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Current Biology : CB Sep 2010The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However,...
The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.
Topics: Antineoplastic Agents; Cell Line; Cell Proliferation; Humans; Leupeptins; Mitosis; Nocodazole; Prometaphase
PubMed: 20832310
DOI: 10.1016/j.cub.2010.08.018 -
The Journal of Cell Biology Oct 2011Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that...
Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.
Topics: Amino Acid Sequence; Animals; Aurora Kinase B; Aurora Kinases; CHO Cells; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Cricetinae; Enzyme Activation; Humans; Kinetochores; Microscopy, Fluorescence; Mitosis; Molecular Sequence Data; Paclitaxel; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Serine
PubMed: 22024163
DOI: 10.1083/jcb.201104023 -
Molecular Biology of the Cell Jan 2004Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic...
Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.
Topics: Anaphase; Animals; Cell Nucleus; Cells, Cultured; Chromatids; Chromosome Positioning; Cloning, Molecular; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Kinesins; Metaphase; Microinjections; Microscopy, Fluorescence; Microtubules; Models, Molecular; Mutation; RNA, Small Interfering; Spindle Apparatus; Telophase
PubMed: 14528012
DOI: 10.1091/mbc.e03-07-0489 -
Molecular Systems Biology Aug 2020The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human...
The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.
Topics: Cell Nucleolus; Chromosomes, Human; HEK293 Cells; Humans; Ki-67 Antigen; Microscopy, Confocal; Mitosis; Nuclear Proteins; Phenotype; Proteomics; Single-Cell Analysis
PubMed: 32744794
DOI: 10.15252/msb.20209469 -
Cell Death and Differentiation Jul 2011Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP...
Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.
Topics: Aneuploidy; Apoptosis; Calcium-Binding Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Chromosome Segregation; Humans; Kinetochores; Mad2 Proteins; Metaphase; Mitosis; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Repressor Proteins
PubMed: 21274008
DOI: 10.1038/cdd.2010.189 -
ELife Oct 2017The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein...
The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.
Topics: Cell Cycle; Cell Line; Flow Cytometry; Humans; Immunohistochemistry; Mass Spectrometry; Proteome; Proteomics
PubMed: 29052541
DOI: 10.7554/eLife.27574 -
The EMBO Journal Dec 2020Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function...
Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.
Topics: Cell Cycle Proteins; Chromosomes; Humans; Kinesins; Metaphase; Microtubules; Mitosis; Spindle Apparatus
PubMed: 33073400
DOI: 10.15252/embj.2020105432 -
Mechanisms of Development Jul 2008The Drosophila Myb protein, DMyb, is a transcription factor important for cell proliferation and development. Unlike the mRNAs produced by mammalian myb genes,...
The Drosophila Myb protein, DMyb, is a transcription factor important for cell proliferation and development. Unlike the mRNAs produced by mammalian myb genes, Drosophila myb transcripts do not fluctuate substantially during the cell cycle. A comprehensive analysis of the localization and degradation of the DMyb protein has now revealed that DMyb is present in nuclei during S phase of all mitotically active tissues throughout embryogenesis and larval development. However, DMyb and Mip130, another member of the Myb complex, are not uniformly distributed throughout the nucleus. Instead, both proteins, which colocalize, appear to be specifically excluded from heterochromatic regions of chromosomes. Furthermore, DMyb and Mip130 are unstable proteins that are degraded during prometaphase of mitosis. The timing of their degradation is reminiscent of Cyclin A, but at least for DMyb, the mechanism differs; although DMyb degradation is dependent on core APC/C components, it does not depend on the Fizzy or Fizzy-related adaptor proteins. DMyb levels are also high in actively endoreplicating polyploid cells, but there is no indication of cyclical degradation. We conclude that cell cycle specific degradation of DMyb and Mip130 is likely to be utilized as a key regulatory mechanism in down-regulating their levels and the activity of the Myb complex.
Topics: Animals; Carrier Proteins; Cell Cycle Proteins; Drosophila Proteins; Drosophila melanogaster; Euchromatin; Gene Expression Regulation, Developmental; Prometaphase; Proto-Oncogene Proteins c-myb
PubMed: 18424081
DOI: 10.1016/j.mod.2008.02.005 -
G3 (Bethesda, Md.) May 2020The physical connections established by recombination are normally sufficient to ensure proper chromosome segregation during female Meiosis I. However, nonexchange...
The physical connections established by recombination are normally sufficient to ensure proper chromosome segregation during female Meiosis I. However, nonexchange chromosomes (such as the Muller element or "dot" chromosome in can still segregate accurately because they remain connected by heterochromatic tethers. A recent study examined female meiosis in the closely related species and , and found a nearly twofold difference in the mean distance the obligately nonexchange dot chromosomes were separated during Prometaphase. That study proposed two speculative hypotheses for this difference, the first being the amount of heterochromatin in each species, and the second being the species' differing tolerance for common inversions in natural populations. We tested these hypotheses by examining female meiosis in 12 additional species. While neither hypothesis had significant support, we did see 10-fold variation in dot chromosome sizes, and fivefold variation in the frequency of chromosomes out on the spindle, which were both significantly correlated with chromosome separation distances. In addition to demonstrating that heterochromatin abundance changes chromosome behavior, this implies that the duration of Prometaphase chromosome movements must be proportional to the size of the element in these species. Additionally, we examined , a species that lacks a free dot chromosome. We observed that chromosomes still moved out on the meiotic spindle, and the element was always positioned closest to the spindle poles. This result is consistent with models where one role of the dot chromosomes is to help organize the meiotic spindle.
Topics: Animals; Chromosome Segregation; Drosophila; Drosophila melanogaster; Female; Meiosis; Spindle Apparatus
PubMed: 32217631
DOI: 10.1534/g3.120.400867 -
Cell Cycle (Georgetown, Tex.) Oct 2021To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal...
To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH's functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.
Topics: Anaphase; Centromere; Chromatids; Chromosome Segregation; Genomic Instability; Humans; Mitosis
PubMed: 34530686
DOI: 10.1080/15384101.2021.1970877