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ELife Nov 2017The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we...
The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.
Topics: CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line; Chromosome Segregation; Cyclin A; Humans; Kinetochores; Microtubules; Myosin-Light-Chain Phosphatase; Phosphorylation; Prometaphase; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 29154753
DOI: 10.7554/eLife.29303 -
PloS One 2011During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our...
BACKGROUND
During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes.
METHODOLOGY/PRINCIPAL FINDINGS
It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest.
CONCLUSIONS/SIGNIFICANCE
These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.
Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cyclin B; Cyclin B1; Cyclin-Dependent Kinases; Female; Flow Cytometry; Humans; Microscopy, Fluorescence; Mitosis; Nocodazole; Prometaphase; RNA, Small Interfering
PubMed: 21918689
DOI: 10.1371/journal.pone.0024312 -
Mutation Research Jul 2008The aim of this study was to determine the effect of alloxan, an inhibitor of N-acetylglucosaminyl transferase that acts during the G2/M transition, on the course of...
The aim of this study was to determine the effect of alloxan, an inhibitor of N-acetylglucosaminyl transferase that acts during the G2/M transition, on the course of mitosis in murine bone marrow cells. Mitotic cells from animals treated with different doses of alloxan were analyzed for the frequency of prometaphasic and metaphasic chromosomes based on their morphology and length. The results indicate that alloxan treatment substantially increases the frequency of prometaphase chromosomes. This suggests that N-acetylglucosaminyl transferase is also involved in the G2/M transition in bone marrow cells. Alloxan treatment also provides a method for obtaining large chromosomes for the analysis of chromosome bands, FISH and sister-chromatid exchanges.
Topics: Alloxan; Animals; Bone Marrow Cells; Chromosomes; Male; Mice; Mice, Inbred BALB C; Mitosis; Prometaphase
PubMed: 18606246
DOI: 10.1016/j.mrgentox.2008.06.006 -
Cell Aug 2011Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic...
Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.
Topics: Animals; Cell Line; Centromere; Chromosomes; Humans; Kinetochores; Mice; Microtubules; Models, Molecular; Prometaphase; Spindle Apparatus
PubMed: 21854981
DOI: 10.1016/j.cell.2011.07.012 -
In Vitro Dec 1984Six different techniques were evaluated to define better those technical factors that are most critical for obtaining prometaphase cells for banding analysis. Our... (Comparative Study)
Comparative Study
Six different techniques were evaluated to define better those technical factors that are most critical for obtaining prometaphase cells for banding analysis. Our results demonstrate: colcemid exposures of 30 min or less have no effect on increasing the yield of prometaphase cells, colcemid exposures of greater than 0.1 microgram/ml can be toxic, methotrexate depresses the mitotic index significantly and seems to increase the incidence of prometaphase cells only because it suppresses later forms; and (d) the optimum number of cytogenetically satisfactory prometaphase cells can be obtained with a 4-h exposure to a combination of low concentration actinomycin D (0.5 microgram/ml) and colcemid (0.1 microgram/ml). This technique inhibits chromosome condensation while permitting prometaphase cells to accumulate for 4 h.
Topics: Chromosome Aberrations; Chromosome Banding; Dactinomycin; Demecolcine; Ethidium; Female; Humans; Male; Methotrexate; Mitotic Index; Prophase
PubMed: 6530228
DOI: 10.1007/BF02619667 -
Cell Cycle (Georgetown, Tex.) Oct 2010The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction...
The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.
Topics: Amino Acid Sequence; Anaphase-Promoting Complex-Cyclosome; Antigens, CD; Cadherins; Cdc20 Proteins; Cell Cycle Proteins; Checkpoint Kinase 1; Checkpoint Kinase 2; E2F1 Transcription Factor; HeLa Cells; Humans; Prometaphase; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Small Interfering; Transcription Factor DP1; Ubiquitin-Protein Ligase Complexes
PubMed: 20948288
DOI: 10.4161/cc.9.19.13162 -
Molecular Biology of the Cell Apr 2011Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase...
Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.
Topics: Animals; CDC2 Protein Kinase; Cell Cycle Proteins; Cyclin B; Cyclin-Dependent Kinases; Feedback, Physiological; Female; G2 Phase; Gene Expression; HeLa Cells; Humans; Membrane Proteins; Mitosis; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Prometaphase; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; S Phase; Xenopus Proteins; Xenopus laevis; cdc25 Phosphatases
PubMed: 21325631
DOI: 10.1091/mbc.E10-07-0599 -
Developmental Biology Feb 2021The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental...
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 °C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.
Topics: Animals; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Checkpoints; Cyclin B; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Enzyme Activation; Metaphase; Mitosis; Prometaphase; Prophase; Protein Phosphatase 1; Temperature
PubMed: 33278404
DOI: 10.1016/j.ydbio.2020.11.010 -
Journal of Cell Science Apr 2018During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in...
During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.
Topics: Anaphase; Aurora Kinase A; Azepines; Cell Line, Tumor; Chromatids; Chromosome Segregation; Gene Expression Regulation, Enzymologic; Humans; Kinetochores; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Microtubules; Mitosis; Nocodazole; Paclitaxel; Prometaphase; Pyrimidines; Spindle Apparatus
PubMed: 29555820
DOI: 10.1242/jcs.191353 -
Biochimie Sep 2014Pterocarpans, a family of isoflavonoids found in the diverse Fabaceae, display potent cytotoxic activity over a panel of tumor cell lines, and among those tested, 2,3,9-...
Pterocarpans, a family of isoflavonoids found in the diverse Fabaceae, display potent cytotoxic activity over a panel of tumor cell lines, and among those tested, 2,3,9- trimethoxypterocarpan displays the most potent activity. This study evaluates the effects of 2,3,9-trimethoxypterocarpan and its related derivatives on cell cycle progression and microtubule function in select breast cancer cell lines (MCF7, T47d and HS578T). The pterocarpans, with the exception of 3,4-dihydroxy-9-methoxipterocarpan, induced increased frequencies of mitotic cells by inducing arrest in prometaphase. While microtubule organization in interphase cells was not modified during treatment, mitotic cells exhibited high frequencies of monastral spindles surrounded by condensed chromosomes. Immunofluorescence staining with an anti-γ-tubulin antibody showed double-dot labeling in the spindle polar region, suggesting that pterocarpan treatment blocked centrosome segregation. We found that this mitotic arrest was reversible when the cells were treated for up to 24 h followed by recovery in drug-free medium, but not after 48-h treatment followed by incubation in drug-free medium. In that case, treated cells typically underwent cell multinucleation and apoptosis.
Topics: Antineoplastic Agents; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Humans; Membrane Potential, Mitochondrial; Prometaphase; Prophase; Pterocarpans; Time Factors
PubMed: 24952350
DOI: 10.1016/j.biochi.2014.06.005