Bod1, a novel kinetochore protein required for chromosome biorientation

We have combined the proteomic analysis of Xenopus laevis in vitro-assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule-kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.     

Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.     

Mutual Dependence of Mob1 and the Chromosomal Passenger Complex for Localization during Mitosis

The spatial and temporal coordination of chromosome segregation with cytokinesis is essential to ensure that each daughter cell receives the correct complement of chromosomal and cytoplasmic material. In yeast, mitotic exit and cytokinesis are coordinated by signaling cascades whose terminal components include a nuclear Dbf2-related family kinase and a noncatalytic subunit, Mps one binding (Mob) 1. There are five human Mob1 isoforms, all of which display redundant localization patterns at the spindle poles and kinetochores in early mitosis, and the spindle midzone during cytokinesis. Mob1 shares similar localization patterns to Polo-like kinase (Plk1) and the chromosomal passenger complex (CPC), and although depletion of Plk1 resulted in a loss of Mob1 from the spindle poles, Mob1 recruitment to kinetochores was unaffected. Conversely, disruption of CPC signaling resulted in a loss of Mob1 from kinetochores without disrupting recruitment to the spindle poles. In Mob1-depleted cells, the relocalization of the CPC and mitotic kinesin-like protein (MKLP) 2 to the spindle midzone was delayed during early anaphase, and as a consequence, the midzone recruitment of MKLP1 also was affected. Together, these results suggest that Mob1 and the other mammalian orthologues of the mitotic exit network regulate mitotic progression by facilitating the timely mobilization of the CPC to the spindle midzone.     

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

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

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.     

Metaphase arrest with centromere separation in polo mutants of Drosophila

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.     

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.     

Connecting up and clearing out: how kinetochore attachment silences the spindle assembly checkpoint

With the goal of creating two genetically identical daughter cells, cell division culminates in the equal segregation of sister chromatids. This phase of cell division is monitored by a cell cycle checkpoint known as the spindle assembly checkpoint (SAC). The SAC actively prevents chromosome segregation while one or more chromosomes, or more accurately kinetochores, remain unattached to the mitotic spindle. Such unattached kinetochores recruit SAC proteins to assemble a diffusible anaphase inhibitor. Kinetochores stop production of this inhibitor once microtubules (MTs) of the mitotic spindle are bound, but productive attachment of all kinetochores is required to satisfy the SAC, initiate anaphase, and exit from mitosis. Although mechanisms of kinetochore signaling and SAC inhibitor assembly and function have received the bulk of attention in the past two decades, recent work has focused on the principles of SAC silencing. Here, we review the mechanisms that silence SAC signaling at the kinetochore, and in particular, how attachment to spindle MTs and biorientation on the mitotic spindle may turn off inhibitor generation. Future challenges in this area are highlighted towards the goal of building a comprehensive molecular model of this process.     

Budding yeast mitotic chromosomes have an intrinsic bias to biorient on the spindle

BACKGROUND: Chromosomes must biorient on the mitotic spindle, with the two sisters attached to opposite spindle poles. The spindle checkpoint detects unattached chromosomes and monitors biorientation by detecting the lack of tension between two sisters attached to the same pole. After the spindle has been depolymerized and allowed to reform, budding yeast sgo1 mutants fail to biorient their sister chromatids and die as cells divide. RESULTS: In sgo1 mutants, chromosomes attach to microtubules normally but cannot reorient if both sisters attach to the same pole. The mutants' fate depends on the position of the spindle poles when the chromosomes attach to microtubules. If the poles have separated, sister chromatids biorient, but if the poles are still close, sister chromatids often attach to the same pole, missegregate, and cause cell death. CONCLUSIONS: These observations argue that budding yeast mitotic chromosomes have an intrinsic, geometric bias to biorient on the spindle. When the poles have already separated, attaching one kinetochore to one pole predisposes its sister to attach to the opposite pole, allowing the cells to segregate the chromosomes correctly. When the poles have not separated, the second kinetochore eventually attaches to either of the two poles randomly, causing orientation errors that are corrected in the wild-type but not in sgo1 mutants. In the absence of spindle damage, sgo1 cells divide successfully, suggesting that kinetochores only make stable attachments to microtubules after the cells have entered mitosis and separated their spindle poles.     

The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.     

Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.     

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.     

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.     

Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A

BACKGROUND: Metaphase is thought to be a force-equilibrium state of "tug of war," in which poleward forces are pulling kinetochores and counteracting the cohesive forces between the centromeres. Unlike conventional kinesins, members of the Kin I family are microtubule-depolymerizing enzymes, which are expected to be molecules that could generate poleward forces. RESULTS: We have characterized mitotic roles of two Kin I homologs, Klp5 and Klp6, in fission yeast. Klp5 and Klp6 colocalize to the mitotic kinetochores and the spindle midzone. These two proteins form a heterocomplex, but not a homocomplex. Albeit not essential, both proteins are required for accurate chromosome segregation and normal morphology of interphase microtubules. Time-lapse live analysis using GFP-alpha-tubulin indicates that these mutants spend a much longer time (2-fold) in mitosis before the initiation of anaphase B. Further observation using kinetochore and centromere markers shows that, in these mutants, sister centromeres move back and forth between the two poles, indicating that entry into anaphase A is delayed. This is supported by live image analysis showing that Cut2 securin is retained during the prolonged mitosis. Furthermore, the mitotic extension is dependent upon the Mad2 spindle checkpoint. CONCLUSIONS: We discuss two models of Kin I function in fission yeast. One proposes that Klp5 and Klp6 are required for efficient capturing of kinetochores by the spindles, while the other proposes that they are required to generate tension upon kinetochore capturing. Kin I, therefore, plays a fundamental role in the establishment of metaphase, probably by generating poleward forces at the kinetochores.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

Inhibition of protein kinase C zeta blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment

The attachment of spindle microtubules to kinetochores is crucial for accurate segregation of chromosomes to daughter cells during mitosis. While a growing number of proteins involving this step are being identified, its molecular mechanisms are still not clear. Here we show that protein kinase C ζ (PKCζ) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCζ was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCζ molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCζ was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCζ at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCζ, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCζ by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCζ inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCζ inhibitor also dislocated the minus-end directed motor protein dynein from kinetochores, but not the mitotic checkpoint proteins Mad2 and CENP-E. Prolonged exposure to the PKCζ inhibitor eventually resulted in cell death. These results suggest a critical role of PKCζ in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.     

Lack of tension at kinetochores activates the spindle checkpoint in budding yeast

The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle. The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis.     

A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cells

Polo kinases play critical roles for proper M-phase progression. They are characterized by the presence of two regions of homology in the C-terminal non-catalytic domain, termed polo-box 1 (PB1) and polo-box 2 (PB2). Here we demonstrate that both PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1/K82M or the C-terminal plk1 Delta N induced a pre-anaphase arrest with elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibited randomly oriented spindle structures, whereas metaphase cells exhibited aberrant bipolar spindles with Mad2 localization at kinetochores of misaligned chromosomes. Microtubule nucleation activity of centrosomes was not compromised. In vivo time-lapse studies revealed that expression of plk1 Delta N resulted in repeated cycles of bipolar spindle formation and disruption, suggestive of a defect in spindle stability. A prolonged arrest frequently led to the generation of micronucleated cells in the absence of sisterchromatid separation and centrosome duplication, indicating that micronucleation is not a result of accumulated cytokinesis failures. Interestingly, bypass of the mitotic arrest by dominant-negative spindle checkpoint components led to a failure in completion of cytokinesis. We propose that, in mammalian cells, the polo-box-dependent Plk1 activity is required for proper metaphase/anaphase transition and for cytokinesis.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.     

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.