Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

• MeSH
• Anaphase * MeSH
• Spindle Apparatus metabolism   MeSH
• Chromatids * metabolism   MeSH
• Mitosis MeSH
• Cell Cycle Proteins metabolism   MeSH
• Chromosome Segregation MeSH
• Separase genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

• MeSH
• Anaphase MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• cdc25 Phosphatases MeSH
• Meiosis * MeSH
• Metaphase MeSH
• Mice MeSH
• Oocytes * metabolism   MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.

• MeSH
• Anaphase MeSH
• Spindle Apparatus * metabolism   MeSH
• Phosphorylation MeSH
• HeLa Cells MeSH
• Humans MeSH
• Mitosis genetics   MeSH
• Cell Cycle Proteins * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.

• MeSH
• Anaphase * MeSH
• Cyclin A1 physiology   MeSH
• Cyclin A2 physiology   MeSH
• Microscopy, Fluorescence MeSH
• Meiosis MeSH
• Metaphase MeSH
• Microinjections MeSH
• Mice MeSH
• Oocytes cytology   MeSH
• Proteasome Endopeptidase Complex physiology   MeSH
• Chromosome Segregation * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.

• MeSH
• Anaphase MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Spindle Apparatus metabolism   MeSH
• Blastocyst MeSH
• Time-Lapse Imaging MeSH
• Centrosome metabolism   MeSH
• Kinetics MeSH
• Kinetochores metabolism   MeSH
• Microscopy, Confocal MeSH
• Mitosis MeSH
• Mice MeSH
• Protein Serine-Threonine Kinases antagonists & inhibitors   metabolism   MeSH
• Cell Cycle Proteins antagonists & inhibitors   metabolism   MeSH
• Proto-Oncogene Proteins antagonists & inhibitors   metabolism   MeSH
• Pteridines pharmacology   MeSH
• Animals MeSH
• Zygote drug effects   metabolism   MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.

• MeSH
• Anaphase drug effects   MeSH
• Spindle Apparatus drug effects   metabolism   MeSH
• Arabidopsis cytology   drug effects   enzymology   MeSH
• Butadienes pharmacology   MeSH
• Cytokinesis drug effects   MeSH
• Extracellular Signal-Regulated MAP Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Stress, Physiological * drug effects   MeSH
• Kinetochores drug effects   metabolism   MeSH
• Meristem cytology   drug effects   metabolism   MeSH
• Microtubules drug effects   metabolism   MeSH
• Mitogen-Activated Protein Kinases metabolism   MeSH
• Nitriles pharmacology   MeSH
• Nitrosation drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Plant Cells drug effects   metabolism   MeSH
• Chromosome Segregation drug effects   MeSH
• Telophase drug effects   MeSH
• Tubulin metabolism   MeSH
• Tyrosine analogs & derivatives   pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs. Copyright © 2015, American Association for the Advancement of Science.

• MeSH
• Anaphase MeSH
• Aneuploidy * MeSH
• Spindle Apparatus * metabolism   MeSH
• Kinetochores metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Meiosis * MeSH
• Mice MeSH
• Oocytes * pathology   MeSH
• Microtubule-Organizing Center metabolism   MeSH
• Microtubule-Associated Proteins genetics   metabolism   MeSH
• ran GTP-Binding Protein metabolism   MeSH
• Chromosome Segregation * MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

• MeSH
• Anaphase MeSH
• Aneuploidy MeSH
• Time-Lapse Imaging methods   MeSH
• Histones genetics   metabolism   MeSH
• Kinetochores metabolism   MeSH
• Ubiquitin-Protein Ligase Complexes genetics   metabolism   MeSH
• Microscopy, Confocal methods   MeSH
• M Phase Cell Cycle Checkpoints MeSH
• Metaphase MeSH
• Microinjections MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oocytes cytology   metabolism   MeSH
• Chromosome Pairing * MeSH
• Proteolysis MeSH
• Chromosomes, Mammalian genetics   metabolism   MeSH
• Mammals MeSH
• Chromosome Segregation * MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Tubulin genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Telomeres, as elaborate nucleo-protein complexes, ensure chromosomal stability. When impaired, the ends of linear chromosomes can be recognised by cellular repair mechanisms as double-strand DNA breaks and can be healed by non-homologous-end-joining activities to produce dicentric chromosomes. During cell divisions, particularly during anaphase, dicentrics can break, thus producing naked chromosome tips susceptible to additional unwanted chromosome fusion. Many telomere-building protein complexes are associated with telomeres to ensure their proper capping function. It has been found however, that a number of repair complexes also contribute to telomere stability. RESULTS: We used Arabidopsis thaliana to study the possible functions of the DNA repair subunit, NBS1, in telomere homeostasis using knockout nbs1 mutants. The results showed that although NBS1-deficient plants were viable, lacked any sign of developmental aberration and produced fertile seeds through many generations upon self-fertilisation, plants also missing the functional telomerase (double mutants), rapidly, within three generations, displayed severe developmental defects. Cytogenetic inspection of cycling somatic cells revealed a very early onset of massive genome instability. Molecular methods used for examining the length of telomeres in double homozygous mutants detected much faster telomere shortening than in plants deficient in telomerase gene alone. CONCLUSIONS: Our findings suggest that NBS1 acts in concert with telomerase and plays a profound role in plant telomere renewal.

• MeSH
• Anaphase MeSH
• Arabidopsis cytology   enzymology   genetics   growth & development   MeSH
• Chromosomal Instability MeSH
• Chromosomes, Plant genetics   metabolism   MeSH
• Cytogenetic Analysis MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Telomere Homeostasis MeSH
• In Situ Hybridization, Fluorescence MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Germination MeSH
• Flowers cytology   genetics   metabolism   MeSH
• Protein Interaction Mapping MeSH
• Meiosis MeSH
• DNA Repair MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Plant Cells enzymology   metabolism   MeSH
• Self-Fertilization MeSH
• Seeds genetics   growth & development   metabolism   MeSH
• Telomerase genetics   metabolism   MeSH
• Telomere genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

• MeSH
• Anaphase MeSH
• Anaphase-Promoting Complex-Cyclosome MeSH
• Cell Cycle MeSH
• Cyclin A * metabolism   MeSH
• Cyclin-Dependent Kinase 2 MeSH
• Cyclin-Dependent Kinases metabolism   MeSH
• Fibroblasts cytology   metabolism   MeSH
• G1 Phase MeSH
• CDC2-CDC28 Kinases * MeSH
• Ubiquitin-Protein Ligase Complexes * MeSH
• Conserved Sequence * MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Ligases genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Tumor Cells, Cultured MeSH
• Protein Serine-Threonine Kinases * metabolism   MeSH
• S Phase MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Ubiquitin-Protein Ligases MeSH
• Ubiquitins metabolism   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH