ABSTRACT
Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination.1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules,2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3BUBR1.3,4 Here we reveal the distinct functions of Mad2 and Mad3BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3BUBR1 associates with the TOGL1 domain of Stu1CLASP, a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3BUBR1-Stu1CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3BUBR1-Stu1CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred.
ABSTRACT
Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.
Subject(s)
Kinetochores , Protozoan Proteins , Kinetochores/metabolism , Kinetochores/chemistry , Protozoan Proteins/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Trypanosoma brucei brucei/metabolism , Trypanosoma brucei brucei/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Models, Molecular , Amino Acid Sequence , Phylogeny , Protein Binding , Crystallography, X-Ray , Chromosome Segregation , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/geneticsABSTRACT
Wee1 is a cell cycle regulator that phosphorylates Cdk1/Cdc2 and inhibits G2/M transition. Loss of Wee1 in fission yeast results in an early onset of mitosis. Interestingly, we found that cells lacking Wee1 require the functional spindle checkpoint for their viability. Genetic analysis indicated that the requirement is not attributable to the early onset of mitosis. Live-cell imaging revealed that some kinetochores are not attached or bioriented in the wee1 mutant. Furthermore, Mad2, a component of the spindle checkpoint known to recognize unattached kinetochores, accumulates in the vicinity of the spindle, representing activation of the spindle checkpoint in the mutant. It appears that the wee1 mutant cannot maintain stable kinetochore-microtubule attachment, and relies on the delay imposed by the spindle checkpoint for establishing biorientation of kinetochores. This study revealed a role of Wee1 in ensuring accurate segregation of chromosomes during mitosis, and thus provided a basis for a new principle of cancer treatment with Wee1 inhibitors.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Kinetochores/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Spindle Apparatus/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Mitosis , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
OBJECTIVE: The most severe type of male infertility is non-obstructive azoospermia (NOA), where there is no sperm in the ejaculate due to failure of spermatogenesis, affecting 10%-20% of infertile men with azoospermia. Genetic studies have identified dozens of NOA genes. The main aim of the present study is to identify a novel monogenic mutation that may cause NOA. MATERIALS AND METHODS: We studied the pedigree of a consanguineous family with three NOA and one fertile brother by a family-based exome-sequencing, segregation analysis, insilico protein modeling and single-cell RNA sequencing data analysis. RESULTS: Bioinformatics analysis followed by sanger sequencing revealed that three NOA brothers were homozygous for a rare missense variant in Cyclin Dependent Kinase Regulatory Subunit Associated Protein 2 (Centrosomin) CDK5RAP2 (NM_018249:exon26:c.A4003T:p.R1335W, rs761196443). Protein modeling demonstrated that CDK5RAP2, Arg1335Trp resided nearby the Microtubule Associated Protein RP/EB Family Member 1 (EB1/MAPRE1) interaction site. As a consequence of the R1335W mutation, the positively charged Arginine was replaced by to the hydrophobic tryptophan residue, possibly leading to local instability in the structure and perturbation in the CDK5RAP2-MAPRE1 interaction. CONCLUSION: Our study reports a novel missense variant of CDK5RAP2 that segregates in homozygosity with male infertility and NOA in a consanguineous family. In silico structural predictions and gene expression data indicate a potential role of the CDK5RAP2 variant in causing defective centrosomic maturation during spermatogenesis.
Subject(s)
Azoospermia , Infertility, Male , Humans , Male , Azoospermia/genetics , Azoospermia/complications , Infertility, Male/genetics , Mutation , Mutation, Missense , Nerve Tissue Proteins/genetics , Cell Cycle Proteins/geneticsABSTRACT
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
ABSTRACT
As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.
Subject(s)
Schizosaccharomyces , Anaphase-Promoting Complex-Cyclosome/genetics , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mammals/metabolism , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Spindle Apparatus/physiologyABSTRACT
The combretastatin A-4/oltipraz hybrid (COH), 5-(3-amino-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiole-3-one (COH-203) is one of the COH compounds synthesized by our previous study, which has been reported to affect a number of cancer cell lines, such as SGC-7901, KB, HT-1080, HepG2, SMMC-7721 and BEL-7402. The sensitivity of human acute leukemia cell lines to COH-203, and the mechanism underlying its anti-proliferative effects remain unknown, which was investigated in the present study. In the present study, it was demonstrated that COH-203 had notable time- and dose-dependent antiproliferative effects on the human acute promyelocytic leukemia HL-60 cell line. Furthermore, COH-203 treatment resulted in cell cycle arrest at G2/M phase in a dose-dependent manner, and subsequently induced apoptosis. Western blot analysis revealed that upregulation of cyclin B was associated with G2/M arrest. In addition, treatment with COH-203 resulted in downregulated expression of Bcl-2. This result revealed that COH-203-induced apoptosis in HL-60 cells may occur via the mitochondrial pathway in a caspase-dependent manner.
ABSTRACT
The balance of phospho-signaling at the outer kinetochore is critical for forming accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. A major player in determining this balance is the PP2A-B56 phosphatase, which is recruited to the kinase attachment regulatory domain (KARD) of budding uninhibited by benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This unleashes a rapid, switch-like phosphatase relay that reverses mitotic phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase. Here, we demonstrate that the C-terminal pseudokinase domain of human BUBR1 is required to promote KARD phosphorylation. Mutation or removal of the pseudokinase domain results in decreased PP2A-B56 recruitment to the outer kinetochore attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. Our data, therefore, elucidate a function for the BUBR1 pseudokinase domain in ensuring accurate and timely exit from mitosis.
Subject(s)
M Phase Cell Cycle Checkpoints/physiology , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Cell Cycle Checkpoints/physiology , Cell Cycle Proteins/metabolism , Chromosomes/metabolism , HeLa Cells , Humans , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints/genetics , Mitosis , Phosphorylation , Protein Binding , Protein Domains/genetics , Protein Serine-Threonine Kinases/genetics , Spindle Apparatus/metabolismABSTRACT
Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.
ABSTRACT
In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of Xenopus and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.
Subject(s)
Invertebrates/embryology , M Phase Cell Cycle Checkpoints/physiology , Spindle Apparatus/metabolism , Animals , Cell Cycle Checkpoints/physiology , Cell Cycle Proteins/metabolism , Chromosome Segregation/physiology , Embryo, Nonmammalian/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Mitosis/physiology , Nocodazole/pharmacology , Signal Transduction/physiologyABSTRACT
Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1 The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1 Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.
Subject(s)
Cell Cycle Proteins/genetics , Gene Expression Regulation, Fungal , Protein Phosphatase 2/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Type C Phospholipases/genetics , Cell Cycle , Cell Cycle Proteins/metabolism , Chromosome Segregation , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Gene Deletion , Gene Regulatory Networks , Genes, Fungal , Protein Phosphatase 2/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Type C Phospholipases/metabolismABSTRACT
Background: Cholangiocarcinoma is a highly lethal neoplasm for which the currently available chemotherapeutic agents are suboptimal. Numerous studies show that alterations in expression of genes related to mitotic spindle and mitotic checkpoint are involved in chromosomal instability and tumor progression in various malignancies. This study aimed to evaluate these genes in cholangiocarcinoma patients. Material and methods: Different public datasets were analyzed to examine the expression of 76 selected mitotic spindle checkpoint genes including Aurora Kinase A (AURKA) in cholangiocarcinoma. Afterwards, cell number counting, CCK-8 assay, and Caspase 3/7 assay were used to explore the antitumor effect of AURKA inhibitor Alisertib in vitro. In addition, xenograft model was used to evaluate the antitumor effect of Alisertib in vivo. Furthermore, siRNA mediated silencing of AURKA was used to verify the function of AURKA in cholangiocarcinoma. Results: Components of the mitotic spindle checkpoint, including AURKA, were broadly dysregulated in human cholangiocarcinoma. High AURKA mRNA expression was associated with poor survival in cholangiocarcinoma patients within different datasets. AURKA specific inhibitor Alisertib, inhibited cell growth, induced cell cycle arrest in G2/M phase, and promoted apoptosis in cholangiocarcinoma cell lines. Additionally, Alisertib also inhibited tumor growth in a cholangiocarcinoma xenograft mouse model. Furthermore, AURKA knockdown by siRNA recapitulated the antitumor effect of Alisertib. AURKA expression was also highly correlated with its interaction proteins Polo-like kinase 1(PLK1) and Targeting protein for xenopus kinesin-like protein2 (TPX2) in different cholangiocarcinoma datasets. Conclusions: Highly expressed AURKA confers poor outcomes in cholangiocarcinoma and may represent a rational therapeutic target.
ABSTRACT
The DNA damage response recognizes DNA lesions and coordinates a cell cycle arrest with the repair of the damaged DNA, or removal of the affected cells to prevent the passage of genetic alterations to the next generation. The mitotic cell division, on the other hand, is a series of processes that aims to accurately segregate the genomic material from the maternal to the two daughter cells. Despite their great importance in safeguarding genomic integrity, the DNA damage response and the mitotic cell division were long viewed as unrelated processes, mainly because animal cells that are irradiated during mitosis continue cell division without repairing the broken chromosomes. However, recent studies have demonstrated that DNA damage proteins play an important role in mitotic cell division. This is performed through regulation of the onset of mitosis, mitotic spindle formation, correction of misattached kinetochore-microtubules, spindle checkpoint signaling, or completion of cytokinesis (abscission), in the absence of DNA damage. In this review, we summarize the roles of DNA damage proteins in unperturbed mitosis, analyze the molecular mechanisms involved, and discuss the potential implications of these findings in cancer therapy.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , Genomic Instability , Mitosis/genetics , Animals , Cell Division , HumansABSTRACT
CHK1 and WEE1 play pivotal roles in G2/M checkpoint following exogenous DNA damage and regulation of DNA replication under normal cellular conditions. Here, we monitored and compared the cell cycle kinetics of mitosis-associated events after CHK1 and WEE1 inhibitor treatments in a human tongue cancer cell line (SAS). A fluorescent ubiquitination-based cell cycle indicator (Fucci) that reflects SCFSKP2 and APCCDH1 E3 ligase activities was used to monitor cell cycle progression. Numerous γH2AX-positive cells were observed within the S phase population of cells following CHK1 inhibitor treatment, and polyploid cells exhibiting DNA damage emerged via abortive mitosis (endomitosis) at 24 h post treatment. While WEE1 inhibitor-treated cells exhibited similar polyploidy via endomitosis at later time points, they possessed fewer γH2AX foci during S phase, and polyploid cells exhibiting DNA damage were scarce. Instead, mitosis duration greatly extended and was accompanied by an abnormal emission of Fucci red fluorescence. Kinetic analysis of Fucci fluorescence revealed that abnormal emission occurred at early M phase in a manner independent of green fluorescence degradation as a marker of APCCDH1 activation. When an inhibitor of the essential spindle checkpoint factor MPS1 was co-treated with a WEE1 inhibitor, the elongated mitosis duration and abnormal red fluorescence were abrogated, and WEE1-induced reduction of clonogenic survival was offset. We demonstrate novel differential effects on mitosis-associated events following CHK1 and WEE1 inhibitor treatments.
Subject(s)
Cell Cycle Proteins/genetics , Checkpoint Kinase 1/genetics , Epithelial Cells/drug effects , Gene Expression Regulation, Neoplastic , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/genetics , Cdh1 Proteins/genetics , Cdh1 Proteins/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Checkpoint Kinase 1/antagonists & inhibitors , Checkpoint Kinase 1/metabolism , DNA Damage , Epithelial Cells/metabolism , Epithelial Cells/pathology , Flow Cytometry , G2 Phase Cell Cycle Checkpoints/drug effects , Genes, Reporter , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Mitosis/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , S Phase/drug effects , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Time-Lapse ImagingABSTRACT
The effects of CHK1 inhibitor on cell cycle kinetics have not been fully investigated yet. In this study, we closely analyzed this kinetics using a CHK1 inhibitor (PF00477736) in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). This system allowed us to visualize cell cycle progression following CHK1 inhibitor treatment in real-time. FACS analysis showed that high levels of DNA damage as determined by γH2AX immunostaining was induced in S phase and that polyploid cells harboring the same levels of DNA damage appeared thereafter. Surprisingly, time-lapse imaging of Fucci fluorescence revealed that many cells entered M phase at once and exhibited prolonged mitosis; eventually progressing to G1 phase not accompanied by cytokinesis; this is an endomitosis-like event. Most of these cells then underwent S/G2 phases at least once, which corroborated the appearance of polyploid cells. However, a small fraction of cells with 2â¯N DNA content still remained 24â¯h after the treatment. When co-treated with MAD2 inhibitor, a core factor constituting spindle checkpoint, the 2â¯N DNA cell fraction disappeared and almost all cells exhibited endomitosis, leading to enhanced sensitivity. Detailed cell cycle analysis revealed that induction of an endomitosis-like event might be associated with CHK1 inhibitor-induced cell death in HeLa cells.
Subject(s)
Benzodiazepinones/pharmacology , Checkpoint Kinase 1/antagonists & inhibitors , Checkpoint Kinase 1/metabolism , Mitosis/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Cell Cycle/drug effects , Cell Survival , DNA Damage , Fluorescence , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints/drug effects , Mad2 Proteins/antagonists & inhibitors , Mad2 Proteins/metabolism , Phosphorylation/drug effects , Polyploidy , Time-Lapse ImagingABSTRACT
In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. Here, we show that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/metabolism , Caenorhabditis elegans/metabolism , Cdc20 Proteins/metabolism , Cyclin B/metabolism , G2 Phase , Mitosis , Proteolysis , Animals , Caenorhabditis elegans Proteins/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinases/metabolism , Fertility , Humans , Models, Biological , Phosphorylation , Protein Binding , Spindle Apparatus/metabolismABSTRACT
The spindle checkpoint monitors kinetochore-microtubule interactions and generates a "wait anaphase" delay when any defects are apparent [1-3]. This provides time for cells to correct chromosome attachment errors and ensure high-fidelity chromosome segregation. Checkpoint signals are generated at unattached chromosomes during mitosis. To activate the checkpoint, Mps1Mph1 kinase phosphorylates the kinetochore component KNL1Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [4-6] via a direct Bub3 interaction with phospho-MELT motifs [7, 8]. Mps1Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-Mad2 complexes to produce a signaling platform [9, 10]. The Bub1-Mad1 platform is thought to recruit Mad3, Cdc20, and Mad2 to produce the mitotic checkpoint complex (MCC), which is the diffusible wait anaphase signal [9, 11, 12]. The MCC binds and inhibits the mitotic E3 ubiquitin ligase, known as Cdc20-anaphase promoting complex/cyclosome (APC/C), and stabilizes securin and cyclin to delay anaphase onset [13-17]. Here we demonstrate, in both budding and fission yeast, that kinetochores and KNL1Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, Mad2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [9], and Bub1's highly conserved N-terminal tetratricopeptide repeat (TPR) domain [18, 19]. We demonstrate that the Bub1 TPR domain is both necessary and sufficient to bind and recruit Mad3. We propose that this brings Mad3 into close proximity to Mad1-Mad2 and Mps1Mph1 kinase, enabling efficient generation of MCC complexes.
Subject(s)
Cell Cycle Proteins/genetics , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Tetratricopeptide Repeat/genetics , Cell Cycle Proteins/metabolism , M Phase Cell Cycle Checkpoints , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Successful mitosis requires that cyclin B1:CDK1 kinase activity remains high until chromosomes are correctly aligned on the mitotic spindle. It has therefore been unclear why, in mammalian oocyte meiosis, cyclin B1 destruction begins before chromosome alignment is complete. Here, we resolve this paradox and show that mouse oocytes exploit an imbalance in the ratio of cyclin B1 to CDK1 to control CDK1 activity; early cyclin B1 destruction reflects the loss of an excess of non-CDK1-bound cyclin B1 in late prometaphase, while CDK1-bound cyclin B1 is destroyed only during metaphase. The ordered destruction of the two forms of cyclin B1 is brought about by a previously unidentified motif that is accessible in free cyclin B1 but masked when cyclin B1 is in complex with CDK1. This protects the CDK1-bound fraction from destruction in prometaphase, ensuring a period of prolonged CDK1 activity sufficient to achieve optimal chromosome alignment and prevent aneuploidy.
Subject(s)
Aneuploidy , CDC2 Protein Kinase/metabolism , Cyclin B1/genetics , Oocytes/metabolism , Animals , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , Female , Meiosis/physiology , Mice , Mitosis/physiology , Spindle Apparatus/metabolismABSTRACT
The spindle checkpoint ensures accurate chromosome segregation during mitosis and guards against aneuploidy. Insulin signaling governs metabolic homeostasis and cell growth, and its dysregulation leads to metabolic disorders, such as diabetes. These critical pathways have been extensively investigated, but a link between the two has not been established until recently. Our recent study reveals a critical role of spindle checkpoint regulators in insulin signaling and metabolic homeostasis through regulating endocytosis of the insulin receptor (IR). These findings have linked spindle checkpoint proteins to metabolic regulation, expanding the connection between cell division and metabolism. Here, we briefly review the unexpected roles of spindle checkpoint regulators in vesicle trafficking and insulin signaling.