In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.

• MeSH
• Anaphase physiology   MeSH
• Anaphase-Promoting Complex-Cyclosome MeSH
• Cell Line MeSH
• Cell Cycle physiology   MeSH
• Cyclin A metabolism   MeSH
• Cyclin B metabolism   MeSH
• Cyclin B1 MeSH
• DNA-Binding Proteins * MeSH
• Phosphorylation MeSH
• Ubiquitin-Protein Ligase Complexes * MeSH
• Humans MeSH
• Ligases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Retinoblastoma Protein metabolism   MeSH
• S Phase MeSH
• Transcription Factor DP1 MeSH
• E2F Transcription Factors MeSH
• Transcription Factors metabolism   MeSH
• Carrier Proteins * MeSH
• Ubiquitin-Protein Ligases MeSH
• Retinoblastoma-Binding Protein 1 MeSH
• Check Tag
• Humans 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

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

• MeSH
• Anaphase-Promoting Complex-Cyclosome MeSH
• Cell Cycle * MeSH
• Cyclin E metabolism   MeSH
• Cyclin-Dependent Kinase 2 MeSH
• Cyclin-Dependent Kinases metabolism   MeSH
• DNA-Binding Proteins * MeSH
• Fluorescent Antibody Technique MeSH
• Interphase drug effects   MeSH
• CDC2-CDC28 Kinases * MeSH
• Ubiquitin-Protein Ligase Complexes * MeSH
• Humans MeSH
• Ligases * metabolism   MeSH
• Macromolecular Substances MeSH
• Mitosis * MeSH
• Tumor Cells, Cultured MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Cdc20 Proteins MeSH
• Drosophila Proteins * MeSH
• Antibodies pharmacology   MeSH
• Flow Cytometry MeSH
• DNA Replication * MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Trans-Activators * MeSH
• Transcription Factor DP1 MeSH
• E2F Transcription Factors MeSH
• Transcription Factors metabolism   MeSH
• Carrier Proteins * MeSH
• Ubiquitin-Protein Ligases MeSH
• Protein Binding MeSH
• Retinoblastoma-Binding Protein 1 MeSH
• Blotting, Western MeSH
• Check Tag
• Humans 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

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Aneuploidy * MeSH
• Cell Culture Techniques methods   MeSH
• Chromosome Aberrations * MeSH
• Meiosis physiology   MeSH
• Mice MeSH
• Oocytes cytology   metabolism   MeSH
• Chromosome Segregation * MeSH
• Temperature * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article 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

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCF(betaTrCP)-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase-specific, SCF(betaTrCP)-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCF(betaTrCP)- but not APC(Cdh1)-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR-Chk1 pathway.

• MeSH
• Adaptor Proteins, Signal Transducing * metabolism   MeSH
• Anaphase-Promoting Complex-Cyclosome MeSH
• Cell Line MeSH
• Phosphorylation MeSH
• G1 Phase MeSH
• Ubiquitin-Protein Ligase Complexes * metabolism   physiology   MeSH
• Humans MeSH
• DNA Damage MeSH
• Protein Kinases metabolism   MeSH
• SKP Cullin F-Box Protein Ligases * metabolism   physiology   MeSH
• Substrate Specificity MeSH
• Ubiquitin Thiolesterase * metabolism   physiology   MeSH
• Ubiquitination physiology   MeSH
• Check Tag
• Humans MeSH

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Arabidopsis cytology   genetics   MeSH
• Models, Biological MeSH
• Chromosomes, Plant genetics   MeSH
• Cyclins genetics   metabolism   MeSH
• Genes, Dominant MeSH
• Phosphorylation MeSH
• Phosphothreonine metabolism   MeSH
• Epistasis, Genetic MeSH
• Genetic Testing MeSH
• Meiosis * MeSH
• Mutation genetics   MeSH
• Protein Subunits metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Tetraploidy MeSH
• Tubulin metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Embryo, Mammalian drug effects   metabolism   MeSH
• Embryonic Development drug effects   MeSH
• M Phase Cell Cycle Checkpoints * MeSH
• Mice MeSH
• Oocytes drug effects   growth & development   metabolism   MeSH
• Prodrugs MeSH
• Cattle MeSH
• Tosylarginine Methyl Ester administration & dosage   pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Cattle MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Kinetochores metabolism   MeSH
• Microscopy, Confocal MeSH
• Meiosis physiology   MeSH
• Mice MeSH
• Oocytes growth & development   MeSH
• Microtubule-Organizing Center metabolism   MeSH
• Image Processing, Computer-Assisted MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Chromosome Segregation physiology   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH