolved how this facilitates MCC formation. For instance, it remains to be determined whether kinetochore-bound or soluble BUBR1 is incorporated into the MCC. The MCC inhibits the activity of the APC/C (anaphase-promoting complex, also known as the cyclosome)– CDC20 complex, and the crucial mitotic targets of the APC/C (cyclin B and securin) are thereby stabilized while the checkpoint is active, which prevents chromosome segregation. CDK1, cyclin-dependent kinase 1.

E3 ubiquitin ligase Enzyme responsible for transfer of ubiquitin to substrates, often targeting them for degradation by the proteasome. E3 ligases transfer ubiquitin from E2 enzymes to their substrates.

Kinetochore activation of the checkpoint Mitotic progression is controlled by the APC/C (anaphasepromoting complex, also known as the cyclosome), which is an E3 ubiquitin ligase, and its co-activator cell division control protein 20 (CDC20). This complex activates anaphase onset and mitotic exit by targeting two key proteins for proteasome-dependent degradation: securin, which protects sister chromatid cohesin from proteolytic cleavage, and cyclin B, the mitotic cyclindependent kinase 1 (CDK1) cofactor 29. The spindle checkpoint inhibits these APC/C functions by inactivating CDC20 through the mitotic checkpoint complex (MCC), which consists of CDC20 in complex with MAD2, BUB1‑related protein (BUBR1; Mad3 in yeast) and BUB3 (REF. 30). The MCC inactivates the APC/C through various mechanisms31–35 and is the biochemical manifestation of the ‘wait anaphase’ signal. The key step in MCC formation is conformational activation of MAD2 from the free ‘open’ form (O-MAD2) to the CDC20‑bound ‘closed’ form (C-MAD2) (FIG. 2b) (for reviews, see REFS 36,37 ). Studies using FRAP

(fluorescence recovery after photobleaching) to examine MAD2 localization dynamics implicated kinetochores as the sites of MAD2 conversion38–40. Conversion is a catalytic process, occurring through the association of soluble O-MAD2 with kinetochore-bound C-MAD2. MAD1 is the receptor for C-MAD2 at the kinetochore (which is distinct from CDC20‑bound C-MAD2) and facilitates MAD2 conformational conversion (for reviews, see REFS 36,37). A major function of the kineto chore, then, is to promote MAD2 conversion through the hierarchical recruitment of checkpoint proteins. This cascade seems to consist of the conserved kinases Aurora B and MPS1 at the top of the hierarchy, followed by recruitment of the BUB1–BUB3 complex, then by recruitment of BUBR1–BUB3, and finally by recruitment of a heterotetramer composed of MAD1 and MAD2 (REFS 39,41–45). CDC20 also localizes to kinetochores in most organisms except budding yeast, which also lacks Mad3 localization42. We focus below on the newly elucidated mechanisms for BUB and MAD protein recruitment.



REVIEWS Box 1 | Conserved checkpoint kinases Early work showed that the checkpoint requires phosphorylation and that kinetochore phosphorylation is directly responsive to microtubule attachment state7,8 (for a review, see REF. 9). Considerable effort has since gone into investigating the role of kinases and identifying their substrates.

CDK1–cyclin B The activity of cyclin-dependent kinase 1 (CDK1)–cyclin B is required for mitotic entry, and anaphase progression depends on degradation of cyclin B and removal of CDK1‑dependent phosphorylation9,29. Cyclin B is a major target of the APC/C (anaphase-promoting complex, also known as the cyclosome), and stabilizing it is a primary outcome of checkpoint activity. However, function of CDK1 is complex, and it is involved in both activating and silencing the checkpoint, at least partly through cell division control protein 20 (CDC20) phosphorylation9,140. Aurora B Aurora B has a crucial role in promoting chromosome bi-orientation by destabilizing incorrect kinetochore–microtubule attachments, a complementary function to the checkpoint. Aurora B is also crucial for the checkpoint to function under certain conditions, such as a lack of tension. Although this may largely be a consequence of the kinetochore–microtubule attachment destabilizing function in yeast141, human Aurora B seems to also be important for loading or stabilizing monopolar spindle 1 (MPS1) on kinetochores115–118,142. MPS1 MPS1 has a widely conserved role in activation of the checkpoint (for a review, see REF. 143). Furthermore, MPS1 is unique in that its overexpression results in constitutive checkpoint activation, identifying it as the central checkpoint kinase144. Like Aurora B, MPS1 is important for promoting bi-orientation, although the underlying mechanism is unclear115,122,145. Kinetochore phosphorylation by MPS1 activates the checkpoint through recruitment of the checkpoint proteins budding uninhibited by benzimidazole 1 (BUB1), BUB3, mitotic arrest-deficient 1 (MAD1) and MAD2 (REFS 48–50,78), and possibly through downstream substrates114,146,147. BUB1 Although BUB1 kinase activity is dispensable for a checkpoint response55, BUB1 phosphorylates histone H2A, which indirectly leads to Aurora B localization56. BUB1 kinase activity therefore has an indirect role in promoting bi-orientation and the checkpoint.

Pseudokinase A protein that is evolutionarily derived from an active kinase that has lost catalytic activity.

BUB protein kinetochore recruitment. Although kineto chore localization of checkpoint proteins is necessary for checkpoint signalling, the precise binding sites and mechanism regulating the binding of checkpoint proteins to the kinetochore was unknown until recently. It has now been established that the core kinetochore protein KNL1 recruits BUB1, BUBR1 and BUB3 (BUB proteins)46–51, although the recruitment mechanisms are complex and not yet fully understood. BUB1 and BUBR1 are related proteins that are universally required for the checkpoint and are important for kinetochore bi-orientation41,52,53 (for a review, see REF. 54). BUB1 is a protein kinase, whereas BUBR1 is a pseudokinase in vertebrates, and the catalytic domains of both proteins are apparently required for their bi-orientation functions but not for the spindle checkpoint 53,55,56. Consistent with this, the yeast Mad3 checkpoint protein is related to BUBR1 but lacks the pseudokinase domain and has no function in bi-orientation57 (FIG. 3A). BUB1 and BUBR1 both form constitutive (that is, stably associated) complexes with the BUB3 protein; these complexes are mutually exclusive because both BUB1 and BUBR1 associate with the same surface of BUB3 (REF. 58). BUB1 and BUBR1 bind to BUB3 through a BUB3‑binding domain (also known as a GLEBS domain) (FIG. 3A).

KNL1 contains several conserved motifs (for reviews, see REFS 17,59,60 ), although they are not conserved throughout its length. Two types of motifs specific to KNL1 are the ‘KI’ motifs and the ‘MELT’like ([M/I/‌L/‌V]-[E/D]-[M/I/L/V]-T) motifs47–50,57. Initial work characterized an interaction between a KI motif on KNL1 and the conserved TPR motif on BUB1 (FIG. 3A), and this was proposed to mediated BUB1 kinetochore recruitment46,47. Surprisingly, however, this interaction is dispensable for kinetochore targeting of BUB1 in vivo61. Additional work determined that MPS1 kinase activity stimulates BUB1 localization and checkpoint activation62, and a trio of studies ultimately determined that MPS1‑mediated phosphorylation of the Thr residues on the MELT-like motifs of KNL1 is required for BUB1 kinetochore localization48–50. Crystallography and additional biochemistry revealed that the phosphorylation is required for a high-affinity interaction between BUB3 and KNL1 (REF. 51), indicating that BUB3 binding to KNL1 is the key step in localizing BUB1–BUB3 to kinetochores. However, BUB1 contributes to stabilizing the BUB3–KNL1 interaction in vitro51,63, consistent with the observation that BUB1 is important for BUB3 localization in vivo44,64. Unexpectedly, the mechanism of BUBR1 recruitment to the kinetochore is different from that of the related BUB1 protein and is still unresolved. BUBR1 does not stabilize the BUB3–MELT inter action51,63, suggesting that it may not localize through BUB3–KNL1 binding. BUBR1 localization depends on BUB1 (REFS 50,65–67), so one possibility is that BUB1 directly recruits BUBR1 through dimerization68. This would explain why ectopically localized fission yeast Bub1 recruits Mad3 (REF. 69) (FIG. 3B). Such a binding mode might leave BUBR1‑associated BUB3 available for interactions aside from MELT-like-motif binding 63,61. Quantitative analysis of BUB1 and BUBR1 kineto chore recruitment has been investigated in human cell lines and in vitro63,70,71. MELT-like motifs within KNL1 exist in multiple copies (of varying number) in different organisms (FIG. 3A), suggesting multiple BUB protein binding sites. Indeed, in vivo analyses of KNL1–BUB protein binding indicated that the number of MELT-like motifs correlates with the amount of BUB1 binding, at least up to several motifs63,70,71. Interestingly, whereas a single MELT-like motif can recruit BUB1 and restore some checkpoint function, additional motifs are necessary for BUBR1 binding to be detectable in vivo and to promote chromosome congression, an effect attributed to BUBR1 (REFS 59,65,66). This raises the possibility that kinetochore-localized BUBR1 is dispensable for the checkpoint and specifically regulates microtubule attachments. BUB protein binding can also be enhanced by KI motifs through a distinct mechanism. In contrast to the MELT-like motifs, KI motifs are conserved only among some vertebrates57, and only two motifs are present in KNL1, although they might be specialized versions of more loosely defined repetitive motifs70. One KI motif (KI1) interacts only with BUB1, whereas the other (KI2) seems to interact only with BUBR1 (REFS 47,61,63,68,72). These motifs enhance BUB protein



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Figure 3 | Checkpoint protein regions and interactions. A | Interacting regions of kinetochore and checkpoint proteins Nature Reviews | Molecular Biology are depicted. Lines between regions indicate established interactions, and phosphoregulated interactions areCell shown in light grey. BUB1‑related protein kinase (BUBR1; mitotic arrest-deficient 3 (Mad3) in yeast) domains vary by organism: the pseudokinase domain is absent in some lineages57. Structures have been determined for interactions of budding uninhibited by benzimidazole 1 (Bub1)–Bub3 and Mad3–Bub3 (REF. 58), BUB1–BUB3–kinetochore null protein 1 (KNL1)51, BUBR1–KNL1 (REF. 72), BUBR1–MAD2 (REFS 32,148) and MAD1–MAD2 (REF. 149). MELT-like ([M/I/L/V]-[E/D]-[M/I/L/V]-T) repeats exhibit poor sequence-level conservation and repeat numbers vary from two in some fungi and plants to 27 in Xenopus tropicalis57,70. Note that a single MELT-like motif interacts with three WD40 domains51. RLK is a MAD1 motif that is required for BUB1 association86, C‑helix is the MAD1 carboxy‑terminal α-helix, CD1 is the ‘conserved domain 1’ of BUB1 (REFS 90,93), and TPR and WD40 are common protein domains. C‑helix, RLK, CD1 and BUB3‑binding regions are all required for the checkpoint. Diagrams are oriented with amino termini on the left. B | Hypothetical BUBR1 kinetochore binding mechanisms. Ba | The KI2 motif of KNL1 interacts directly with BUBR1, possibly facilitating recruitment of a single BUBR1 molecule47,63,70. Bb | BUB1 can also recruit BUBR1, possibly through a BUB1–BUBR1 interaction. BUB3 that is associated with BUBR1 at the kinetochore might therefore be available for other interactions63.

affinity for KNL1 over a MELT-like sequence alone63,70, but they are not strictly required for BUB protein kineto chore recruitment 61. One possibility is that the KI motifs serve as a sensitized switch, recruiting just enough BUB protein to signal the checkpoint without stabilizing microtubule attachments (which is a specific function of BUBR1). This may be the case when kinetochores are attached to microtubules that lack tension and must subsequently be destabilized. In cases in which kinetochores are completely unattached to microtubules, it would be desirable to both activate the checkpoint and stabilize newly formed microtubule attachments, which would be achieved by recruiting a complete complement of BUB proteins. As we discuss below, attached but tensionless

kinetochores seem to elicit a weaker checkpoint response than unattached kinetochores, consistent with the idea that they recruit fewer BUB proteins. KI motifs might therefore serve as modules to activate the checkpoint while preserving maximal error-correction capabilities at the kinetochore in response to a tension defect. MAD1 and MAD2 kinetochore localization. Although BUB1–BUB3 localization is required for the checkpoint, its localization does not always correlate with checkpoint activation. For instance, some BUB1 is retained on early anaphase kinetochores39,44,73, and BUB1 but not MAD1 is present on kinetochores bound to the sides of microtubules, which do not signal the checkpoint 74.



REVIEWS By contrast, kinetochore localization of the MAD1– MAD2 heterotetramer is strictly correlated with checkpoint signalling, indicating that MAD1–MAD2 kinetochore association is the deterministic step in checkpoint activation. This was elegantly demonstrated by tethering MAD1 to kinetochores, which resulted in constitutive checkpoint activation75,76. However, tethering MAD1 to chromosome arms did not activate checkpoint signalling, indicating that at least one additional kinetochore factor is required for activation. Identifying these factors has proved to be a major challenge due to the complexity of the kinetochore and the labile nature of MAD1 association. Recent work showed that the BUB1 protein is a key component in localizing MAD1 to the kinetochore. In budding yeast, Mad1 interacts with Bub1 and requires an RLK motif that is present in Mad1 (REF. 77) (FIG. 3A). Mutating this motif or depleting BUB1 abolished kinetochore localization of MAD1 in budding yeast and Caenorhabditis elegans 78,79. Furthermore, a MAD1‑kinetochore interaction was demonstrated in vitro with kinetochores and was found to require MPS1‑mediated phosphorylation of BUB1 (REF. 78). This phosphorylation is therefore a promising candidate for direct control of checkpoint activation in response to microtubule attachment. Human MAD1 might require an additional receptor because RLK mutation or BUB1 depletion only reduced MAD1 levels at kinetochores by half in HeLa cells80. One candidate for an alternative MAD1 co‑receptor is the Rod–Zwilch–Zw10 complex (RZZ), which is present mainly in metazoans57. KNL1 and its constitutive binding partner Zwint are required to localize RZZ81,82, and RZZ localization may be regulated through Aurora B‑dependent phosphorylation of Zwint83. RZZ is required for stable MAD1 localization in human cells81, and the RZZ-associated protein Spindly interacts with MAD1 by co-immunoprecipitation in C. elegans 84. However, RZZ–Spindly promotes dynein-mediated MAD1 stripping from kinetochores in metazoans, so it is difficult to discern a precise role for this complex in MAD1 recruitment (for a review, see REF. 85). The localization of MAD1 in human cells (as discerned by super-resolution microscopy) is consistent with binding to both BUB1–BUB3 and RZZ, because both complexes localize within a few nanometres of MAD1 (REF. 82). In budding yeast, Mad2 might fulfil this co‑receptor function because it is required for Mad1 localization, in contrast to other organisms42,78,79,86. Kinetochore binding of MAD1–C-MAD2 was thought to be sufficient for O-MAD2 conversion and checkpoint activation87,88, but it now seems that there is an additional, undefined MAD1 requirement. In budding yeast, Mad1 is constitutively associated with Mad2 (REF. 89), whereas the Bub1‑Mad1 interaction is checkpoint specific86. This implies that the Mad1‑Bub1 interaction might be necessary to stimulate Mad2 activation. Several recent studies have confirmed this idea, demonstrating that deletion of the Mad1 carboxyl terminus prevents checkpoint signalling despite correct localization of Bub1 (and presumably Bub3), Mad1 and

Mad2 (REFS 90–92). Furthermore, tethering of C-MAD2 to kinetochores constitutively activates the checkpoint in a MAD1‑dependent manner 91. Both the Mad1 RLK motif and a conserved region of Bub1 are also required for the checkpoint with kinetochore-tethered Mad1 (REFS 90,92,93) . These findings point to the BUB1– MAD1 interaction as the key to activating MAD2 and transducing the checkpoint signal downstream of the kinetochore. Silencing the checkpoint signal at the kinetochore. Silencing the checkpoint signal is crucial for mitotic progression and is regulated by multiple mechanisms, including MCC dissociation and regulation of metazoan MAD2 by the MAD2-binding protein p31Comet. Here we discuss how checkpoint signalling is turned off by removal of checkpoint proteins from the kinetochore (for more detailed reviews, see REFS 85,94–96). Current models imply that loss of MAD1/2 from kineto chores halts MAD2 conversion and MCC formation, thereby extinguishing the checkpoint signal. At least two independent mechanisms lead to MAD1–MAD2 kinetochore dissociation: dynein-mediated stripping of checkpoint proteins and reversal of activating phosphorylation through phosphatase activity (FIG. 4a,b). Dynein localizes to the kinetochore through RZZ–Spindly, and microtubule binding to kinetochores allows dynein to transport RZZ–Spindly to the spindle pole. MAD1– MAD2 and BUBR1 are also removed by dynein during this process, coupling microtubule binding to stripping of kinetochore checkpoint proteins97–100. This elegant mechanism is not universal, however, because dynein does not localize to kinetochores in all eukaryotes. In addition, depletion of Spindly, the dynein receptor, does not completely prevent MAD1–MAD2 removal, suggesting that Spindly might normally supersede an additional conserved removal pathway 98,100. Interestingly, proteins of the centromere-proximal kinetochore subcomplex CCAN (constitutive centromeric-associated network) seem to have a role in opposing dynein stripping because depletion of the CCAN member centro mere protein I (CENP‑I) greatly enhances the rate of MAD1 dissociation101,102. Another possible silencing mechanism is direct displacement of MAD1–MAD2 from kinetochores by microtubule binding, possibly by steric occlusion94 (FIG. 4c). However, MPS1 activity promotes MAD1 kinetochore localization despite microtubule attachment and bi-orientation103,104. Therefore, if this mechanism exists, it must be redundant or suppressed under these conditions. The most widely conserved silencing mechanism is the phosphatase activity of protein phosphatase 1 (PP1; the yeast counterpart of which is Glc7)66,105–109. PP1 localizes to the kinetochore through KNL1, but it may also function through additional proteins, as is the case in fission yeast 107,108,110. Preventing the PP1–KNLl inter action results in constitutive checkpoint activation, and this may be caused by an attachment defect (owing to a failure in counteracting Aurora B‑dependent phosphorylation110) or a checkpoint silencing defect 107,108. PP1 removes BUB1–BUB3 from kinetochores 48,71,



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Figure 4 | Possible spindle checkpoint silencingNature mechanisms the kinetochore. Reviewsat| Molecular Cell Biology Numerous mechanisms seem to be involved in silencing the checkpoint. Removal of mitotic arrest-deficient 1 (MAD1)–MAD2 and inactivation of monopolar spindle 1 (MPS1) might be the key silencing events at the kinetochore. a | In human cells, the motor protein dynein transfers MAD1–MAD2 and the Rod–Zwilch–ZW10 complex (RZZ) from kinetochores to microtubules, trafficking it towards the spindle pole. This is mediated by Spindly, which mediates dynein association with RZZ85. b | Protein phosphatase 1 (PP1) associates with kinetochores and is important for checkpoint silencing71,105–109. PP1 activity promotes removal of budding uninhibited by benzimidazole 1 (BUB1)–BUB3, although it is not known whether it promotes dissociation of BUB1–BUB3 from MAD1–MAD2 (REFS 48,66). Other phosphatases might also be important for silencing. c | Microtubules might silence the checkpoint by physically displacing MAD1 or inducing conformational change at the kinetochore that reduces MAD1 affinity. d | MPS1 activity promotes its own dissociation from the kinetochore57,107. The substrates that mediate removal are unknown. It is also unknown what drives MPS1 (re)association with kinetochores, although Aurora B and checkpoint kinase 2 (CHK2) might be major factors103,115–118,150,151. KNL1, kinetochore null protein 1.

thereby promoting checkpoint silencing upstream of MAD1–MAD2 localization. PP1 might also target other checkpoint substrates. Following silencing of the kinetochore-dependent checkpoint signal, the phosphatase CDC14 has a critical role in maintaining checkpoint silencing during mitotic exit 111,112. Control of MPS1 localization might also be a key step to silencing the kinetochore signalling cascade because the level of MPS1 in the kinetochore is substantially reduced by the time of anaphase onset 39,113 (FIG. 4d). Although the MPS1 recruitment mechanism remains incompletely defined, MPS1 turns over at kinetochores, as determined by FRAP39, and its kinase activity promotes its own dissociation from kinetochores103,114.

To retain checkpoint-signalling function, MPS1 must logically be re‑recruited to the kinetochore. This re‑recruitment might be a major regulatory step and is likely to be mediated by Aurora B103,115–118. After anaphase onset, degradation of the checkpoint proteins MPS1 and BUB1 prevents checkpoint reactivation113,120. Additional checkpoint silencing mechanisms may exist, as KNL1 seems to have a role in checkpoint silencing that is independent of PP1 activity or dynein stripping119. The checkpoint and the nuclear pore. Whereas kinetochores regulate the checkpoint throughout mitosis, the kinetochore does not seem to be necessary for all checkpoint signalling activity. For instance, although most cells do not assemble kinetochores outside mitosis, they nonetheless harbour MCCs before mitotic entry, raising the possibility that kinetochores are not the only catalyst for MCC production. Additionally, MAD1 localizes to the nuclear pore complex (NPC), suggesting that these large protein assemblies act as alternative, pre-mitotic (interphase) checkpoint signalling scaffolds. A new study has validated this hypothesis121, demonstrating that MAD1 localization to the NPC is required for a characteristic checkpoint-dependent delay in mitosis known as the ‘mitotic timer’ (REFS 121–124). Because BUB1–BUB3 has not been reported to localize to the NPC, it remains unclear whether NPCs use the same MAD2 activation mechanism as kinetochores or whether NPCs continuously activate MAD2 until they are disassembled at nuclear envelope breakdown. Cells defective in MAD1 NPC targeting exhibit increased levels of lagging chromosomes at anaphase, indicative of merotelic attachments persisting through mitosis despite a functional kinetochore checkpoint 121. Therefore, it seems that NPC-localized MAD1–MAD2 assists the kinetochorebased mechanisms in promoting correct segregation. Presumably, this occurs by facilitating MCC production before mitotic entry, when kinetochores assemble, and by allowing more time for chromosomes to bi-orient 121. In addition to the mitotic timer, NPCs might have additional checkpoint-related functions. The conserved human nuclear-pore protein translocated promoter region (TPR) helps to stabilize the checkpoint proteins MAD1 and MAD2. Consistently, TPR depletion has a moderate effect on the checkpoint response125. MAD1 also cycles between kinetochores and NPCs in checkpoint-active budding yeast, which retain their nuclear envelope during mitosis104. In these cells, NPC-localized MAD1 is proposed to facilitate checkpoint activity by blocking nuclear import of the checkpoint-silencing phosphatase PP1 (REF. 104; for a review, see REF. 126).

Checkpoint response strength and timing It is imperative to elucidate how the checkpoint functions at its limits to understand why signalling failures lead to mis-segregation and aneuploidy in a natural context. Because cancer cells are typically aneuploid and highly dependent on checkpoint function, this might shed light on ways to target the checkpoint for chemotherapy (for reviews, see REFS 127,128). Classic studies established that both attachment and tension defects



REVIEWS can lead to checkpoint arrest, but these defects had not been systematically analysed for their molecular output until recently. New studies have expanded on previous work by analysing the timing from initial signalling to the stabilization of APC/C targets. Variation in checkpoint signalling strength. A single unattached kinetochore can halt the cell cycle20, so it seems that a minimal attachment defect is capable of a full checkpoint response. A logical conclusion is that the checkpoint response is binary — either completely on or off. However, hints that more severe attachment defects result in a stronger checkpoint have emerged over time. For example, it was observed that cells can undergo ‘mitotic slippage’ — a bypass of cell-cycle arrest despite an active checkpoint — and that this can happen faster under certain circumstances21,22. Recent reports have quantified variations in the strength of the checkpoint response23,24,129 (FIG. 5a). Checkpoint strength was analysed using low doses of nocodazole, a drug that destabilizes microtubules and generates unattached kinetochores; a correlation was found between high numbers of unattached kinetochores (as measured by chromosomes displaced from the metaphase plate) and slow degradation rates of securin, an APC/C substrate, indicating enhanced checkpoint function23. Another study measured the destruction of cyclin A, which competes with checkpoint proteins for binding to CDC20 and subsequent ubiquitylation and degradation24,130. The study used cyclin A degradation as a quantitative readout for checkpoint activity, and it found that different spindle poisons activate the checkpoint to different degrees. Paclitaxel (Taxol; Bristol-Myers-Squibb), a microtubulestabilizing drug that activates the checkpoint by generating tension defects, gave a significantly weaker response than nocodazole. Furthermore, checkpoint activity correlated with the amount of MAD2 localized to kineto chores, indicating the difference in duration is due to upstream signalling events at the kinetochore24. Remarkably, the amount of MAD2 that localized to individual kinetochores, and not just the number of kinetochores that bind to MAD2, was increased with greater microtubule destabilization24. This suggests that individual kinetochores can vary the strength of the checkpoint signal they generate. As discussed above, the BUB proteins have multiple binding sites at each kinetochore that could lead to variation in the checkpoint response, and the number of BUB proteins at the kinetochore is likely to correlate with the strength of the checkpoint signal63,70,71. The variation in BUB protein numbers could be caused by changes in upstream MPS1 activity (FIG. 5b). This is particularly likely in the case of paclitaxel treatment because tensionless kinetochores might have a distinct or partial mechanism to activate the checkpoint relative to unattached kinetochores (for a review, see REF. 4). Different microtubulebinding modes of the kinetochore could conceivably trigger this variation, as microtubule binding can occur through the lattice (side) or end of a microtubule, and there are multiple microtubule-binding elements within the kinetochore131,132. In any case, increased amounts of

active CDC20 may be the effector for reduced checkpoint efficiency. Stronger checkpoint response was correlated with greater levels of MCC-associated CDC20, indicating that more total CDC20 was inactivated24. Furthermore, altering expression levels of CDC20 and MAD2 by promoter swapping indicated that the ratio of CDC20 to MAD2 in the cell must be carefully regulated for proper checkpoint function129. It is currently unclear why the checkpoint response varies depending on the spindle defect, although it is reasonable for cells to risk undergoing segregation eventually because extended mitotic arrest can lead to inviability. Cells may therefore balance the risk of missegregation with that of extended arrest by modulating the strength of the checkpoint response. However, a weak checkpoint response is likely to be elicited early in mitosis, when kinetochores are newly formed (having had little time to accumulate MCCs) and microtubule attachments are present but not necessarily correct (leading to weak signalling). Therefore, cells may buffer this weak response by accumulating interphase MCCs through the mitotic timer 121. This proposal is supported by the observations that mis-localizing MAD1 from nuclear pores sensitizes cells to weakened kinetochore signalling by Aurora B inhibition121, and that weak checkpoint activation is correlated with reduced levels of intact MCCs24. Thus, cells might prepare for weak signalling at mitotic onset, but become more sensitive to mitotic slippage as the risks associated with prolonged arrest increase. Checkpoint kinetics and the anaphase response. A critical feature of the checkpoint is that it must respond to lowtension kinetochores until metaphase, but this must not continue through anaphase, when sister chromosomes are no longer held together by cohesin and kineto chores are therefore under little tension (FIG. 1). This is the so‑called ‘anaphase problem’, and although multiple factors are involved in overcoming it (for a review, see REF. 112), we focus here on recent studies that relate to the checkpoint kinetics and activity of CDK1 kinase, the mitotic master regulator. Silencing during anaphase is mediated by phosphatase activity to reverse mitotic phosphorylation71,105–108,110 (for a review, see REF. 133) in conjunction with proteolysis of checkpoint proteins upon mitotic exit113,120 (for a review, see REF. 134). For instance, forced expression of MPS1 in an anaphase arrest was sufficient to induce checkpoint signalling 113, suggesting that the checkpoint must stay silenced throughout anaphase. Additionally, the onset of cyclin B degradation, which indicates checkpoint silencing, correlates precisely with the completion of chromosome congression135. Recent temporal analysis of checkpoint reactivation has helped to clarify the anaphase checkpoint silencing process further. In vivo laser ablation of microtubules in metaphase human cells revealed that checkpoint signalling, measured by ensuing MAD2 recruitment, was intact throughout metaphase23. However, a subset of cells initiated anaphase within a brief window following MAD2 kinetochore recruitment, suggesting that cells already prepared for



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Nature Reviews | Molecularactivity. Cell Biology Figure 5 | The graded checkpoint response. a | Various conditions result in different degrees of checkpoint Weak checkpoint activation results from minor defects, such as a lack of tension caused by paclitaxel (Taxol; Bristol-MyersSquibb) treatment, which eliminates microtubule dynamics, or dimethylenastron (DMA) treatment, which collapses the mitotic spindle while preserving microtubule attachments. A weak checkpoint results in reduced mitotic duration (mitotic slippage), faster cyclin B degradation and low levels of mitotic arrest-deficient 2 (MAD2) at kinetochores. Complete disruption of microtubules results in a strong checkpoint response with an increased length of mitosis, slower cyclin B degradation and increased levels of MAD2 at kinetochores24. The rate of securin degradation — a readout for the activity of the APC/C (anaphase-promoting complex, also known as the cyclosome), and therefore checkpoint inactivity — correlates inversely with the number of unattached kinetochores23. b | Hypothetical model for modulation of checkpoint strength through monopolar spindle protein 1 (MPS1) activity. More-severe perturbations can enhance or stabilize MPS1‑mediated phosphorylation of its target proteins, which include Bub1 and the KNL1 homologue in yeast. This in turn leads to greater kinetochore localization of MAD2 and a stronger checkpoint response, indicated by higher levels of mitotic checkpoint complex (MCC)24 and less free cell division control protein 20 (CDC20). Titration of MPS1 activity by inhibitor addition to checkpoint-arrested cells resulted in a proportionally attenuated checkpoint response, similarly to microtubule-directed drug treatments24. Activity of the opposing phosphatase protein phosphatase 1 (PP1) may also be responsive to microtubule attachment. KNL1, kinetochore null protein 1.

anaphase could not arrest. Consistent with this, the rate of securin degradation (indicating APC/C activity and an incomplete checkpoint response) was only slightly reduced at 0–5 minutes after checkpoint protein kinetochore recruitment, whereas it was reduced by up to 60‑fold between 5 and 10 minutes after kinetochore recruitment23. Similar kinetics were also observed in fission yeast 25 and in earlier studies that analysed anaphase onset following the completion of chromosome congression38,136. Thus, a temporal gap exists between checkpoint protein recruitment and APC/C inactivation, meaning the checkpoint is functionally off during this time.

Several additional studies have analysed the anaphase response further by using non-degradable cyclin B, the cofactor that activates CDK1 towards its mitotic targets, in fission yeast 25, mice27 and human cells26,28. Stabilizing cyclin B permitted securin degradation and sister chromatid separation with a corresponding loss of kineto chore tension 25–28. Nonetheless, stabilized cyclin B allowed checkpoint proteins to relocalize to kinetochores with the onset of anaphase, as had been previously obs erved25,26,28,29,111,134,137. This indicated that loss of tension at anaphase does indeed promote checkpoint signalling, but this response is normally repressed by CDK1



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Figure 6 | Control of the timing of checkpoint signalling. Interphase: before mitosis, nuclear pore complexes (NPCs) Nature Reviewsinhibitor | Molecular CellasBiology promote production of the APC/C (anaphase-promoting complex, also known as the cyclosome) known the mitotic checkpoint complex (MCC). In a similar way to kinetochores, NPCs promote MCC production through mitotic arrest-deficient 1 (MAD1)–MAD2, although any potential upstream activation signals are unknown121. Prophase/ prometaphase: in most cell types, kinetochore–microtubule attachments are established following nuclear envelope breakdown. Kinetochores, initially unattached, signal the checkpoint and thereby inhibit the APC/C. Metaphase: upon bi-orientation at metaphase, kinetochore checkpoint signalling is silenced, leading to APC/C activity and the beginning of cyclin B degradation (arrow). Early anaphase: at anaphase, attached kinetochores are no longer under tension. Rapid inactivation of cyclin-dependent kinase 1 (CDK1)–cyclin B by the APC/C prevents these unattached kinetochores from signalling the checkpoint25–27,112. Late anaphase: following segregation, APC/C activity destroys checkpoint proteins, including budding uninhibited by benzimidazole 1 (BUB1) and monopolar spindle protein 1 (MPS1), fully silencing checkpoint control over the APC/C113,120,134. INM, inner nuclear membrane; ONM, outer nuclear membrane.

inactivation. Because CDK1 activity is required for the checkpoint (for a review, see REF. 9), its silencing by cyclin B degradation ensures that the checkpoint is not reactivated26–28,129. It was further demonstrated that an anaphase checkpoint response is too slow to inhibit the APC/C, and this slow response might serve as a failsafe mechanism to prevent interruption of anaphase by checkpoint activation25,28. Therefore, transient satis faction of the checkpoint is sufficient to activate the APC/C, and the fast kinetics of APC/C degradation of securin and cyclin B preclude functional checkpoint reactivation during anaphase (FIG. 6).

Conclusions and perspectives Substantial advances have recently been made in understanding how the checkpoint operates, including the identification and structural analyses of precise protein interactions between core kinetochore components and checkpoint proteins, the reconstitution of checkpoint activation steps in vitro, the characterization of NPCs as the source of the mitotic timer, and the description of the checkpoint as a graded and kinetically limited response. Given the complexity of the kinetochore, reconstituting in vitro checkpoint signalling events that occur around this complex structure has been a major obstacle. As we describe, biochemical reconstitution of BUB and MAD protein binding to core kinetochore proteins has now been achieved, and it may soon be possible to entirely reconstitute the checkpoint in vitro to fully understand the signalling cascade. As discussed above, it seems that the canonical model for MAD1–MAD2 activation is

incomplete90–92, and it will therefore be critical to determine what additional event (or events) is required for formation of the MCC downstream of MAD1 recruitment. Advances in reconstitution will also permit structural studies that will be crucial for gaining a detailed understanding of checkpoint mechanisms. Cumulatively, recent work has established that MPS1 is the central checkpoint kinase at both kinetochores and NPCs121,122. The identification of NPCs as a mitotic timer raises new questions about whether this timing mechanism is regulated and precisely why chromosome mis-segregation is increased in its absence. It is possible that initial kinetochore signalling is too weak or slow to delay the cell cycle in response to moderate kinetochore– microtubule attachment defects without pre-formed MCCs derived from NPCs121. Because BUB1–BUB3 has not been localized to the nuclear pore, an intriguing possibility is that the RAE1–NUP98 nuclear pore subcomplex functions analogously to BUB1–BUB3 in activating MAD1–MAD2. Not only does RAE1 bear a striking similarity to BUB3 in terms of structure51,58, RAE1–NUP98 is also necessary, like BUB1–BUB3, for accurate segregation and securin stabilization138. Regarding kinetochore checkpoint activation, a critical future question is what controls the kinase cascade. Ultimately, this activation signal must be opposed by tension-bearing microtubule attachments. MPS1 activity towards BUB1 might be specifically regulated because BUB1 and MAD1 kinetochore localization are separable (see, for example, REF. 74), and this might be the critical step controlled by microtubule attachment or tension.



REVIEWS When tethered to a kinetochore, MPS1 can recruit checkpoint proteins and activate the checkpoint at the same time as preserving bi-oriented attachments62,103,104, implying that checkpoint silencing requires either the removal or inactivation of kinetochore-bound MPS1. Although PP1 opposes MPS1 activity 48,71, phosphatase activity does not seem to be controlled by tension10, suggesting that another mechanism might regulate MPS1 activity towards BUB1. MPS1 actively cycles at kinetochores and promotes its own dissociation, so one possibility is that its re‑recruitment could be controlled by microtubule attachments. Determining exactly how MPS1 output is connected to kinetochore–microtubule attachment is a major challenge for the future. Additional important questions to address are the underlying mechanism of BUBR1 kinetochore recruitment and the precise details of MCC formation. The demonstration that the kinetochore response is variable in strength and is kinetically limited raises new questions about aneuploidy and cancer. Weak activation and slow kinetics can possibly circumvent the checkpoint and lead to aneuploidy. It will be informative to learn how frequently mis-segregation results from mitotic slippage and late-arising attachment errors. Moreover, the sensitivity of the checkpoint to relative 1. 2. 3. 4.

5. 6.

7.

8. 9.

10.

11.

12. 13.

Gordon, D. J., Resio, B. & Pellman, D. Causes and consequences of aneuploidy in cancer. Nature Rev. Genet. 13, 189–203 (2012). Ricke, R. M. & van Deursen, J. M. Aneuploidy in health, disease, and aging. J. Cell Biol. 201, 11–21 (2013). McIntosh, J. R. Motors or dynamics: What really moves chromosomes? Nature Cell Biol. 14, 1234–1234 (2012). Maresca, T. J. & Salmon, E. D. Welcome to a new kind of tension: translating kinetochore mechanics into a wait-anaphase signal. J. Cell Sci. 123, 825–835 (2010). Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517 (1991). Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519–531 (1991). References 5 and 6 established the existence of the spindle checkpoint and identified upstream checkpoint signalling genes through genetic screens. Nicklas, R. B., Ward, S. C. & Gorbsky, G. J. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Biol. 130, 929–939 (1995). Minshull, J., S. H., Tonks, N. K. & Murray, A. W. A. MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. 79, 475–486 (1994). Funabiki, H. & Wynne, D. J. Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122, 135–158 (2013). Li, X. & Nicklas, R. B. Tension-sensitive kinetochore phosphorylation and the chromosome distribution checkpoint in praying mantid spermatocytes. J. Cell Sci. 110, 537–545 (1997). Nicklas, R. B., Campbell, M. S., Ward, S. C. & Gorbsky, G. J. Tension-sensitive kinetochore phosphorylation in vitro. J. Cell Sci. 111, 3189–3196 (1998). This classic study used innovative biophysical methods to conclusively demonstrate a role for tension in checkpoint satisfaction. Gorbsky, G. J. & Ricketts, W. A. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J. Cell Biol. 122, 1311–1321 (1993). Maresca, T. J. & Salmon, E. D. Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity. J. Cell Biol. 184, 373–381 (2009).

checkpoint-protein abundance129 suggests that aneuploid cancer cells can evade the checkpoint in part through moderate perturbation of checkpoint-protein levels (for a review, see REF. 139). A better understanding of quantitative protein ratios might therefore assist in targeting therapeutics to cancer cells. Additional knowledge about the graded checkpoint response will also entail identifying the underlying mechanisms that account for variability. Because MAD2 levels at individual kinetochores vary with the attachment defect 24, upstream events are likely to tune the checkpoint response. In the coming years, further advances are likely to be made in our understanding of activation mechanisms, and one of the most important advances will be discovering the long-sought biochemical and structural basis for converting defects in forces at kinetochores into a phosphorylation signalling cascade. Structural studies will also inform how the BUB and MAD proteins interact at successive steps in the checkpoint. Advances in areas such as checkpoint silencing and the kinetics of individual checkpoint steps are also likely to be made in the near future, and will enhance our understanding of the dynamic limits of the checkpoint. Such work will lead to a greater understanding of genome stability and will improve our ability to target this crucial pathway for the treatment of cancer.

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Acknowledgements

The authors are grateful to the reviewers and M. Miller for thoughtful comments on the manuscript and apologize to those who were not cited owing to space limitations. N.L. was supported by a US National Institutes of Health (NIH) centre interdisciplinary training grant (T32 CA080416), and work in the Biggins laboratory is supported by NIH grants GM064386 and GM078069 to S.B.

Competing interests statement

The authors declare no competing interests.


The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling.

Mad1 contribution to spindle assembly checkpoint signalling goes beyond presenting Mad2 at kinetochores.

Slipping past the spindle assembly checkpoint.

A role for the spindle assembly checkpoint in the DNA damage response.

DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

The spindle checkpoint and chromosome segregation in meiosis.

Spindle assembly checkpoint silencing and beyond.

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

How the kinetochore switches off the spindle assembly checkpoint.

The spindle assembly checkpoint: More than just keeping track of the spindle.

TRAIP is a regulator of the spindle assembly checkpoint.

Spindle assembly checkpoint acquisition at the mid-blastula transition.

Complex Commingling: Nucleoporins and the Spindle Assembly Checkpoint.

The spindle assembly checkpoint: progress and persistent puzzles.

Sustaining the spindle assembly checkpoint to improve cancer therapy.

Regulation of mitotic progression by the spindle assembly checkpoint.

The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis.

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint.

Cenp-meta is required for sustained spindle checkpoint.

Signalling dynamics in vertebrate segmentation.

Formation of multiprotein assemblies in the nucleus: the spindle assembly checkpoint.

A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions.