SMART MODE:

NORMAL GENOMIC

• Edgerton H, Paolillo V, Oakley BR
• Spatial regulation of the spindle assembly checkpoint and anaphase-promoting complex in Aspergillus nidulans.
• Mol Microbiol. 2015; 95: 442-57
• Display abstract

• The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of gamma-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase-promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the gamma-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.

• Marston AL
• Chromosome segregation in budding yeast: sister chromatid cohesion and related mechanisms.
• Genetics. 2014; 196: 31-63
• Display abstract

• Studies on budding yeast have exposed the highly conserved mechanisms by which duplicated chromosomes are evenly distributed to daughter cells at the metaphase-anaphase transition. The establishment of proteinaceous bridges between sister chromatids, a function provided by a ring-shaped complex known as cohesin, is central to accurate segregation. It is the destruction of this cohesin that triggers the segregation of chromosomes following their proper attachment to microtubules. Since it is irreversible, this process must be tightly controlled and driven to completion. Furthermore, during meiosis, modifications must be put in place to allow the segregation of maternal and paternal chromosomes in the first division for gamete formation. Here, I review the pioneering work from budding yeast that has led to a molecular understanding of the establishment and destruction of cohesion.

• Wang Y, Jin F, Higgins R, McKnight K
• The current view for the silencing of the spindle assembly checkpoint.
• Cell Cycle. 2014; 13: 1694-701
• Display abstract

• Chromosome bipolar attachment is achieved when sister kinetochores are attached by microtubules emanating from opposite spindle poles, and this process is essential for faithful chromosome segregation during anaphase. A fundamental question in cell biology is how cells ensure that chromosome segregation only occurs after bipolar attachment. It is well documented that unattached kinetochores activate the spindle assembly checkpoint (SAC) to delay chromosome segregation. Therefore, the silencing of the SAC is thought to trigger anaphase onset, but how correct chromosome attachment is coupled with SAC silencing and the subsequent anaphase onset is poorly understood. The establishment of chromosome bipolar attachment not only results in the occupancy of kinetochores by microtubules but also applies tension on sister kinetochores. A long-standing debate is whether the kinetochore attachment (occupancy) or the tension silences the SAC. Recent work in budding yeast reveals the SAC silencing network SSN that prevents SAC silencing prior to tension generation at kinetochores. Therefore, this signaling pathway ensures that SAC silencing and the subsequent anaphase onset occur only after chromosome bipolar attachment applies tension on chromosomes. This review will summarize the recent advances in the understanding of the SAC silencing process.

• Tipton AR et al.
• Monopolar spindle 1 (MPS1) kinase promotes production of closed MAD2 (C-MAD2) conformer and assembly of the mitotic checkpoint complex.
• J Biol Chem. 2013; 288: 35149-58
• Display abstract

• MPS1 kinase is an essential component of the spindle assembly checkpoint (SAC), but its functioning mechanisms are not fully understood. We have shown recently that direct interaction between BUBR1 and MAD2 is critical for assembly and function of the human mitotic checkpoint complex (MCC), the SAC effector. Here we report that inhibition of MPS1 kinase activity by reversine disrupts BUBR1-MAD2 as well as CDC20-MAD2 interactions, causing premature activation of the anaphase-promoting complex/cyclosome. The effect of MPS1 inhibition is likely due to reduction of closed MAD2 (C-MAD2), as expressing a MAD2 mutant (MAD2(L13A)) that is locked in the C conformation rescued the checkpoint defects. In the presence of reversine, exogenous C-MAD2 does not localize to unattached kinetochores but is still incorporated into the MCC. Contrary to a previous report, we found that sustained MPS1 activity is required for maintaining both the MAD1.C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. Additionally, mitotic phosphorylation of BUBR1 is also affected by MPS1 inhibition but seems dispensable for MCC assembly. Our results support the notion that MPS1 kinase promotes C-MAD2 production and subsequent MCC assembly to activate the SAC.

• Biggins S
• The composition, functions, and regulation of the budding yeast kinetochore.
• Genetics. 2013; 194: 817-46
• Display abstract

• The propagation of all organisms depends on the accurate and orderly segregation of chromosomes in mitosis and meiosis. Budding yeast has long served as an outstanding model organism to identify the components and underlying mechanisms that regulate chromosome segregation. This review focuses on the kinetochore, the macromolecular protein complex that assembles on centromeric chromatin and maintains persistent load-bearing attachments to the dynamic tips of spindle microtubules. The kinetochore also serves as a regulatory hub for the spindle checkpoint, ensuring that cell cycle progression is coupled to the achievement of proper microtubule-kinetochore attachments. Progress in understanding the composition and overall architecture of the kinetochore, as well as its properties in making and regulating microtubule attachments and the spindle checkpoint, is discussed.

• Nijenhuis W et al.
• A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.
• J Cell Biol. 2013; 201: 217-31
• Display abstract

• The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

• Cane S, McGilvray PT, Maresca TJ
• Insights from an erroneous kinetochore-microtubule attachment state.
• Bioarchitecture. 2013; 3: 69-76
• Display abstract

• Faithful distribution of the genome requires that sister kinetochores, which assemble on each chromatid during cell division, interact with dynamic microtubules from opposite spindle poles in a configuration called chromosome biorientation. Biorientation produces tension that increases the affinity of kinetochores for microtubules via ill-defined mechanisms. Non-bioriented kinetochore-microtubule (kt-MT) interactions are prevalent but short-lived due to an error correction pathway that reduces the affinity of kinetochores for microtubules. Interestingly, incorrect kt-MT interactions can be stabilized by experimentally applying force to misoriented chromosomes. Here, a live-cell force assay is utilized to characterize the molecular composition of a common type of improper kt-MT attachment. Our force-related studies are also discussed in the context of current models for tension-dependent stabilization of kt-MT interactions.

• Wang M et al.
• BRK1, a Bub1-related kinase, is essential for generating proper tension between homologous kinetochores at metaphase I of rice meiosis.
• Plant Cell. 2012; 24: 4961-73
• Display abstract

• Bub1 (for budding uninhibited by benzimidazole 1), one of the main spindle checkpoint kinases, acts as a kinetochore scaffold for assembling other checkpoint proteins. Here, we identify a plant Bub1-related kinase 1 (BRK1) in rice (Oryza sativa). The brk1 mutants are sterile due to the precocious separation of sister chromatids at the onset of anaphase I. The centromeric recruitment of SHUGOSHIN1 and phosphorylation of histone H2A at Thr-134 (H2A-pT134) depend on BRK1. Although the homologs can faithfully separate from each other at the end of meiosis I, the uncorrected merotelic attachment of paired sister kinetochores at the early stage of metaphase I in brk1 reduces the tension across homologous kinetochores, causes the metaphase I spindle to be aberrantly shaped, and subsequently affects the synchronicity of homolog separation at the onset of anaphase I. In addition, the phosphorylation of inner centromeric histone H3 at Ser-10 (H3-pS10) during diakinesis depends on BRK1. Therefore, we speculate that BRK1 may be required for normal localization of Aurora kinase before the onset of metaphase I, which is responsible for correcting the merotelic attachment.

• Jin F, Liu H, Li P, Yu HG, Wang Y
• Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint.
• PLoS Genet. 2012; 8: 1002492-1002492
• Display abstract

• The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.

• Barford D
• Structural insights into anaphase-promoting complex function and mechanism.
• Philos Trans R Soc Lond B Biol Sci. 2011; 366: 3605-24
• Display abstract

• The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.

• Lara-Gonzalez P, Scott MI, Diez M, Sen O, Taylor SS
• BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner.
• J Cell Sci. 2011; 124: 4332-45
• Display abstract

• The spindle assembly checkpoint (SAC) is a signalling network that delays anaphase onset until all the chromosomes are attached to the mitotic spindle through their kinetochores. The downstream target of the spindle checkpoint is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that targets several anaphase inhibitors for proteolysis, including securin and cyclin B1. In the presence of unattached kinetochores, the APC/C is inhibited by the mitotic checkpoint complex (MCC), a tetrameric complex composed of three SAC components, namely BubR1, Bub3 and Mad2, and the APC/C co-activator Cdc20. The molecular mechanisms underlying exactly how unattached kinetochores catalyse MCC formation and how the MCC then inhibits the APC/C remain obscure. Here, using RNAi complementation and in vitro ubiquitylation assays, we investigate the domains in BubR1 required for APC/C inhibition. We observe that kinetochore localisation of BubR1 is required for efficient MCC assembly and SAC response. Furthermore, in contrast to previous studies, we show that the N-terminal domain of BubR1 is the only domain involved in binding to Cdc20-Mad2 and the APC/C. Within this region, an N-terminal KEN box (KEN1) is essential for these interactions. By contrast, mutation of the second KEN box (KEN2) of BubR1 does not interfere with MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for inhibition of APC/C when activated by Cdc20 (APC/C(Cdc20)). Indeed, we show that this second KEN box promotes SAC function by blocking the recruitment of substrates to the APC/C. Thus, we propose a model in which the BubR1 KEN boxes play two very different roles, the first to promote MCC assembly and the second to block substrate recruitment to APC/C(Cdc20).

• Kops GJ et al.
• APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome.
• J Cell Sci. 2010; 123: 1623-33
• Display abstract

• Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.

• Malureanu LA, Jeganathan KB, Hamada M, Wasilewski L, Davenport J, van Deursen JM
• BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase.
• Dev Cell. 2009; 16: 118-31
• Display abstract

• BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, we demonstrate that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. We find that the BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, our results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates.

• Li M et al.
• Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis.
• PLoS One. 2009; 4: 7701-7701
• Display abstract

• In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1-3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

• Essex A, Dammermann A, Lewellyn L, Oegema K, Desai A
• Systematic analysis in Caenorhabditis elegans reveals that the spindle checkpoint is composed of two largely independent branches.
• Mol Biol Cell. 2009; 20: 1252-67
• Display abstract

• Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1-three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1(MDF-1)/Mad2(MDF-2)-based signal, with a largely independent Mad3(SAN-1)/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2(MDF-2) levels bypasses the requirement for BUB-3 and Mad3(SAN-1) in kinetochore-dependent checkpoint activation. Mad3(SAN-1) does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2(MDF-2). We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1(MDF-1)/Mad2(MDF-2) signal generated at kinetochores is integrated with a separate cytoplasmic Mad3(SAN-1)/BUB-3-based pathway.

• Ibrahim B, Diekmann S, Schmitt E, Dittrich P
• In-silico modeling of the mitotic spindle assembly checkpoint.
• PLoS One. 2008; 3: 1555-1555
• Display abstract

• BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (M)SAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (M)SAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.

• Burton JL, Solomon MJ
• Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint.
• Genes Dev. 2007; 21: 655-67
• Display abstract

• Inappropriate attachment/tension between chromosomal kinetochores and the kinetochore microtubules activates the spindle assembly checkpoint, which delays anaphase by blocking the ubiquitin-mediated degradation of securin/Pds1p by APCCdc20. The checkpoint proteins Mad2 and Mad3/BubR1 bind to Cdc20, although how they inhibit APCCdc20 is unclear. We investigated the roles of two evolutionarily conserved KEN boxes and a D box within Mad3/BubR1. Although such motifs usually mediate APC-substrate recognition and ubiquitination, they have no apparent role in Mad3p turnover in Saccharomyces cerevisiae. Instead, these motifs are important for Mad3p function in the checkpoint and for binding to Cdc20p. We show that the Mad3p D box and KEN boxes function together to mediate Cdc20p-Mad3p interaction and that Mad3p and an anaphase-promoting complex (APC) substrate, Hsl1p, compete for Cdc20p binding in a D-box- and KEN-box-dependent manner. In vivo, we observed an increased binding of Cdc20p to Mad3p and decreased binding to Hsl1p upon checkpoint activation. Furthermore, we demonstrate that Mad2p stimulates the association between Mad3p and Cdc20p and that this stimulated binding requires KEN box 1 within Mad3p. These findings implicate Mad3p as a pseudosubstrate inhibitor of APCCdc20, competing with APC substrates for Cdc20p binding. We present a model aimed at unifying previous analyses of checkpoint function by focusing on the Mad3-Cdc20 interaction.

• Davenport J, Harris LD, Goorha R
• Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1.
• Exp Cell Res. 2006; 312: 1831-42
• Display abstract

• The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.

• Scott RJ, Lusk CP, Dilworth DJ, Aitchison JD, Wozniak RW
• Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae.
• Mol Biol Cell. 2005; 16: 4362-74
• Display abstract

• In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.

• Lee MS, Spencer FA
• Bipolar orientation of chromosomes in Saccharomyces cerevisiae is monitored by Mad1 and Mad2, but not by Mad3.
• Proc Natl Acad Sci U S A. 2004; 101: 10655-60
• Display abstract

• The spindle checkpoint governs the timing of anaphase separation of sister chromatids. In budding yeast, Mad1, Mad2, and Mad3 proteins are equally required for arrest in the presence of damage induced by antimicrotubule drugs or catastrophic loss of spindle structure. We find that the MAD genes are not equally required for robust growth in the presence of more subtle kinetochore and microtubule damage. A mad1Delta synthetic lethal screen identified 16 genes whose deletion in cells lacking MAD1 results in death or slow growth. Eleven of these mad1Delta genetic interaction partners encode proteins at the kinetochore-microtubule interface. Analysis of the entire panel revealed similar phenotypes in combination with mad2Delta. In contrast, 13 panel mutants exhibited a less severe phenotype in combination with mad3Delta. Checkpoint arrest in the absence of bipolar orientation and tension (induced by replication block in a cdc6 mutant) was lacking in cells without MAD1 or MAD2. Cells without MAD3 were checkpoint-proficient. We conclude that Mad1 and Mad2 are required to detect bipolar orientation and/or tension at kinetochores, whereas Mad3 is not.

• Larsen NA, Harrison SC
• Crystal structure of the spindle assembly checkpoint protein Bub3.
• J Mol Biol. 2004; 344: 885-92
• Display abstract

• Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal. These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids. To explore the molecular interactions mediated by Bub3, we have determined the crystal structure of the Saccharomyces cerevisiae protein Bub3p at 2.35 A resolution. Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members. Several loops are substantially elongated, but extra domains or insertions are not present at the termini. In particular, two extended loops project from the top face of the propeller, forming a cleft. Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins. We propose that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1. Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces. Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding.

• Vanoosthuyse V, Valsdottir R, Javerzat JP, Hardwick KG
• Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p.
• Mol Cell Biol. 2004; 24: 9786-801
• Display abstract

• Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

• Poddar A, Daniel JA, Daum JR, Burke DJ
• Differential kinetochore requirements for establishment and maintenance of the spindle checkpoint are dependent on the mechanism of checkpoint activation in Saccharomyces cerevisiae.
• Cell Cycle. 2004; 3: 197-204
• Display abstract

• 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.

• Reynolds N, Ohkura H
• Polo boxes form a single functional domain that mediates interactions with multiple proteins in fission yeast polo kinase.
• J Cell Sci. 2003; 116: 1377-87
• Display abstract

• Polo kinases play multiple roles in cell cycle regulation in eukaryotic cells. In addition to the kinase domain, conservation at the primary sequence level is also found in the non-catalytic region mainly in three blocks, namely the polo boxes. Although several studies have implicated the polo boxes in protein localisation, no systematic study to elucidate the roles of individual polo boxes has been carried out. Here we show, by using fission yeast, that the polo boxes form a single functional unit that is essential for both cellular function and cell-cycle-regulated localisation to the spindle pole bodies. Various polo box mutations abolish the mitotic arrest seen upon overexpression of plo1 but do not prevent the untimely septation seen under these conditions, showing that the functions of Plo1 may be separated. Plo1 interacts with multiple proteins including cell cycle regulators in a polo-box-dependent manner. Isolation of mutants that differentially disrupt these interactions revealed a role for the polo boxes in mediating protein-protein interactions.

• Yamaguchi S, Decottignies A, Nurse P
• Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint.
• EMBO J. 2003; 22: 1075-87
• Display abstract

• Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.

• Castillo AR, Meehl JB, Morgan G, Schutz-Geschwender A, Winey M
• The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p.
• J Cell Biol. 2002; 156: 453-65
• Display abstract

• Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1-8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.

• Jaspersen SL, Giddings TH Jr, Winey M
• Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p.
• J Cell Biol. 2002; 159: 945-56
• Display abstract

• Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

• Mulvihill DP, Hyams JS
• Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint.
• J Cell Sci. 2002; 115: 3575-86
• Display abstract

• In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation. Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries. We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2(+), is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells. Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A. Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation. Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1. The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN). The delay was also abolished in cells lacking Zfs1, a component of the previously described S. pombe cytokinesis checkpoint. Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner. Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation. We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.

• Yu H
• Regulation of APC-Cdc20 by the spindle checkpoint.
• Curr Opin Cell Biol. 2002; 14: 706-14
• Display abstract

• 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 anaphase-promoting 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, Mad2 and Cdc20 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.

• Hoyt MA
• A new view of the spindle checkpoint.
• J Cell Biol. 2001; 154: 909-11
• Display abstract

• Previous studies of the spindle checkpoint suggested that its ability to prevent entry into anaphase was mediated by the inhibition of the anaphase-promoting complex (APC) ubiquitin ligase by Mad2. Two new studies challenge that view by demonstrating that another checkpoint protein, BubR1, is a far more potent inhibitor of APC function.

• Stern BM, Murray AW
• Lack of tension at kinetochores activates the spindle checkpoint in budding yeast.
• Curr Biol. 2001; 11: 1462-7
• Display abstract

• 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.

• Shimizu Y, Akashi T, Okuda A, Kikuchi A, Fukui K
• NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.
• Gene. 2000; 246: 395-404
• Display abstract

• Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

• Tanaka K et al.
• Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase.
• Mol Cell Biol. 2000; 20: 3459-69
• Display abstract

• Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

• Li R
• Bifurcation of the mitotic checkpoint pathway in budding yeast.
• Proc Natl Acad Sci U S A. 1999; 96: 4989-94
• Display abstract

• The coordination of mitotic events is ensured through the spindle assembly checkpoint. BFA1 is required for this checkpoint in budding yeast because its disruption abolishes the mitotic arrest when spindle assembly is inhibited. Analysis of the genetic interaction of BFA1 with known mitotic checkpoint genes suggest that Bfa1 functions in the same pathway with Bub2 but not with Mad1 or Mad2. Both Bfa1 and Bub2 localize to spindle poles, and overexpression of Bfa1 arrests the cell cycle in anaphase. These findings suggest a bifurcation of the spindle assembly checkpoint: whereas one branch of the pathway, consisting of Mad1-3, Bub1 and 3, and Mps1, may prevent premature disjunction of sister chromosomes, the other, consisting of Bfa1 and Bub2, may function at spindle poles to prevent cytokinesis before the completion of chromosome segregation.

• Kim JH, Kang JS, Chan CS
• Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae.
• J Cell Biol. 1999; 145: 1381-94
• Display abstract

• The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.

• Tinker-Kulberg RL, Morgan DO
• Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage.
• Genes Dev. 1999; 13: 1936-49
• Display abstract

• The separation of sister chromatids in anaphase is followed by spindle disassembly and cytokinesis. These events are governed by the anaphase-promoting complex (APC), which triggers the ubiquitin-dependent proteolysis of key regulatory proteins: anaphase requires the destruction of the anaphase inhibitor Pds1, whereas mitotic exit requires the destruction of mitotic cyclins and the inactivation of Cdk1. We find that Pds1 is not only an inhibitor of anaphase, but also blocks cyclin destruction and mitotic exit by a mechanism independent of its effects on sister chromatid separation. Pds1 is also required for the mitotic arrest and inhibition of cyclin destruction that occurs after DNA damage. Even in anaphase cells, where Pds1 levels are normally low, DNA damage stabilizes Pds1 and prevents cyclin destruction and mitotic exit. Pds1 blocks cyclin destruction by inhibiting its binding partner Esp1. Mutations in ESP1 delay cyclin destruction; overexpression of ESP1 causes premature cyclin destruction in cells arrested in metaphase by spindle defects and in cells arrested in metaphase and anaphase by DNA damage. The effects of Esp1 are dependent on Cdc20 (an activating subunit of the APC) and on several additional proteins (Cdc5, Cdc14, Cdc15, Tem1) that form a regulatory network governing mitotic exit. We speculate that the inhibition of cyclin destruction by Pds1 may contribute to the ordering of late mitotic events by ensuring that mitotic exit is delayed until after anaphase is initiated. In addition, the stabilization of Pds1 after DNA damage provides a mechanism to delay both anaphase and mitotic exit while DNA repair occurs.

• Alexandru G, Zachariae W, Schleiffer A, Nasmyth K
• Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.
• EMBO J. 1999; 18: 2707-21
• Display abstract

• In yeast, anaphase entry depends on Pds1 proteolysis, while chromosome re-duplication in the subsequent S-phase involves degradation of mitotic cyclins such as Clb2. Sequential proteolysis of Pds1 and mitotic cyclins is mediated by the anaphase-promoting complex (APC). Lagging chromosomes or spindle damage are detected by surveillance mechanisms (checkpoints) which block anaphase onset, cytokinesis and DNA re-replication. Until now, the MAD and BUB genes implicated in this regulation were thought to function in a single pathway that blocks APC activity. We show that spindle damage blocks sister chromatid separation solely by inhibiting APCCdc20-dependent Pds1 proteolysis and that this process requires Mad2. Blocking APCCdh1-mediated Clb2 proteolysis and chromosome re-duplication does not require Mad2 but a different protein, Bub2. Our data imply that Mad1, Mad2, Mad3 and Bub1 regulate APCCdc20, whereas Bub2 regulates APCCdh1.

• Chan GK, Schaar BT, Yen TJ
• Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1.
• J Cell Biol. 1998; 143: 49-63
• Display abstract

• We have identified a 350-amino acid domain in the kinetochore motor CENP-E that specifies kinetochore binding in mitosis but not during interphase. The kinetochore binding domain was used in a yeast two-hybrid screen to isolate interacting proteins that included the kinetochore proteins CENP-E, CENP-F, and hBUBR1, a BUB1-related kinase that was found to be mutated in some colorectal carcinomas (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Wilson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303). CENP-F, hBUBR1, and CENP-E assembled onto kinetochores in sequential order during late stages of the cell cycle. These proteins therefore define discrete steps along the kinetochore assembly pathway. Kinetochores of unaligned chromosome exhibited stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E and hBUBR1 remain colocalized at kinetochores until mid-anaphase when hBUBR1 localized to portions of the spindle midzone that did not overlap with CENP-E. As CENP-E and hBUBR1 can coimmunoprecipitate with each other from HeLa cells, they may function as a motor-kinase complex at kinetochores. However, the complex distribution pattern of hBUBR1 suggests that it may regulate multiple functions that include the kinetochore and the spindle midzone.

• Fang G, Yu H, Kirschner MW
• The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation.
• Genes Dev. 1998; 12: 1871-83
• Display abstract

• The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1. We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC.

• Gorbsky GJ, Chen RH, Murray AW
• Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase.
• J Cell Biol. 1998; 141: 1193-205
• Display abstract

• In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.

• He X, Patterson TE, Sazer S
• The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex.
• Proc Natl Acad Sci U S A. 1997; 94: 7965-70
• Display abstract

• The spindle checkpoint monitors mitotic spindle integrity and the attachment of kinetochores to the spindle. Upon sensing a defect the checkpoint blocks cell cycle progression and thereby prevents chromosome missegregation. Previous studies in budding yeast show that the activated spindle checkpoint inhibits the onset of anaphase by an unknown mechanism. One possible target of the spindle checkpoint is anaphase promoting complex (APC), which controls all postmetaphase events that are blocked by spindle checkpoint activation. We have isolated mad2, a spindle checkpoint component in fission yeast, and shown that mad2 overexpression activates the checkpoint and causes a cell cycle arrest at the metaphase-to-anaphase transition. In addition to the observation that mad2-induced arrest can be partially relieved by mitosis-promoting factor inactivation, we present genetic evidence consistent with the hypothesis that the spindle checkpoint imposes a cell cycle arrest by inhibiting APC-dependent proteolysis.

• Li Y, Gorbea C, Mahaffey D, Rechsteiner M, Benezra R
• MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity.
• Proc Natl Acad Sci U S A. 1997; 94: 12431-6
• Display abstract

• Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

• Nicklas RB
• How cells get the right chromosomes.
• Science. 1997; 275: 632-7
• Display abstract

• When cells divide, the chromosomes must be delivered flawlessly to the daughter cells. Missing or extra chromosomes can result in birth defects and cancer. Chance events are the starting point for chromosome delivery, which makes the process prone to error. Errors are avoided by diverse uses of mechanical tension from mitotic forces. Tension stabilizes the proper chromosome configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.

• Yamamoto A, Guacci V, Koshland D
• Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s).
• J Cell Biol. 1996; 133: 99-110
• Display abstract

• We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA-damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.

• Wells WA
• The spindle-assembly checkpoint: aiming for a perfect mitosis, every time.
• Trends Cell Biol. 1996; 6: 228-34
• Display abstract

• Checkpoints reduce the frequency of errors in cell division by delaying the progress of the cell cycle until certain processes are complete. The spindle-assembly checkpoint prevents the onset of anaphase until a bipolar spindle is present and all chromosomes are attached to the spindle. Evidence from yeast and mammalian cells suggests that kinetochores are at least one source of the signal that stops the cell cycle. Recent studies in budding yeast have begun to define the signal-transduction pathway involved in the spindle-assembly checkpoint, but details of the endpoint of the pathway, where these signals interact with the cell-cycle machinery, remain to be characterized.

• Hardwick KG, Murray AW
• Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast.
• J Cell Biol. 1995; 131: 709-20
• Display abstract

• The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.

• Li R, Murray AW
• Feedback control of mitosis in budding yeast.
• Cell. 1991; 66: 519-31
• Display abstract

• We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.

© 2021 EMBL | Privacy Policy