Regulation of APC Cdc20 by the spindle checkpoint Hongtao Yu

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1 706 Regulation of AP by the spindle checkpoint Hongtao Yu The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphasepromoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, and directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex. Addresses Department of Pharmacology, University of Texas Southwestern Medical enter, 5323 Harry Hines Boulevard, Dallas, TX , USA; hongtao.yu@utsouthwestern.edu urrent Opinion in ell Biology 2002, 14: /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. Published online 1 October 2002 Abbreviations AP anaphase-promoting complex M mitotic checkpoint complex Introduction During the eukaryotic cell division cycle, cells first replicate their DNA in S phase, package the DNA into sister chromatids in mitosis, and then segregate the chromosomes evenly into daughter cells [1]. The accurate separation of sister chromatids relies on the delicate balance of two opposing processes: the cohesion between sister chromatids and the pulling force at the chromosomes exerted by the mitotic spindle (Figure 1). The cohesion of the sister chromatids is established by the cohesin protein complex during DNA replication and persists until chromosome segregation [1]. In mitosis, the sister chromatids attach to the mitotic spindle at kinetochores that consist of protein complexes associated with centromeric DNA. It is vital for the sister chromatids to attach to microtubules emanating from the two opposite poles of the mitotic spindle (bi-orientation), thus establishing tension across the two kinetochores of a sister chromatid pair [1,2]. After all sister chromatids have achieved bi-orientation and their kinetochores are under tension, a large ubiquitin ligase called the anaphase-promoting complex (AP) or cyclosome, in association with one of its substrate-binding co-factors,, tags the securin protein with polyubiquitin chains [3]. Degradation of the ubiquitinated securin in turn activates the separase. Proteolytic cleavage of a subunit of the cohesin complex, Scc1, by separase destroys the cohesion between the sister chromatids and triggers the onset of anaphase. The spindle then pulls the two sets of chromatids toward opposite poles of the cell. As a result, each daughter cell receives identical genetic information. Therefore, AP -mediated degradation of securin indirectly causes the loss of chromosome cohesion and initiates chromosome segregation [1,3]. Premature separation of sister chromatids leads to the loss or gain of chromosomes in daughter cells (aneuploidy), a prevalent form of genetic instability of human cancer [4]. hromosome mis-segregation in human female meiosis leads to severe birth defects [5]. To avoid these disastrous consequences, cells employ a surveillance mechanism called the spindle checkpoint to ensure the high-fidelity transmission of their genetic material [6 9]. Not surprisingly, AP is a molecular target of the spindle checkpoint [6 9]. Inhibition of AP by the checkpoint stabilises securin and prevents the separation of sister chromatids until the proper attachment of all kinetochores to the spindle [6 9]. The molecular components of the spindle checkpoint were identified initially in S. cerevisiae [6 9]. Homologues of these checkpoint proteins were later found in other organisms, including mammals (Table 1). These include Mad1,, Mad3/BubR1, Bub1, Bub2, Bub3 and Mps1. With the exception of Bub2, these proteins form a complex intracellular signalling network to block the action of AP (Figure 1). In budding yeast, Bub2 does not appear to be involved in the regulation of AP and chromosome segregation [10]. Instead, it negatively regulate the functions of the mitotic exit network (MEN), leading to inhibition of AP dh1, which mediates the degradation of mitotic cyclins and other important cell cycle regulators [10]. Although several members of mammalian MEN have been identified, no functional homologues of Bub2 have been found in vertebrates. In this review, I will discuss recent advances in our understanding of the checkpoint-mediated inhibition of AP in response to spindle damage. Sensing the spindle defect: microtubule attachment and tension A cell cycle checkpoint typically consists of three essential elements: the sensors that monitor defects; the signal transducers; and the targets or effectors. AP is a critical target of the spindle checkpoint and some of the Mad and Bub proteins are involved in transducing the signals in this system. Although the sensors that monitor the spindle defect have not been established, the spindle defects that activate the spindle checkpoint are better understood [6]. Elegant experiments in mammalian cells unequivocally demonstrated that the spindle checkpoint senses the existence of unattached kinetochores [11]. It is also

2 The spindle checkpoint Yu 707 clear that the checkpoint responds to the absence of microtubule occupancy at the unattached kinetochores [12 ]. What is less clear is whether the checkpoint also senses the consequent lack of tension at these sites because of the absence of the spindle pulling force. Lack of tension at the kinetochores activates the spindle checkpoint during meiosis of insect spermatocytes [13,14]. Unfortunately, a single kinetochore in insect and mammalian cells contains multiple microtubule-binding sites (on average, 30 for a mammalian kinetochore) and tension between the kinetochores of a bi-oriented pair of sister chromatids increases the number of attached microtubules [15]. Owing to this interdependency of tension and microtubule occupancy, it is difficult to separate their individual contributions to checkpoint activation in metazoan cells. In fact, there are conflicting reports regarding the role of tension in checkpoint activation in mammalian cells [12,16,17,18,19 ]. Figure 1 ENP-E BubR1(Mad3) Bub3 Lack of occupancy tension at kinetochores Aurora B/Ipl1 Rod Zw10?? Mps1 Bub1 Bub3 Mad1 P To resolve this controversy, Murray and co-workers [20,21 ] took advantage of the fact that each kinetochore in budding yeast binds a single microtubule, thus eliminating the possibility of partial microtubule occupation at a given kinetochore. Using yeast mutants defective in meiotic recombination or DNA replication, they showed that microtubule-attached kinetochores not under tension activate the spindle checkpoint during meiosis and mitosis in budding yeast [20,21 ]. Recently, Tanaka et al. [2,22 ] showed that the yeast Aurora-like kinase Ipl1 is required for establishing bi-orientation and tension of sister chromatids. Interestingly, Ipl1 is also involved in the spindle checkpoint [23,24 ]. In particular, Ipl1 might sense the lack of tension at kinetochores during checkpoint signalling [23,24 ]. It is intriguing that the same protein positively required for the bi-orientation process is also responsible for monitoring the status of bi-orientation at a later stage of the cell cycle [23]. These findings lend strong support to the notion that lack of tension at kinetochores is sensed by the spindle checkpoint in budding yeast. Very recently, Aurora B (the mammalian homologue of Ipl1) has been shown to be required for bipolar attachment of chromosomes and for the proper function of the spindle checkpoint in mammalian cells, although the exact role of Aurora B in the checkpoint is not known [25,26,27]. onsidering the remarkable conservation of the underlying mechanisms of many cell cycle events between yeast and mammals, the spindle checkpoint in metazoans may also monitor tension at the kinetochores during mitosis and meiosis. Finally, tension sensing is the only possible way to detect and correct the harmful situation of mono-oriented sister chromatids with kinetochores attached to the same spindle pole in mitosis. It makes sense that the spindle checkpoint monitors both microtubule occupancy and tension at the kinetochores. It has become increasingly clear that the spindle checkpoint senses the lack of tension at the kinetochores. The tension-sensing mechanism may involve the kinesinlike motor ENP-E [28]. Moreover, the lack of tension BubR1 Bub3 AP Securin Ub Ub Ub Separase ohe M Degradation by the proteasome Sister-chromatid separation urrent Opinion in ell Biology The spindle checkpoint pathway. The spindle checkpoint is activated by the lack of microtubule occupancy and tension at the kinetochores. The defect-sensing mechanism of the checkpoint is unknown. However, it may involve Aurora B/Ipl1 and ENP-E. The functions of Rod and Zw10 are unclear and they are tentatively placed at the top of the pathway. ENP-E interacts with BubR1 physically, but it is unknown whether and how it affects the function of BubR1. In budding yeast, Aurora B/Ipl1 may act upstream of Mps1 in sensing tension. Whether ENP-E or Aurora B/Ipl1 regulates Bub1 Bub3 has not been established. In budding yeast, Mps1 phosphorylates Mad1. Similarly, human Bub1 phosphorylates human Mad1 in vitro. Thus, Mps1 and Bub1 Bub3 may lie upstream of Mad1. Upon checkpoint activation, both BubR1 Bub3 and interact with directly and inhibit its ability to activate AP. The transient interaction between BubR1 Bub3 and complexes leads to the formation of the BubR1 Bub3 complex (the M), which is a more efficient inhibitor of AP. When all kinetochores achieve bipolar attachment to the mitotic spindle, the checkpoint is inactivated. The active AP then ubiquitinates securin. Degradation of securin allows the activation of separase, which cleaves Scc1, a subunit of the cohesin protein complex. Loss of cohesion of sister chromatids triggers chromosome segregation and the onset of anaphase. Ub, ubiquitin. sin

3 708 ell division, growth and death Table 1 Molecular components of the spindle checkpoint. Veterbrates S. cerevisiae S. pombe Kinetochore localisation Domains and motifs Interactions and functions Mad1 Mad1 Mad1 Yes oiled-coil Binds to and recruits to Kinetochores; phosphorylated by Mps1 and Bub1 in vitro. Yes HORMA Binds to and inhibits AP; binds to Mad1 BubR1 Mad3 Mad3 Yes TPR*; GLEBS; kinase Binds to Bub3 and ; inhibits AP in vitro; binds to the mitotic motor ENP-E Bub1 Bub1 Bub1 Yes TPR*; GLEBS; kinase Binds to Bub3; binds to Mad1 and in S. cerevisiae; phosphorylates human Mad1 in vitro Bub2 cdc16 GAP Negatively regulates Tem1 and the mitotic exit network in S. cerevisiae Bub3 Bub3 Bub3 Yes WD-40 Binds to Bub1 and BubR1(Mad3) Mps1 Mps1 mph1 Yes TPR*; kinase Phosphorylates Mad1 in S. cerevisiae Aurora B Ipl1 ark1 Yes Kinase Binds to INENP/Sli15/Pic1; promotes bi-orientation Rod Yes Binds to Zw10 Zw10 Yes Binds to Rod and Zwint-1 (a coiled-coil protein) dc55 ASBD Regulatory B subunit of PP2A; regulates Swe1 degradation Slp1 Yes -box; D-box; Binds to, BubR1(Mad3), Emi1, AP, and AP substrates; KEN-box; WD-40 activates AP; ubiquitinated by AP-dh1; phosphorylated by dk1 in mammals *The amino-terminal 150 residues of Bub1, BubR1, Mad3 and Mps1 from all organisms share low sequence homology and contain structural motifs similar to the tetratrico peptide repeats (TPRs) (H Yu, unpublished data). The kinase domain of BubR1 lacks key catalytic residues and may not be a functional kinase. may also activate a specific subset of checkpoint proteins, such as Ipl1/Aurora B and Mps1 [24 ]. It is not known how these proteins communicate with the downstream components of the checkpoint. Spindle-damaging agents, such as nocodazole and benomyl, destroy the mitotic spindle and leave all kinetochores unoccupied, and can thus be used to mimic extreme cases of spindle defects. Under these conditions, yeast mutants deleted for any of the checkpoint genes singly fail to arrest in mitosis [29,30]. Mouse cells deleted for the genes encoding or Bub3 or mammalian cells treated with Mad1 short interfering RNA (sirna) also escape from the mitotic arrest in the presence of nocodazole [31,32,33 ]. Thus, the proper function of every single spindle checkpoint gene appears to be essential for establishing and maintaining a mitotic arrest in cases of severe spindle damage. The spindle checkpoint is also required for the fidelity of chromosome segregation during the normal cell cycle. After all, unattached kinetochores and kinetochores not under tension exist during the pro-metaphase of every single cell-division cycle, which are expected to activate the checkpoint. onsistent with this notion, both mad2- null budding yeast and mammalian cells show higher frequency of chromosome mis-segregation in the absence of spindle-disrupting agents [29,31]. Establishing the checkpoint: the diffusible wait anaphase signal A single unattached kinetochore within a cell is sufficient to activate the spindle checkpoint; therefore, it must produce an inhibitory signal that diffuses away to block the activity of the AP throughout the cell. Recent genetic, biochemical, cell biological and structural studies have begun to shed light on the nature of this elusive diffusible checkpoint signal that inhibits AP. urrently, there are two related, yet different, views of how the checkpoint regulates AP. In both models, the AP is inhibited through direct stoichiometric binding of the mitotic checkpoint complex (M), which contains BubR1 (Mad3 in yeast), Bub3, and or the BubR1 Bub3 and sub-complexes [34 36,37,38,39,40 ]. The two views differ in the mechanism of M assembly. In the first model (Figure 2), the unattached kinetochores catalyse the formation of these inhibitory checkpoint protein complexes, which then diffuse away to inhibit the AP. The diffusible signal is thus the M or its sub-complexes, such as and BubR1 Bub3. Different diffusible signals may be generated, depending on the types of spindle damage [16,18]. In the second model, the M is present throughout the cell cycle and its formation does not occur at the kinetochores. Upon checkpoint activation, a yet unidentified diffusible signal turns over at the kinetochores and sensitises the AP for its prolonged inhibition by the M. I next discuss the evidence supporting the first model and try to reconcile the differences between the two models. onsistent with the essential role of kinetochores in generating the inhibitory checkpoint signals, the known vertebrate checkpoint proteins, including Mad1,, Bub1, BubR1, Bub3, Mps1, Zw10 and Rod, localise to

4 Unattached kinetochore Attached kinetochore The spindle checkpoint Yu 709 Figure 2 Bub3 Bub1 + Bub3 Bub1 Mad1 Mad1 ENP-E P Mad1 Mad1 P Bub3 BubR1 ENP-E Microtubule Bub3 BubR1 Bub3 BubR1 AP M? AP + Bub3 BubR1 + Inactive Active Prometaphase Metaphase Anaphase urrent Opinion in ell Biology A model for the formation of AP-inhibitory complexes at the kinetochores. Nearly all known checkpoint proteins are enriched at the unattached kinetochores. In this model, is recruited to the kinetochore by Mad1, and is brought to the kinetochore by BubR1 Bub3. Mad1 also triggers a conformational change of. The mechanism by which dissociate from Mad1 remains to be determined. Phosphorylation of Mad1 by Bub1 or Mps1 might play a role in this process. dissociated from Mad1 might retain a conformation more suitable for binding to that is already bound to BubR1 Bub3, resulting in the formation of the M. This mitotic checkpoint complex, or its sub-complexes, might then diffuse away from the kinetochores to associate with AP and block it activity. When the kinetochores are captured by microtubules, Mad1 and no longer localise to kinetochores. The concentrations of BubR1 Bub3 and other checkpoint proteins at the kinetochores also decrease. M ceases to form. The break-up of M leads to the activation of AP, degradation of securin, and the separation of sister chromatids. kinetochores in mitosis [18,28,41,42,43,44,45 ]. More interestingly, some of the checkpoint proteins, such as Mad1 and, only localise to unattached, untense kinetochores [12,41]. The concentrations of Bub1, BubR1 and Bub3 at the kinetochores also decrease after microtubule attachment and the establishment of kinetochore tension [12,16,18,19 ]. These findings, along with the fact that (the target of checkpoint inhibition) is also enriched at kinetochores, suggest that the checkpoint complexes might be assembled at the unattached kinetochores [34,46]. Furthermore, as predicted by the first model, and several checkpoint proteins, including and BubR1, turn over rapidly at the kinetochores in mammalian cells ([47]; ED Salmon, personal communication). The co-localisation of many checkpoint proteins at the kinetochores also suggests that these proteins interact with each other physically. Indeed, numerous checkpoint protein complexes have been detected in yeast and vertebrates. Among them, the Mad1, Bub1 Bub3 and BubR1(Mad3) Bub3 complexes are constitutive and exist throughout the cell cycle [35,38,48,49]. Upon

5 710 ell division, growth and death Figure 3 Apo Ligand N The conformational change of upon binding to or Mad1. The top panel shows the ribbon diagram of the apo- and the ligand complexes. The bottom panel is a schematic drawing of the rearrangement of the secondary structural elements upon - or Mad1-binding N B A N 1 or Mad1 B A β β β β αb αa α αb αa α N β7 β β β β β1 11 N 173 β7 169 β1 or Mad1 157 β6 149 N 82 β β β β8 182 urrent Opinion in ell Biology checkpoint activation, there are higher concentrations of and BubR1 Bub3 complexes, leading to the formation of the larger M complex BubR1(Mad3) Bub3 [38,39,40 ]. In yeast, a Bub1 Bub3 Mad1 complex is also observed in checkpoint-activated cells [49]. The model shown in Figure 2 is further supported by a plethora of biochemical and structural data. and BubR1 directly bind to and inhibit the ligase activity of the AP collaboratively [34,38,40 ]. The purified M from HeLa cells inhibits the AP much more efficiently than alone [39 ]. A ternary complex containing AP, and exists in nocodazolearrested HeLa cell lysates [34]. BubR1 has also been shown to interact physically with the AP [39 ]. Therefore, the M or its subcomplexes bind to the AP directly in response to spindle damage. Within the M, the BubR1 and interactions are dependent on checkpoint signalling [38,40,50,51]. In particular, the formation of the interaction involves a large conformational change of (Figure 3), which may be the rate-limiting step for the formation of the M [33,52]. To facilitate the binding of to, is brought to the unattached kinetochores by Mad1 [33,41,48]. Because Mad1 and contain similar -binding peptide motifs, binding of Mad1 to also triggers the same large conformational change of as does [33,53 ]. It is possible that dissociated from Mad1 at the kinetochores transiently retains a conformation more amenable for binding than apo-, thus leading to the efficient assembly of the M [33 ]. There are important differences among published reports on the nature of the inhibitory complexes of the AP in vertebrates and the mechanisms of their formation [38,39,40 ]. This has led to the two different views of how the checkpoint operates, as outlined above. First, although both Tang et al. [38 ] and Fang [40 ] have readily detected and BubR1 Bub3 interactions in checkpoint-active HeLa cells, they failed to observe the existence of the M (BubR1 Bub3 ), as described by Sudakin et al.

6 The spindle checkpoint Yu 711 [39 ]. This discrepancy might be caused by the use of different purification protocols. onsidering the fact that a similar Mad3 Bub3 complex exists in budding and fission yeast, there is little doubt that the M forms in vertebrate cells [35,37 ] (see Update). Both Tang et al. [38 ] and Fang [40 ] use similar protocols for the purification of the M, and it is very likely that the M breaks up during the purification into two sub-complexes containing BubR1 Bub3 and [38,40 ]. However, their results suggest that the BubR1 Bub3 and subcomplexes might be more stable in checkpoint-activated cells, and the two sub-complexes only transiently associate to yield the intact but fragile M [38,40 ]. Another unresolved issue is the possibility of the checkpoint-enhanced assembly of the M at the kinetochores. Sudakin et al. [39 ] showed that the M is present throughout the cell cycle, yet it only associates with mitotic AP. Moreover, in yeast, functional kinetochores are not required for the formation of the M [36]. These findings are at odds with data from many laboratories, which demonstrate kinetochore localisation of nearly all checkpoint proteins and enhanced and BubR1 Bub3 interactions in checkpoint-active vertebrate cells [38,40,51]. This discrepancy can be easily reconciled by postulating that basal levels of the M can form in interphase cells. Upon checkpoint activation, unattached kinetochores merely increase the efficiency of M assembly and its cellular concentration. As suggested by Sudakin et al. [39 ], modifications of the AP in mitosis then increase its affinity toward the M, thus establishing a stable AP M complex. Future studies are needed to resolve these differences. Inhibiting AP The M is unlikely to inhibit the AP in a catalytic fashion, due to the following observations. First, BubR1 and within the M are responsible for inhibition of the AP, and they appear to inhibit the AP in a stoichiometric fashion in vitro [34,38,39,40 ]. Second, BubR1 is the only protein within M that contains a domain with potential enzymatic activity. However, unlike BubR1, its yeast homologue Mad3 does not contain a kinase domain. Furthermore, the kinase domain of BubR1 may not be functional because it lacks key catalytic residues conserved in other kinases (H Yu, unpublished data). Finally, even if BubR1 can act as a kinase, mutation of a critical conserved residue in BubR1 that is expected to abrogate its kinase activity does not affect the ability of BubR1 to inhibit the AP [38 ]. In fact, a BubR1 mutant lacking the entire kinase domain is still functional in AP inhibition [38 ]. Therefore, M may inhibit the AP through direct association, in an equal molar fashion. The M or its subcomplexes appear to be stoichiometric inhibitors of the AP. This raises the question of whether a single unattached kinetochore is sufficient to generate enough M for inhibition of the AP. A definitive answer to this question requires accurate quantitative measurements of the number of -, BubR1-, and -binding sites at each kinetochore, the turnover rate of these proteins at the kinetochores, the break-up rate of the M in the cytosol, and the cellular concentrations of all the relevant components. However, it is conceivable that the M might bind the AP more tightly than does free (i.e. the association of the M with the AP might prevent the binding of free to the AP). This would reduce the amount of the M needed to inhibit the AP. It is also worth pointing out that AP is regulated by additional non-checkpoint mechanisms. For example, binding of Emi1 to and phosphorylation of by dk1 also inhibit AP at pro-metaphase [54,55]. Thus, the spindle checkpoint might not need to inhibit the entire population of AP. The hypothesis that the spindle checkpoint only inhibits selective pools of AP is also consistent with the fact that the degradation of certain AP substrates, such as cyclin A, is not inhibited by the spindle checkpoint [56 ]. The mechanism by which the M or its subcomplexes inhibit the AP is not clear. To further complicate the issue, only limited knowledge is available about how the AP works as a ubiquitin ligase. The AP contains a so-called ullin RING (AP2 AP11) catalytic core [57]. The -like proteins may be responsible for substrate recruitment [58]. Even though and BubR1(Mad3) bind directly to, the spindle checkpoint does not seem to block the ability of to recruit substrates [59,60 ]. Instead, the vertebrate appears to block substrate release from in vitro [61 ]. These surprising results suggest that the spindle checkpoint and M may interfere with the ability of the AP to interact with its substrates in a productive way. Dismantling the checkpoint The co-localisation of checkpoint proteins and at the unattached kinetochores appears to be required for the assembly of the M and the diffusible APinhibitory signal. Thus, the inactivation of the checkpoint might be initiated by the loss of kinetochore localisation of Mad1 and and by the partial loss of BubR1, Bub1, Bub3, ENP-E and at the kinetochores. These checkpoint proteins may be depleted through two mechanisms: free diffusion into the cytosol; and motorassisted transport to the spindle poles along the microtubules [62 ]. Once the checkpoint proteins leave the kinetochores upon microtubule attachment and establishment of kinetochore tension, M and its subcomplexes might not form efficiently. The dissociation of the critical and BubR1 interactions may then lead to the activation of AP. It is unclear whether the break-up of these checkpoint complexes is a spontaneous process or if it is assisted by an as yet unidentified mechanism. The

7 712 ell division, growth and death existence of an active mechanism for the break-up of checkpoint inhibitory complexes is suggested indirectly by two unrelated findings. First, structural studies reveal that the dissociation of the complex requires the partial unfolding of, which poses a kinetic barrier for the break-up of M [33,53 ]. This suggests that the unassisted break-up of the M might be slow. Alternatively, by observing the mitotic progression of PtK1 cells possessing two spindles, Rieder et al. [63] found that the presence of unattached kinetochores in one spindle did not prevent a neighbouring spindle from entering anaphase. This can be explained by one of two possibilities: first, the inhibitory checkpoint signal is associated with the spindle and is not freely diffusible; or second, the checkpoint inhibitory complexes can only travel a limited distance before they fall apart [63]. Taking into account that the spontaneous break-up of the M might be slow, the latter possibility is consistent with the existence of an active mechanism for extinguishing the checkpoint signal in the cytosol. onclusions Recent studies have provided insight into the nature of the spindle defect that activates the checkpoint and the biochemical functions of several spindle checkpoint proteins. The spindle checkpoint is activated by kinetochores not yet attached by microtubules and not under tension. These kinetochores mediate the assembly of checkpoint complexes containing BubR1(Mad3), Bub3,,, or subsets of these proteins, which are responsible for the inhibition of AP. Despite the progress made, many significant questions remain. First, we need to know more about how the M is assembled and how it inhibits AP. In particular, the mechanism by which is relayed from Mad1 to remains to be established. Second, it will be crucial to understand the mechanism of the regulated kinetochore localisation of various checkpoint proteins, such as Mad1, Bub1 and BubR1. This is the key for the activation and inactivation of the checkpoint. In the end, we have to explain how the spindle checkpoint translates an imbalance of force at the kinetochores into an AP-inhibitory signal. Much more work is needed. May the force be with us. Update Recent work has shown that a M containing BubR1, Bub3, and only forms in checkpoint-active Xenopus egg extracts, not in interphase extracts [64]. This is consistent with the model that unattached kinetochores stimulate the formation of the M. Acknowledgements I thank Bonnie Howell, Andy Hoyt, Marc Kirschner, Ted Salmon, Tim Yen and Hui Zou for reading the manuscript critically and for providing insightful comments. I also thank Xuelian Luo, Jose Rizo-Rey and members of my laboratory for helpful discussions. The work in my laboratory is supported by the National Institutes of Health, the Packard Foundation, the Burroughs Wellcome Fund and the Welch Foundation. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Nasmyth K, Peters JM, Uhlmann F: Splitting the chromosome: cutting the ties that bind sister chromatids. Science 2000, 288: Tanaka TU: Bi-orienting chromosomes on the mitotic spindle. urr Opin ell Biol 2002, 14: Peters JM: The anaphase-promoting complex. Proteolysis in mitosis and beyond. Mol ell 2002, 9: Jallepalli PV, Lengauer : hromosome segregation and cancer: cutting through the mystery. Nat Rev ancer 2001, 1: Hunt P, Hassold T: Sex matters in meiosis. Science 2002, 296: Millband DN, ampbell L, Hardwick KG: The awesome power of multiple model systems: interpreting the complex nature of spindle checkpoint signaling. Trends ell Biol 2002, 12: Gorbsky GJ: The mitotic spindle checkpoint. urr Biol 2001, 11:R Shah JV, leveland DW: Waiting for anaphase: and the spindle assembly checkpoint. ell 2000, 103: Hoyt MA: A new view of the spindle checkpoint. J ell Biol 2001, 154: Gardner RD, Burke DJ: The spindle checkpoint: two transitions, two pathways. Trends ell Biol 2000, 10: Rieder L, ole RW, Khodjakov A, Sluder G: The checkpoint delaying anaphase in response to chromosome mono-orientation is mediated by an inhibitory signal produced by unattached kinetochores. J ell Biol 1995, 130: Hoffman DB, Pearson G, Yen TJ, Howell BJ, Salmon ED: Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. Mol Biol ell 2001, 12: Using quantitative digital imaging and immunofluorescence microscopy of PtK1 cells, the authors measure the concentrations of BubR1,, 3F3 antigen, ENP-E, dynein and REST antigen at kinetochores under several conditions: at pro-metaphase, in the presence of spindle-damaging drugs, and at metaphase. They find that, upon microtubule attachment, the kinetochore concentration of drops more sharply than those of BubR1, ENP-E and dynein. However, the amounts of, BubR1, ENP-E and dynein at kinetochores do not increase with taxol treatment, which presumably leads to loss of tension at kinetochores without disrupting microtubule attachment. 13. Li X, Nicklas RB: Mitotic forces control a cell-cycle checkpoint. Nature 1995, 373: Nicklas RB: How cells get the right chromosomes. Science 1997, 275: Nicklas RB, Waters J, Salmon ED, Walter S: heckpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential. J ell Sci 2001, 114: Skoufias DA, Andreassen PR, Lacroix FB, Wilson L, Margolis RL: Mammalian mad2 and bub1/bubr1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc Natl Acad Sci USA 2001, 98: The authors find that low concentrations of vinblastine arrest HeLa cells in mitosis, with all kinetochores attached to microtubules but not under tension. These kinetochores recruit Bub1 and BubR1, but not. Higher concentrations of vinblastine destroy the mitotic spindle and the kinetochores are not attached by microtubules. Under these conditions, is recruited to the kinetochores. Zhou et al. (2002) [19 ] describe similar findings in HeLa cells arrested in mitosis with noscapine. This drug does not affect microtubule polymerisation, but alters the dynamics of microtubule assembly and causes loss of tension at the kinetochores. They show that and Bub1/BubR1 behave differently in response to loss of microtubule attachment or tension at the kinetochores. These results suggest that specific subsets of checkpoint proteins may be activated by different spindle defects. 17. Kapoor TM, Mayer TU, oughlin ML, Mitchison TJ: Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J ell Biol 2000, 150:

8 The spindle checkpoint Yu Taylor SS, Hussein D, Wang Y, Elderkin S, Morrow J: Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells. J ell Sci 2001, 114: Zhou J, Panda D, Landen JW, Wilson L, Joshi H: Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint. J Biol hem 2002, 277: See annotation Skoufias et al. (2001) [16 ]. 20. Shonn MA, Mcarroll R, Murray AW: Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis. Science 2000, 289: Stern BM, Murray AW: Lack of tension at kinetochores activates the spindle checkpoint in budding yeast. urr Biol 2001, 11: Budding yeast cells with the D6 gene deleted do not replicate their DNA, but are able to duplicate spindle pole bodies and enter mitosis. The unreplicated chromosomes in these cells appear to attach to the mitotic spindle normally. Obviously, without the opposing sister chromatids, the kinetochores in these cells lack tension. The authors then used the degradation kinetics of Pds1 (a known AP substrate) to monitor the status of the spindle checkpoint. They show that the degradation of Pds1 is delayed in D6-null cells, and this delay in the kinetics of Pds1 degradation requires the function of. Thus, tension-lacking kinetochores activate the spindle checkpoint in yeast. 22. Tanaka TU, Rachidi N, Janke, Pereira G, Galova M, Schiebel E, Stark MJ, Nasmyth K: Evidence that the Ipl1 Sli15 (Aurora kinase INENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. ell 2002, 108: Before DNA replication, the kinetochores of yeast chromosomes are attached to the microtubules from the lone spindle pole in G1. As the spindle poles duplicate, the old spindle pole moves to the daughter cell while the newly duplicated one remains in the mother. In the D6-null yeast cells, the unreplicated chromosomes are initially all attached to the old spindle pole. As these cells enter mitosis, the unreplicated chromosomes detach from the old spindle pole and re-attach to the new one with equal frequency, resulting in more or less equal chromosome segregation into mother and daughter cells. However, inactivation of Ipl1 in yeast cells depleted of dc6 causes all the unreplicated chromosomes to segregate to the daughter cell, suggesting that the kinetochores of ipl1/cdc6 double mutant cells fail to detach from the old spindle pole. Therefore, Ipl1 is required for establishing chromosome bi-orientation by promoting the turnover of the microtubule connections between the kinetochores and the spindle poles. 23. Stern BM: Mitosis: Aurora gives chromosomes a healthy stretch. urr Biol 2002, 12:R Biggins S, Murray AW: The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev 2001, 15: Despite the importance of Ipl1 in establishing chromosome bi-orientation, ipl1 mutant cells do not activate the spindle checkpoint, suggesting that the defects in ipl1 mutant cells are not sufficient to activate the checkpoint or Ipl1 itself is a part of the tension-sensing mechanism of the checkpoint. The authors show that Ipl1 is not required for the mitotic arrest induced by nocodazole. However, it is required for the mitotic arrest caused by Mps1 overexpression. Furthermore, yeast mutants lacking dc6 or Scc1/Mcd1 (cohesin) functions activate the checkpoint, due to lack of tension at chromosomes, and delay the degradation of the anaphase inhibitor Pds1 in a checkpoint-dependent manner. Ipl1 is required for the checkpoint-mediated stabilisation of Pds1 in D6 or MD1 mutants. The Ipl1 protein also localises to the vicinity of kinetochores. The authors argue that Ipl1 is involved in the spindle checkpoint. Specifically, Ipl1 might monitor tension at the kinetochores and act upstream of Mps Murata-Hori M, Tatsuka M, Wang YL: Probing the dynamics and functions of Aurora B kinase in living cells during mitosis and cytokinesis. Mol Biol ell 2002, 13: Kallio MJ, Mcleland ML, Stukenberg PT, Gorbsky GJ: Inhibition of Aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. urr Biol 2002, 12: Injection of antibodies against Xenopus Aurora B into Xenopus tissueculture cells causes defects in chromosome movement to the metaphase plate, and mitotic exit in the absence of anaphase and cytokinesis. It also causes alteration of the microtubule network in the mitotic spindle, and a failure of the injected cells to arrest in mitosis in the presence spindle-damaging drugs. These observations are consistent with the vertebrate Aurora B protein playing a role in establishing chromosome bi-orientation and in the spindle checkpoint. However, unlike in budding yeast, Aurora B appears to be essential for the checkpoint-mediated mitotic arrest induced by severe spindle defects. 27. Murata-Hori M, Wang Y: The kinase activity of Aurora B Is required for kinetochore microtubule interactions during mitosis. urr Biol 2002, 12: Abrieu A, Kahana JA, Wood KW, leveland DW: ENP-E as an essential component of the mitotic checkpoint in vitro. ell 2000, 102: Li R, Murray AW: Feedback control of mitosis in budding yeast. ell 1991, 66: Hoyt MA, Totis L, Roberts BT: S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. ell 1991, 66: Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK: hromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein. ell 2000, 101: Kalitsis P, Earle E, Fowler KJ, hoo KH: Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes Dev 2000, 14: Luo X, Tang Z, Rizo J, Yu H: The spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or. Mol ell 2002, 9: Mad1 and, two otherwise unrelated proteins, contain similar short peptide motifs that bind to. Binding of Mad1 and to is mutually exclusive. Overexpression of Mad1 antagonises the checkpoint function of. Upon binding to a peptide that mimics the -binding motifs of Mad1 and, undergoes a major conformational change with extensive reshuffling of secondary structural elements. They also provide evidence that undergoes similar structural rearrangements when bound to the native Mad1 and peptides. Despite the apparent antagonism between Mad1- and -binding to, Mad1 might convert to a form more suitable for -binding under the right circumstances. 34. Fang G, Yu H, Kirschner MW: The checkpoint protein MAD2 and the mitotic regulator D20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev 1998, 12: Hardwick KG, Johnston R, Smith DL, Murray AW: MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, p, and p. J ell Biol 2000, 148: Fraschini R, Beretta A, Sironi L, Musacchio A, Lucchini G, Piatti S: Bub3 interaction with, Mad3 and is mediated by WD40 repeats and does not require intact kinetochores. EMBO J 2001, 20: Millband DN, Hardwick KG: Fission yeast Mad3p is required for p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner. Mol ell Biol 2002, 22: ombining biochemistry and genetics elegantly in fission yeast, this work confirms and extends the authors previous findings in budding yeast. They show that Mad3, Bub3, and Slp1() form a complex in fission yeast. The kinetochore localisation of Mad3 depends on Bub1, Bub3 and Mph1, but not Mad1 and. 38. Tang Z, Bharadwaj R, Li B, Yu H: -independent inhibition of AP by the mitotic checkpoint protein BubR1. Dev ell 2001, 1: BubR1 Bub3 and interactions are enhanced upon checkpoint activation. In the absence of, a purified recombinant BubR1 Bub3 complex inhibits AP in vitro. Furthermore, BubR1 inhibits AP much more efficiently than. The kinase domain of BubR1 is dispensable for its AP inhibitory activity. These data, along with data from Sudakin et al. (2001) [39 ] and Fang (2002) [40 ], suggest that BubR1 and inhibit AP collaboratively. 39. Sudakin V, han GK, Yen TJ: heckpoint inhibition of the AP/ in HeLa cells is mediated by a complex of BUBR1, BUB3, D20, and MAD2. J ell Biol 2001, 154: A mitotic checkpoint complex (M) containing BubR1, Bub3,, and was purified from HeLa cells. M binds to the AP directly and inhibits it much more efficiently than alone. Active M is also isolated from interphase cells, suggesting that unattached kinetochores are not absolutely required for the assembly of M. Interestingly, M only inhibits the activity of mitotic AP. Mitotic chromosomes also suppress AP activity, although the mechanism of inhibition is not established. 40. Fang G: heckpoint Protein BubR1 Acts Synergistically with to Inhibit Anaphase-promoting omplex. Mol Biol ell 2002, 13: See annotations Tang et al. (2001) [38 ] and Sudakin et al. (2001) [39 ].

9 714 ell division, growth and death 41. hen RH, Shevchenko A, Mann M, Murray AW: Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J ell Biol 1998, 143: Abrieu A, Magnaghi-Jaulin L, Kahana JA, Peter M, astro A, Vigneron S, Lorca T, leveland DW, Labbe J: Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. ell 2001, 106: This is the first demonstration that the vertebrate homologue of yeast Mps1 is required for the spindle checkpoint. The Xenopus Mps1 localises to kinetochores. The kinase activity of Mps1 is essential for its checkpoint function. Mps1 is also required for the kinetochore localisation of ENP-E, Mad1 and. 43. Stucke VM, Sillje HH, Arnaud L, Nigg EA: Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication. EMBO J 2002, 21: The mammalian homologue of Mps1 is required for the spindle checkpoint. Antibody injection and RNA-interference-mediated depletion of Mps1 from mammalian cells cause cells to escape from the mitotic arrest induced by spindle-damaging drugs. 44. han GK, Jablonski SA, Starr DA, Goldberg ML, Yen TJ: Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores. Nat ell Biol 2000, 2: Sharp-Baker H, hen RH: Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1,, Bub3, and ENP-E, independently of its kinase activity. J ell Biol 2001, 153: This is one of several elegant studies on the spindle checkpoint in Xenopus from hen and co-workers. Xenopus SF extracts supplemented with sperm nuclei and nocodazole establish an active spindle checkpoint. Immunodepletion of Bub1 from these extracts abolish the kinetochore localisation of Mad1,, Bub3 and ENP-E. Unlike Mps1, the kinase activity of Bub1 is not required for its function in recruiting or keeping other checkpoint proteins at the kinetochores. 46. Kallio M, Weinstein J, Daum JR, Burke DJ, Gorbsky GJ: Mammalian p55d mediates association of the spindle checkpoint protein with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J ell Biol 1998, 141: Howell BJ, Hoffman DB, Fang G, Murray AW, Salmon ED: Visualization of dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J ell Biol 2000, 150: hung E, hen RH: Spindle checkpoint requires Mad1-bound and Mad1-free. Mol Biol ell 2002, 13: Brady DM, Hardwick KG: omplex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. urr Biol 2000, 10: Wu H, Lan Z, Li W, Wu S, Weinstein J, Sakamoto KM, Dai W: p55d/hd20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase. Oncogene 2000, 19: Zhang Y, Lees E: Identification of an overlapping binding domain on for and anaphase-promoting complex: model for spindle checkpoint regulation. Mol ell Biol 2001, 21: Luo X, Fang G, oldiron M, Lin Y, Yu H, Kirschner MW, Wagner G: Structure of the spindle assembly checkpoint protein and its interaction with. Nat Struct Biol 2000, 7: Sironi L, Mapelli M, Knapp S, Antoni AD, Jeang KT, Musacchio A: rystal structure of the tetrameric Mad1 core complex: implications of a safety belt binding mechanism for the spindle checkpoint. EMBO J 2002, 21: The authors determine the crystal structure of in complex with a 100-residue -binding domain of Mad1. ompared with the apo- structure [52], undergoes a dramatic conformational change upon binding to Mad1. The Mad1 portion of the complex forms a dimer, with the -binding region adopting an extended conformation and threading through the protein. As revealed by the structure, the dissociation of from this complex requires partial unfolding of. 54. Reimann JD, Freed E, Hsu JY, Kramer ER, Peters JM, Jackson PK: Emi1 is a mitotic regulator that interacts with and inhibits the anaphase promoting complex. ell 2001, 105: Yudkovsky Y, Shteinberg M, Listovsky T, Brandeis M, Hershko A: Phosphorylation of /fizzy negatively regulates the mammalian cyclosome/ap in the mitotic checkpoint. Biochem Biophys Res ommun 2000, 271: Geley S, Kramer E, Gieffers, Gannon J, Peters JM, Hunt T: Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J ell Biol 2001, 153: This paper shows that cyclin A is degraded by AP at pro-metaphase, before chromosome segregation. Surprisingly, cyclin A degradation is not inhibited by the spindle checkpoint. This remains one of many mysteries of regulated protein degradation involving the AP. 57. Tang Z, Li B, Bharadwaj R, Zhu H, Ozkan E, Hakala K, Deisenhofer J, Yu H: AP2 ullin protein and AP11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Mol Biol ell 2001, 12: Vodermaier H: ell cycle: waiters serving the destruction machinery. urr Biol 2001, 11:R Hilioti Z, hung YS, Mochizuki Y, Hardy F, ohen-fix O: The anaphase inhibitor Pds1 binds to the AP/-associated protein in a destruction box-dependent manner. urr Biol 2001, 11: This paper demonstrates the direct binding between yeast and the AP substrate, Pds1, which has long been suspected from indirect observations. This suggests that the role of in the AP is to recruit substrate. The Pds1 interaction is not blocked by the spindle checkpoint. 60. Pfleger M, Lee E, Kirschner MW: Substrate recognition by the and dh1 components of the anaphase-promoting complex. Genes Dev 2001, 15: The authors show that and dh1 bind to several AP substrates directly, establishing a role for and dh1 in substrate recruitment in AP-mediated ubiquitination reactions. 61. Pfleger M, Salic A, Lee E, Kirschner MW: Inhibition of dh1 AP by the MAD2-related protein MAD2L2: a novel mechanism for regulating dh1. Genes Dev 2001, 15: This paper describes a novel mechanism for the inhibition of the AP. does not block the ability of to recruit substrates. Instead, it appears to prevent the substrate release of AP. 62. Howell BJ, McEwen BF, anman J, Hoffman DB, Farrar EM, Rieder L, Salmon ED: ytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. J ell Biol 2001, 155: In addition to passive diffusion, dynein/dynactin-mediated transport along the microtubules is involved in the removal of checkpoint proteins such as from the kinetochores. This mechanism might be important for turning off the checkpoint. 63. Rieder L, Khodjakov A, Paliulis LV, Fortier TM, ole RW, Sluder G: Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. Proc Natl Acad Sci USA 1997, 94: hen, RH: BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J ell Biol 2002, 158: