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Pathology International Dec 2022
Topics: Humans; Metaphase; Mitotic Index; Mitosis; Mucous Membrane; Colchicine
PubMed: 36205481
DOI: 10.1111/pin.13276 -
Annual Review of Biophysics May 2018The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins,... (Review)
Review
The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.
Topics: Humans; Metaphase; Spindle Apparatus
PubMed: 29792816
DOI: 10.1146/annurev-biophys-060414-034107 -
Nature Communications Nov 2022Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often...
Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.
Topics: Humans; Embryo, Mammalian; Chromosome Segregation; Mosaicism; Metaphase; Karyotype; Blastocyst; Aneuploidy
PubMed: 36347869
DOI: 10.1038/s41467-022-34294-6 -
Cytometry. Part a : the Journal of the... Apr 2021Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to... (Review)
Review
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
Topics: Cell Cycle; Chromosomes, Plant; Flow Cytometry; Metaphase; Plants
PubMed: 33615737
DOI: 10.1002/cyto.a.24324 -
Yakugaku Zasshi : Journal of the... 2022Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process...
Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process with high accuracy and precision, various motor proteins work in a concerted manner. Especially in the metaphase, mitotic chromosomes are delivered by the motor protein of centromere-associated protein E (CENP-E) to the cell equatorial plane (metaphase plate) along mitotic spindles. However, the critical functional failure of CENP-E can activate the spindle assembly checkpoint through the misalignment of chromosomes at the metaphase plate. In this symposium review, the reversibly photoswitchable CENP-E inhibitor PCEI-HU (5) is reported. Compound 5 exhibited almost quantitative trans-cis photoisomerization of the arylazopyrazole photoswitch by illuminating light at 365 nm and 510 nm. Depending on the photoisomerization, CENP-E activity was regulated not only in vitro but also in cells. We successfully established a novel technique using 5 to dynamically photocontrol the CENP-E-dependent chromosome movement and mitotic progression in a living cell.
Topics: Cell Division; Metaphase; Spindle Apparatus
PubMed: 35491157
DOI: 10.1248/yakushi.21-00203-5 -
The Journal of Cell Biology Apr 2019Chromosome alignment is a hallmark of mitosis in metazoans, but the physiological relevance of this orderly behavior has remained unclear. In this issue, Fonseca et al....
Chromosome alignment is a hallmark of mitosis in metazoans, but the physiological relevance of this orderly behavior has remained unclear. In this issue, Fonseca et al. (2019. https://doi.org/10.1083/jcb.201807228) show that chromosome alignment ensures mitotic fidelity by promoting interchromosomal compaction during anaphase.
Topics: Anaphase; Chromosomes; Metaphase; Mitosis
PubMed: 30858193
DOI: 10.1083/jcb.201902041 -
Methods in Molecular Biology (Clifton,... 2017Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle...
Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.
Topics: Anaphase; Cell Culture Techniques; Cell Cycle; Fluorescent Antibody Technique; HeLa Cells; Humans; Metaphase; Mitosis; Prometaphase; Telophase
PubMed: 27815896
DOI: 10.1007/978-1-4939-6603-5_4 -
Current Opinion in Cell Biology Dec 2022In an active, crowded cytoplasm, eukaryotic cells construct metaphase spindles from conserved building blocks to segregate chromosomes. Yet, spindles execute their... (Review)
Review
In an active, crowded cytoplasm, eukaryotic cells construct metaphase spindles from conserved building blocks to segregate chromosomes. Yet, spindles execute their function in a stunning variety of cell shapes and sizes across orders of magnitude. Thus, the current challenge is to understand how unique mesoscale spindle characteristics emerge from the interaction of molecular collectives. Key components of these collectives are tubulin dimers, which polymerise into microtubules. Despite all conservation, tubulin is a genetically and biochemically complex protein family, and we only begin to uncover how tubulin diversity affects microtubule dynamics and thus spindle assembly. Moreover, it is increasingly appreciated that spindles are dynamically intertwined with the cytoplasm that itself exhibits cell-type specific emergent properties with yet mostly unexplored consequences for spindle construction. Therefore, on our way toward a quantitative picture of spindle function, we need to understand molecular behaviour of the building blocks and connect it to the entire cellular context.
Topics: Metaphase; Tubulin; Cell Cycle; Microtubules; Cytoplasm
PubMed: 36436307
DOI: 10.1016/j.ceb.2022.102143 -
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 -
Genes & Development Jun 2008Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. Here we characterize the functions...
Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. Here we characterize the functions of CDK activity associated with the mitotic cyclins Clb1, Clb2, and Clb3. We show that Clb-CDKs are important for the activation of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C)-Cdc20 that triggers the metaphase-anaphase transition. Furthermore, we define an essential role for Clb-CDK activity in anaphase spindle elongation. Thus, mitotic CDKs serve not only to initiate M phase, but are also needed continuously throughout mitosis to trigger key mitotic events such as APC/C activation and anaphase spindle elongation.
Topics: Anaphase; Cell Cycle; Cyclin-Dependent Kinases; Metaphase; Mitosis; Saccharomyces cerevisiae; Spindle Apparatus
PubMed: 18519644
DOI: 10.1101/gad.1638308