The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.     

Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae

Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.     

Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.     

Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation

Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression. However, it is unknown whether Mad1-Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases) have checkpoint functions downstream of Mad1-Mad2 recruitment or whether they act upstream to quench the primary error signal. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division.     

The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C)

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.     

Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism.     

Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner

The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Here we present the fission yeast homologue of Mad3p. Cells devoid of mad3(+) are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory "wait anaphase" signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, we have systematically studied the dependency of Mad3/BubR1 protein recruitment to kinetochores. We find Mad3-GFP kinetochore localization to be dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. We discuss the implications of these findings in the context of our current understanding of spindle checkpoint function.     

Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.     

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.     

Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2-Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1-Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2-Cdc20 complex, and propose that the Mad1-Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1-Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1-Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1-Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2-ligand complexes.     

Checkpoint signalling: Mad2 conformers and signal propagation

The spindle checkpoint protein Mad2 has a tendency to form multimers and adopts at least two structural conformations. New work highlights the importance of the Mad2-Mad2 interaction, and suggests how spindle checkpoint signals are propagated away from kinetochores.     

Mps1 phosphorylation by MAP kinase is required for kinetochore localization of spindle-checkpoint proteins

The spindle checkpoint delays anaphase onset until all chromosomes have achieved bipolar attachment to the spindle microtubules. Unattached kinetochores activate the spindle checkpoint by recruiting several spindle-checkpoint proteins, including Mps1, Mad1, Mad2, Bub1, Bub3, and BubR1 (Mad3 in yeast). In vertebrate cells, active MAP kinase (MAPK) is also enriched at unattached kinetochores and is required for the spindle checkpoint. It has been shown that the kinase activity of Mps1 is required for the spindle checkpoint and for kinetochore localization of Bub1, Bub3, Mad1, and Mad2 . We herein demonstrate that MAPK phosphorylates Mps1 at S844 in Xenopus egg extracts. Interestingly, changing S844 to unphosphorylatable alanine (S844A) has no effect on the kinase activity of Mps1, although it abolishes the checkpoint function of Mps1. Biochemical and immunofluorescence studies show that S844A mutation perturbs kinetochore localization of Mps1 and other spindle-checkpoint proteins, whereas the phosphorylation-mimicking S844D mutant restores their functions. Our studies suggest that Mps1 phosphorylation by MAPK at S844 might create a phosphoepitope that allows Mps1 to interact with kinetochores. In addition, our results indicate that active Mps1 must localize to kinetochores in order to execute its checkpoint function.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability

The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1-c-Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1-c-Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate-limiting step in the assembly of anaphase-inhibitory complexes. SAC proficiency further requires Mad1-c-Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill-defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1-c-Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1-c-Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted.     

Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes

The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.     

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

The spindle assembly checkpoint inhibits anaphase until all chromosomes have become attached to the mitotic spindle. A complex between the checkpoint proteins Mad1 and Mad2 provides a platform for Mad2:Mad2 dimerization at unattached kinetochores, which enables Mad2 to delay anaphase. Here, we show that mutations in Bub1 and within the Mad1 C‐terminal domain impair the kinetochore localization of Mad1:Mad2 and abrogate checkpoint activity. Artificial kinetochore recruitment of Mad1 in these mutants co‐recruits Mad2; however, the checkpoint remains non‐functional. We identify specific mutations within the C‐terminal head of Mad1 that impair checkpoint activity without affecting the kinetochore localization of Bub1, Mad1 or Mad2. Hence, Mad1 potentially in conjunction with Bub1 has a crucial role in checkpoint signalling in addition to presenting Mad2.     

Regulation of kinetochore recruitment of two essential mitotic spindle checkpoint proteins by Mps1 phosphorylation

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.     

MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1-Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.     

Spindle checkpoint signaling requires the mis6 kinetochore subcomplex, which interacts with mad2 and mitotic spindles

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.     

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.