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Seminars in Cell & Developmental Biology Sep 2021The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful... (Review)
Review
The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.
Topics: Humans; Mitosis; Prometaphase; Spindle Apparatus
PubMed: 34127384
DOI: 10.1016/j.semcdb.2021.06.004 -
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 -
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 -
Biochemical Pharmacology Sep 2019Mitosis ensures accurate segregation of duplicated DNA through tight regulation of chromosome condensation, bipolar spindle assembly, chromosome alignment in the... (Review)
Review
Mitosis ensures accurate segregation of duplicated DNA through tight regulation of chromosome condensation, bipolar spindle assembly, chromosome alignment in the metaphase plate, chromosome segregation and cytokinesis. Poly(ADP-ribose) polymerases (PARPs), in particular PARP1, PARP2, PARP3, PARP5a (TNKS1), as well as poly(ADP-ribose) glycohydrolase (PARG), regulate different mitotic functions, including centrosome function, mitotic spindle assembly, mitotic checkpoints, telomere length and telomere cohesion. PARP depletion or inhibition give rise to various mitotic defects such as centrosome amplification, multipolar spindles, chromosome misalignment, premature loss of cohesion, metaphase arrest, anaphase DNA bridges, lagging chromosomes, and micronuclei. As the mechanisms of PARP1/2 inhibitor-mediated cell death are being progressively elucidated, it is becoming clear that mitotic defects caused by PARP1/2 inhibition arise due to replication stress and DNA damage in S phase. As it stands, entrapment of inactive PARP1/2 on DNA phenocopies replication stress through accumulation of unresolved replication intermediates, double-stranded DNA breaks (DSBs) and incorrectly repaired DSBs, which can be transmitted from S phase to mitosis and instigate various mitotic defects, giving rise to both numerical and structural chromosomal aberrations. Cancer cells have increased levels of replication stress, which makes them particularly susceptible to a combination of agents that compromise replication fork stability. Indeed, combining PARP1/2 inhibitors with genetic deficiencies in DNA repair pathways, DNA-damaging agents, ATR and other cell cycle checkpoint inhibitors has yielded synergistic effects in killing cancer cells. Here I provide a comprehensive overview of the mitotic functions of PARPs and PARG, mitotic phenotypes induced by their depletion or inhibition, as well as the therapeutic relevance of targeting mitotic cells by directly interfering with mitotic functions or indirectly through replication stress.
Topics: Animals; DNA Damage; DNA Repair; Humans; Mitosis; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases
PubMed: 30910692
DOI: 10.1016/j.bcp.2019.03.028 -
Annual Review of Biochemistry Jun 2016Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential... (Review)
Review
Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.
Topics: Animals; Centrosome; Chromosome Segregation; Drosophila melanogaster; Gene Expression Regulation; Humans; Kinesins; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Signal Transduction; Spindle Apparatus; Tubulin; Xenopus Proteins; Xenopus laevis; Zygote
PubMed: 27145846
DOI: 10.1146/annurev-biochem-060815-014528 -
Cold Spring Harbor Perspectives in... Feb 2015The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic... (Review)
Review
The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to "resolve" sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Chromosomes; Cytokinesis; DNA-Binding Proteins; Mitosis; Multiprotein Complexes; Sister Chromatid Exchange; Cohesins
PubMed: 25722466
DOI: 10.1101/cshperspect.a015792 -
Cold Spring Harbor Symposia on... 2015How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes, and go... (Review)
Review
How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes, and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The solution to this question presented here rests on the correlations that were found to hold between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase. The key points are the following: (1) The transition from the looped domains and subdomains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through their unfolding into an extended chromatin structure (probably a 10-nm "beads-on-a-string" structure); (2) the architectural proteins of interphase chromatin, such as CTCF and cohesin subunits, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; and (3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the reversibility of the interphase to mitosis process and the "mitotic memory" of interphase architecture.
Topics: Animals; Base Sequence; Cell Nucleus; Chromatin Assembly and Disassembly; Chromosomes; DNA; Genome; Humans; Interphase; Isochores; Metaphase; Mitosis; Prophase
PubMed: 26801160
DOI: 10.1101/sqb.2015.80.027318 -
ELife Apr 2023During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size....
During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.
Topics: Animals; Xenopus laevis; Cell Nucleus; Chromosomes; Cytoplasm; Spindle Apparatus; Cell Size; Mitosis
PubMed: 37096661
DOI: 10.7554/eLife.84360 -
Advances in Experimental Medicine and... 2017Mitosis is the last phase of the cell cycle and it leads to the formation of two daughter cells with the same genetic information. This process must occurr in a very... (Review)
Review
Mitosis is the last phase of the cell cycle and it leads to the formation of two daughter cells with the same genetic information. This process must occurr in a very precise way and this task is essential to preserve genetic stability and to maintain cell viability. Accurate chromosome segregation during mitosis is brought about by an important cellular organelle: the mitotic spindle. This structure is made of microtubules, polymers of alpha and beta tubulin, and it is highly dynamic during the cell cycle: it emanates from two microtubules organizing centers (Spindle Pole Bodies, SPBs, in yeast) that are essential to build a short bipolar spindle, and it undergoes two steps of elongation during anaphase A and anaphase B in order to separate sister chromatids. Several proteins are involved in the control of mitotic spindle dynamics and their activity is tightly coordinated with other cell cycle events and with cell cycle progression.
Topics: Cell Proliferation; Chromosome Segregation; Gene Expression Regulation, Fungal; Kinesins; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus; Tubulin
PubMed: 27722958
DOI: 10.1007/5584_2016_74 -
Mathematical Biosciences May 2024This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase...
This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.
PubMed: 38795952
DOI: 10.1016/j.mbs.2024.109219