PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.     

Differential Kinetochore Requirements for Establishment and Maintenance of the Spindle Checkpoint Are Dependent on the Mechanism of Checkpoint Activation in Saccharomyces cerevisiae

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.     

Differential kinetochore requirements for establishment and maintenance of the spindle checkpoint are dependent on the mechanism of checkpoint activation in Saccharomyces cerevisiae

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.     

NEK2A interacts with MAD1 and possibly functions as a novel integrator of the spindle checkpoint signaling

Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling.     

RED, a spindle pole-associated protein, is required for kinetochore localization of MAD1, mitotic progression, and activation of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.     

Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.     

Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.     

Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle

To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.     

Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis.

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.     

Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3Δ cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.     

Saccharomyces cerevisiae BUB2 prevents mitotic exit in response to both spindle and kinetochore damage

The spindle assembly checkpoint-mediated mitotic arrest depends on proteins that signal the presence of one or more unattached kinetochores and prevents the onset of anaphase in the presence of kinetochore or spindle damage. In the presence of either damage, bub2 cells initiate a preanaphase delay but do not maintain it. Inappropriate sister chromatid separation in nocodazole-treated bub2 cells is prevented when mitotic exit is blocked using a conditional tem1(c) mutant, indicating that the preanaphase failure in bub2 cells is a consequence of events downstream of TEM1 in the mitotic exit pathway. Using a conditional bub2(tsd) mutant, we demonstrate that the continuous presence of Bub2 protein is required for maintaining spindle damage-induced arrest. BUB2 is not required to maintain a DNA damage checkpoint arrest, revealing a specificity for spindle assembly checkpoint function. In a yeast two-hybrid assay and in vitro, Bub2 protein interacts with the septin protein Cdc3, which is essential for cytokinesis. These data support the view that the spindle assembly checkpoint encompasses regulation of distinct mitotic steps, including a MAD2-directed block to anaphase initiation and a BUB2-directed block to TEM1-dependent exit.     

Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle–regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NΔ253), or the conserved Spindly box (ΔSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.     

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background

In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle.

Methodology/Principal Findings

We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of ‘wait anaphase’, plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants.

Conclusions/Significance

We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division.     

MAD2蛋白在蚕豆细胞周期中的同源定位

细胞周期是一个高度有序的过程,其运行过程受到多个检验点(checkpoint)的严格监控.MAD2是动物细胞纺锤体检验点(spindle checkpoint)中一种重要的蛋白。我们利用免疫印记法从蚕豆组织中检测到一种MAD2的同源蛋白,该蛋白的分子量在26KD左右,其亚细胞分布方式与动物细胞大致相同,都随细胞周期的进行而变化:在间期细胞中,MAD2分布于整个细胞中,并且在核膜外侧有优先聚集;在有丝分裂前期细胞中,MAD2在核膜破裂后开始在动粒处聚集;到早中期(prometaphase),MAD2定位于动粒;随着微管的延伸,MAD2逐渐减少直至消失,在中期,晚中期及后期细胞中,动粒处已无MAD2的分布。     

Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast.

Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.     

CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.     

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.     

The first mitosis of the mouse embryo is prolonged by transitional metaphase arrest

The first mitosis of the mouse embryo is almost twice as long as the second. The mechanism of the prolongation of the first mitosis remains unknown, and it is not clear whether prometaphase or metaphase or both are prolonged. Prometaphase is characterized by dynamic chromosome movements and spindle assembly checkpoint activity, which prevents anaphase until establishment of stable kinetochore-microtubule connections. The end of prometaphase is correlated with checkpoint inactivation and disappearance of MAD2L1 (MAD2) and RSN (CLIP-170) proteins from kinetochores. Spindle assembly checkpoint operates during the early mouse mitoses, but it is not clear whether it influences their duration. Here, we determine the length of prometaphases and metaphases during the first two embryonic mitoses by time-lapse video recording of chromosomes and by immunolocalization of MAD2L1 and RSN proteins. We show that the duration of the two prometaphases does not differ and that MAD2L1 and RSN disappear from kinetochores very early during each mitosis. The first metaphase is significantly longer than the second one. Therefore, the prolongation of the first embryonic mitosis is due to a prolonged metaphase, and the spindle assembly checkpoint cannot be involved in this process. We show also that MAD2L1 staining disappears gradually from kinetochores of oocytes arrested at metaphase of the second meiotic division. This shows a striking similarity between the first embryonic mitosis and metaphase arrest in oocytes. We postulate that the first embryonic mitosis is prolonged by a transient metaphase arrest that is independent of the spindle assembly checkpoint and is similar to metaphase II arrest. The molecular mechanism of this transient arrest remains to be elucidated.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase

Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.