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Methods in Molecular Biology (Clifton,... 2023After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent...
After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.
Topics: Chromosomes; DNA; DNA Damage; Interphase; Metaphase
PubMed: 36066713
DOI: 10.1007/978-1-0716-2433-3_10 -
Methods in Molecular Biology (Clifton,... 2023In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule... (Review)
Review
In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.
Topics: Kinesins; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus; Tubulin
PubMed: 36773229
DOI: 10.1007/978-1-0716-2867-6_9 -
Cell Death & Disease Jun 2022CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the...
CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the physiological functions of CCAR2 in regulating cell proliferation in the absence of external stimuli. The present study shows that CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of mitotic phase was perturbed. This disturbance of mitotic progression resulted from premature loss of cohesion with the centromere, and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. It resulted in the formation of lagging chromosomes during anaphase, leading ultimately to the activation of the abscission checkpoint to halt cytokinesis. The CCAR2-dependent mitotic progression was related to spatiotemporal regulation of active Aurora B. In conclusion, the results suggest that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability.
Topics: Aurora Kinase B; Cell Cycle Proteins; Centromere; Chromosome Segregation; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 35672287
DOI: 10.1038/s41419-022-04990-8 -
Frontiers in Cell and Developmental... 2022Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the...
Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog . While depletion of readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55 and PP2A-B56, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56 in a mechanism independent of CDK.
PubMed: 35923846
DOI: 10.3389/fcell.2022.876810 -
Molecular Reproduction and Development Dec 2015Post-translational modifications regulate mitosis and meiosis. For example, O-GlcNAcylation –the addition of a single N acetyl-glucosamine (O-GlcNAc) to serine or... (Review)
Review
Post-translational modifications regulate mitosis and meiosis. For example, O-GlcNAcylation –the addition of a single N acetyl-glucosamine (O-GlcNAc) to serine or threonine residues– is required for mitotic spindle formation and chromosome segregation; here we show that O-GlcNAcylation occurs in mouse oocytes during meiosis. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which respectively adds and removes this modification, are expressed and active during meiosis. Proteins modified with O-GlcNAc (green) are concentrated at the nuclear envelope at prophase I (top cells) and can be found throughout the cytoplasm at metaphase II (bottom cells), with an enrichment immediately adjacent to the condensed chromosomes. OGT (yellow) is distributed throughout the oocyte at prophase I, but is later concentrated at the metaphase-II spindle. In contrast, OGA (cyan) is enriched primarily at the cortex throughout meiosis. These distinct spatial distributions of OGT and OGA suggest that O-GlcNAcylation is maintained at discrete regions of the oocyte – e.g. the meiotic spindle. Studies investigating the precise function of O-GlcNAc, OGT, and OGA during meiotic maturation will provide new insights into the impact of post-translational modifications on gamete quality.
PubMed: 26331270
DOI: 10.1002/mrd.22577 -
Journal of Cell Science Jun 2023The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to...
The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.
Topics: Kinesins; Saccharomyces cerevisiae; Cell Cycle; Anaphase; Metaphase
PubMed: 37305999
DOI: 10.1242/jcs.260621 -
Cell Division Jan 2021Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs)...
BACKGROUND
Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters.
RESULTS
In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact.
CONCLUSIONS
Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.
PubMed: 33514388
DOI: 10.1186/s13008-021-00070-z -
Environmental Research Oct 2022The present study is an attempt to assess the cytogenotoxic effect of untreated and methyl orange treated with Oedogonium subplagiostomum AP1 on Allium cepa roots. On...
The present study is an attempt to assess the cytogenotoxic effect of untreated and methyl orange treated with Oedogonium subplagiostomum AP1 on Allium cepa roots. On the fifth day, root growth, root length, mitotic index, mitotic inhibition/depression, and chromosomal abnormalities were measured in root cells of Allium cepa subjected to untreated and treated methyl orange dye solutions. Roots exposed to treated dye solution exhibited maximum root growth, root length and mitotic index, whereas roots exposed to untreated dye solution had the most mitotic inhibition and chromosomal abnormalities. Allium cepa exposed to untreated dye solution revealed chromosomal aberrations such as disoriented and abnormal chromosome grouping, vagrant and laggard chromosomes, chromosomal loss, sticky chain and disturbed metaphase, pulverised and disturbed anaphase, chromosomal displacement in anaphase, abnormal telophase, and chromosomal bridge at telophase, spindle disturbances and binucleate cells. The comet test was used to quantify DNA damage in the root cells of A. cepa subjected to untreated and treated methyl orange solutions in terms of tail DNA (percent) and tail length. The results concluded that A. cepa exposed to methyl orange induced DNA damage whereas meager damage was noted in the treated dye solution. As a result, the research can be used as a biomarker to detect the genotoxic effects of textile dyes on biota.
Topics: Allium; Azo Compounds; Chromosome Aberrations; DNA Damage; Mitotic Index; Onions; Plant Roots
PubMed: 35716816
DOI: 10.1016/j.envres.2022.113612 -
Biochemical and Biophysical Research... Jan 2022Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional...
Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional proteins but also generates abnormal protein function, which causes several diseases. Several pre-mRNA splicing factors are reported to regulate mitosis directly at mitotic structures and/or indirectly through controlling the pre-mRNA splicing for mitotic proteins. In this study, we described the mitotic functions of SF3B14, a component of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), which we identified as a candidate involved in mitosis based on the large-scale RNA interference (RNAi) screen of the nucleolar proteome database. We observed that SF3B14 depletion caused prolonged mitosis and several mitotic defects, such as monopolar spindle and chromosome misalignment during metaphase. Although SF3B14 was found in the nucleolar proteome database, our immunofluorescent stainings demonstrated that SF3B14 was predominantly localized in the nucleoplasm and excluded from the nucleolus during interphase. In addition, SF3B14 did not colocalize with specific mitotic structures during mitosis, which is not in line with its direct mitotic function. Notably, we found that the SF3B14 depletion reduced protein levels of TUBGCP6, required for centrosome regulation, and increased the unspliced/spliced ratio of its mRNA. Taken together, we propose that the pre-mRNA of TUBGCP6 is one of the targets for SF3B14 splicing through which SF3B14 controls mitotic chromosome behavior.
Topics: Centrosome; Chromosomes, Human; HeLa Cells; Humans; Microtubule-Associated Proteins; Mitosis; Phosphoproteins; RNA Precursors; RNA Splicing; RNA Splicing Factors
PubMed: 34954520
DOI: 10.1016/j.bbrc.2021.12.059 -
Methods in Molecular Biology (Clifton,... 2016The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. Kinetochores are composed...
The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. Kinetochores are composed of more than 100 proteins that transiently assemble during mitosis at a single epigenetically defined region on each chromosome, known as the centromere. Kinetochore assembly and activity must be tightly regulated to ensure proper microtubule interaction and faithful chromosome segregation. Kinetochore malfunction can result in chromosome segregation defects leading to aneuploidy and cell death. As such, cell free and reconstituted systems to analyze kinetochore formation and function are invaluable in probing the biochemical activities of kinetochores. In vitro approaches to studying kinetochores have enabled the manipulation of kinetochore protein structure, function, interactions, and regulation that are not possible in cells. Here we outline a cell-free approach for the assembly of centromeres and recruitment of functional kinetochores that enables their manipulation and analysis.
Topics: Animals; Cell Cycle Checkpoints; Cell Extracts; Centromere; Centromere Protein A; Chromatin; Female; Gene Expression; Histones; Kinetochores; Metaphase; Microtubules; Mitosis; Nucleosomes; Oocytes; Protein Multimerization; Spindle Apparatus; Xenopus
PubMed: 27193846
DOI: 10.1007/978-1-4939-3542-0_8