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Cell Reports Jul 2018The key mitotic regulator Polo-like kinase 1 (Plk1) is activated during G2 phase by Aurora A kinase (AurkA)-mediated phosphorylation of its activation loop, which is...
The key mitotic regulator Polo-like kinase 1 (Plk1) is activated during G2 phase by Aurora A kinase (AurkA)-mediated phosphorylation of its activation loop, which is important for timely mitotic entry. The mechanism for Plk1 activation remains incompletely understood. Here, we report that the activation of Plk1 requires WAC, a WW domain-containing adaptor protein with a coiled-coil region that predominantly localizes to the nucleus in interphase. Cyclin-dependent kinase 1 (Cdk1) phosphorylates WAC, priming its direct interaction with the polo-box domain of Plk1. Knockdown of WAC compromises Plk1 activity and delays mitotic entry. These defects are rescued by exogenous expression of wild-type WAC, but not the Plk1-binding-deficient mutant. WAC also binds AurkA and can enhance Plk1 phosphorylation by AurkA in vitro. Taken together, these results indicate an important role for WAC in promoting Plk1 activation and the timely entry into mitosis.
Topics: Adaptor Proteins, Signal Transducing; CDC2 Protein Kinase; Cell Cycle Proteins; Enzyme Activation; G2 Phase; HeLa Cells; Humans; Metaphase; Mitosis; Phosphorylation; Protein Binding; Protein Domains; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 30021153
DOI: 10.1016/j.celrep.2018.06.087 -
Nature Apr 2006A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this...
A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.
Topics: Animals; CDC2 Protein Kinase; Cell Line; Cells, Cultured; Cyclin B; Cytokinesis; Flavonoids; G1 Phase; HeLa Cells; Humans; Keratinocytes; Metaphase; Mitosis; Models, Biological; Nocodazole; Piperidines; Proteasome Endopeptidase Complex; Xenopus
PubMed: 16612388
DOI: 10.1038/nature04652 -
Virology Sep 2016In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant....
In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy-electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division.
Topics: Antigens, Viral; Cell Line; Centromere; Chromosome Mapping; Chromosomes, Human; DNA, Viral; Fluorescent Antibody Technique, Indirect; Genome, Viral; Herpesvirus 8, Human; Humans; Metaphase; Microscopy, Confocal; Mitosis; Nuclear Proteins; Virus Latency
PubMed: 27254595
DOI: 10.1016/j.virol.2016.05.020 -
Developmental Cell Apr 2019During mitosis, motor proteins associate with microtubules to exert pushing forces that establish a mitotic spindle. These pushing forces generate opposing tension in...
During mitosis, motor proteins associate with microtubules to exert pushing forces that establish a mitotic spindle. These pushing forces generate opposing tension in the chromatin that connects oppositely attached sister chromatids, which may then act as a mechanical signal to ensure the fidelity of chromosome segregation during mitosis. However, the role of tension in mitotic cellular signaling remains controversial. In this study, we generated a gradient in tension over multiple isogenic budding yeast cell lines by genetically altering the magnitude of motor-based spindle forces. We found that a decreasing gradient in tension led to an increasing gradient in the rates of kinetochore detachment and anaphase chromosome mis-segregration, and in metaphase time. Simulations and experiments indicated that these tension responses originate from a tension-dependent kinetochore phosphorylation gradient. We conclude that the cell is exquisitely tuned to the magnitude of tension as a signal to detect potential chromosome segregation errors during mitosis.
Topics: Centromere; Chromatids; Chromatin; Chromosome Segregation; Kinetochores; Mechanical Phenomena; Metaphase; Microtubules; Mitosis; Saccharomyces cerevisiae; Spindle Apparatus
PubMed: 30799228
DOI: 10.1016/j.devcel.2019.01.018 -
The Journal of Cell Biology Mar 2013Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a...
Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150(glued), a subunit of the dynein-dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.
Topics: Anaphase; Cell Cycle Proteins; Dynactin Complex; Dyneins; HeLa Cells; Humans; Metaphase; Microfilament Proteins; Microtubule-Associated Proteins; Microtubules; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Spindle Apparatus; Polo-Like Kinase 1
PubMed: 23509069
DOI: 10.1083/jcb.201207050 -
Molecular and Cellular Biology Apr 2009Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is a nonredundant and essential gene in all eukaryotes. During the mitotic cell cycle,...
Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is a nonredundant and essential gene in all eukaryotes. During the mitotic cell cycle, ODC exhibits two activity peaks: one at the G(1)/S transition and one during the G(2)/M transition. The physiological role of this cell cycle-dependent ODC activity dynamic is not clear. Previous studies have reported a significant elevation of ODC activity during Xenopus oocyte maturation, which resembles mitotic G(2)/M transition. In order to study the roles of ODC activity in the oocytes, we utilized antisense morpholino (xODC mo) oligonucleotides to inhibit ODC translation. We report here that xODC mo abolished ODC activity increase during oocyte maturation. xODC mo-injected oocytes underwent germinal vesicle breakdown, emitted the first polar body, and reached metaphase II, thus completing nuclear maturation. However, the metaphase II oocytes exhibited high levels of reactive oxygen species and became apoptotic. When transferred to host frogs and subsequently ovulated, these eggs were fertilized but exhibited embryo fragmentation. Translation of ODC is therefore integral to cytoplasmic maturation, protecting metaphase II oocytes from reactive oxygen species-induced apoptosis.
Topics: Animals; Apoptosis; Cytochromes c; Embryo, Nonmammalian; Female; Metaphase; Oligonucleotides, Antisense; Oocytes; Oogenesis; Ornithine Decarboxylase; Polyamines; Protein Biosynthesis; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Xenopus
PubMed: 19158268
DOI: 10.1128/MCB.01815-08 -
Proceedings of the National Academy of... Nov 2021DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions....
DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.
Topics: Cell Line, Tumor; Chromatin; Chromosomes; DNA; HCT116 Cells; Humans; Metaphase; X-Ray Diffraction; X-Rays
PubMed: 34750262
DOI: 10.1073/pnas.2109921118 -
PLoS Genetics Nov 2020In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins...
In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Animals; Animals, Genetically Modified; Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; CDC2 Protein Kinase; Cdc20 Proteins; Cell Line; Chromosome Segregation; Cyclin B; Drosophila Proteins; Drosophila melanogaster; Female; Loss of Function Mutation; Mad2 Proteins; Male; Metaphase; Models, Animal; Mutagenesis; Phosphoprotein Phosphatases; Phosphorylation
PubMed: 33137813
DOI: 10.1371/journal.pgen.1009184 -
Cell Cycle (Georgetown, Tex.) Aug 2020The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in...
UNLABELLED
The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events.
ABBREVIATIONS
AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.
Topics: Anaphase; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chromatin; Chromosomes, Mammalian; Guanine Nucleotide Exchange Factors; Indoleacetic Acids; Mammals; Metaphase; Mitosis; Models, Biological; Neoplasm Proteins; Nuclear Proteins; Protein Binding; Proteolysis; Signal Transduction; Spindle Apparatus; Xenopus laevis; ran GTP-Binding Protein
PubMed: 32594833
DOI: 10.1080/15384101.2020.1782036 -
Journal of Clinical Pathology Dec 2004Aneuploidy is frequently noted in malignant tumours. There is much controversy about its cause and effect in relation to malignant tumours. Failure of the spindle... (Review)
Review
Aneuploidy is frequently noted in malignant tumours. There is much controversy about its cause and effect in relation to malignant tumours. Failure of the spindle checkpoint caused by mutation of the responsible genes may be one of the important factors for the development of aneuploidy. Telomere dysfunction may also be a possible source of failure of cytokinesis resulting in aneuploidy. Evidence such as tumour specific aneuploidy, presence of aneuploidy in various preneoplastic conditions, increased frequency of genetic instability in aneuploid cell lines compared with diploid cells, and mutation of mitotic checkpoint genes suggests that aneuploidy possibly plays an active role in carcinogenesis. In this brief review, the various aspects of aneuploidy with special emphasis on its mechanism of development and impact on progression of cancer are discussed.
Topics: Aneuploidy; Cytokinesis; DNA, Neoplasm; Genes, Tumor Suppressor; Humans; Interphase; Metaphase; Mitosis; Mutation; Neoplasms; Oncogenes; Phenotype; Precancerous Conditions; Telomere
PubMed: 15563660
DOI: 10.1136/jcp.2004.018952