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Methods in Molecular Biology (Clifton,... 2022The cell cycle is the series of events that take place in a cell that drives it to divide and produce two new daughter cells. Through more than 100 years of efforts by... (Review)
Review
The cell cycle is the series of events that take place in a cell that drives it to divide and produce two new daughter cells. Through more than 100 years of efforts by scientists, we now have a much clearer picture of cell cycle progression and its regulation. The typical cell cycle in eukaryotes is composed of the G1, S, G2, and M phases. The M phase is further divided into prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis. Cell cycle progression is mediated by cyclin-dependent kinases (Cdks) and their regulatory cyclin subunits. However, the driving force of cell cycle progression is growth factor-initiated signaling pathways that controls the activity of various Cdk-cyclin complexes. Most cellular events, including DNA duplication, gene transcription, protein translation, and post-translational modification of proteins, occur in a cell-cycle-dependent manner. To understand these cellular events and their underlying molecular mechanisms, it is desirable to have a population of cells that are traversing the cell cycle synchronously. This can be achieved through a process called cell synchronization. Many methods have been developed to synchronize cells to the various phases of the cell cycle. These methods could be classified into two groups: synchronization methods using chemical inhibitors and synchronization methods without using chemical inhibitors. All these methods have their own merits and shortcomings.
Topics: Anaphase; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinases; Cyclins; Mitosis
PubMed: 36045194
DOI: 10.1007/978-1-0716-2736-5_1 -
Journal of Cell Science Sep 2020Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer...
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Topics: Humans; Kinetochores; Mitosis; Nocodazole; Prometaphase; Retinal Pigments
PubMed: 32878943
DOI: 10.1242/jcs.247940 -
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 -
Nature Communications Dec 2022Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated...
Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes.
Topics: Animals; Mice; Anaphase-Promoting Complex-Cyclosome; Cell Cycle Proteins; Chromosome Segregation; Dynamins; Kinetochores; Meiosis; Oocytes; Securin; Separase
PubMed: 36513638
DOI: 10.1038/s41467-022-35461-5 -
Cells May 2022The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to... (Review)
Review
The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis.
Topics: Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35563837
DOI: 10.3390/cells11091531 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2022The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate,...
The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.
Topics: Anaphase; HeLa Cells; Humans; Metaphase; Mitosis; Telophase
PubMed: 36045201
DOI: 10.1007/978-1-0716-2736-5_8 -
Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051 -
Frontiers in Cell and Developmental... 2023Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that... (Review)
Review
Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle.
PubMed: 36994100
DOI: 10.3389/fcell.2023.1139367