-
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 -
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 -
Cell Discovery May 2022Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show...
Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show that rRNA species associate with condensed chromosomes during mitosis. In particular, pre-rRNAs such as 45S, 32S, and 30S are highly enriched on mitotic chromosomes. Immediately following nucleolus disassembly in mitotic prophase, rRNAs are released and associate with and coat each condensed chromosome at prometaphase. Using unbiased mass spectrometry analysis, we further demonstrate that chromosome-bound rRNAs are associated with Ki-67. Moreover, the FHA domain and the repeat region of Ki-67 recognize and anchor rRNAs to chromosomes. Finally, suppression of chromosome-bound rRNAs by RNA polymerase I inhibition or by using rRNA-binding-deficient Ki-67 mutants impair mitotic chromosome dispersion during prometaphase. Our study thus reveals an important role of rRNAs in preventing chromosome clustering during mitosis.
PubMed: 35637200
DOI: 10.1038/s41421-022-00400-7 -
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 -
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 -
Cell Cycle (Georgetown, Tex.) Jul 2020To maintain genome stability, chromosomes must be equally distributed among daughter cells at the end of mitosis. The accuracy of chromosome segregation requires... (Review)
Review
To maintain genome stability, chromosomes must be equally distributed among daughter cells at the end of mitosis. The accuracy of chromosome segregation requires sister-kinetochores to stably attach to microtubules emanating from opposite spindle poles. However, initial kinetochore-microtubule interactions are able to turnover so that defective attachment configurations that typically arise during early mitosis may be corrected. Growing evidence supports a role for the RZZ complex in preventing the stabilization of erroneous kinetochore-microtubule attachments. This inhibitory function of RZZ toward end-on attachments is relieved by DYNEIN-mediated transport of the complex as chromosomes congress and appropriate interactions with microtubules are established. However, it remains unclear how DYNEIN is antagonized to prevent premature RZZ removal. We recently described a new mechanism that sheds new light on this matter. We found that POLO kinase phosphorylates the DYNEIN adaptor SPINDLY to promote the uncoupling between RZZ and DYNEIN. Elevated POLO activity during prometaphase ensures that RZZ is retained at kinetochores to allow the dynamic turnover of kinetochore-microtubule interactions and prevent the stabilization of erroneous attachments. Here, we discuss additional interpretations to explain a model for POLO-dependent regulation of the RZZ-SPINDLY-DYNEIN module during mitosis.
Topics: Animals; Cell Cycle Proteins; Chromosome Segregation; Dyneins; Humans; Kinetochores; Mitosis
PubMed: 32544383
DOI: 10.1080/15384101.2020.1780382 -
Frontiers in Cell and Developmental... 2021The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from... (Review)
Review
The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from the early onset to the very last phase. Despite the tight control, errors in mitotic division occur frequently and they may result in various chromosomal instabilities and malignancies. The flow of events during mitotic progression where the chromosomes condensate and rearrange with the help of the cytoskeletal network has been described in great detail. Plasma membrane dynamics and endocytic vesicle movement upon deadhesion and reattachment of dividing cells are also demonstrated to be functionally important for the mitotic integrity. Other cytoplasmic organelles, such as autophagosomes and lysosomes, have until recently been considered merely as passive bystanders in this process. Accordingly, at the onset of nuclear envelope breakdown in prometaphase, the number of autophagic structures and lysosomes is reduced and the bulk autophagic machinery is suppressed for the duration of mitosis. This is believed to ensure that the exposed nuclear components are not unintentionally delivered to autophagic degradation. With the evolving technologies that allow the detection of subtle alterations in cytoplasmic organelles, our understanding of the small-scale regulation of intracellular organelles has deepened rapidly and we discuss here recent discoveries revealing unexpected roles for autophagy and lysosomes in the preservation of genomic integrity during mitosis.
PubMed: 34485308
DOI: 10.3389/fcell.2021.727538