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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 Division Feb 2012The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually...
BACKGROUND
The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.
RESULTS
Here we describe mitotic slippage in yeast bub2Δ mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.
CONCLUSIONS
The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.
PubMed: 22321970
DOI: 10.1186/1747-1028-7-4 -
Molecular Cell Apr 2024The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free...
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Topics: DNA Topoisomerases, Type II; Chromosomes; Mitosis; Interphase; Polymers
PubMed: 38521067
DOI: 10.1016/j.molcel.2024.02.025 -
ELife Jul 2021Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key...
Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.
Topics: CDC2 Protein Kinase; Endosomal Sorting Complexes Required for Transport; HeLa Cells; Humans; Membrane Proteins; Microtubules; Mitosis; Nuclear Envelope; Nuclear Proteins; Telophase
PubMed: 34286694
DOI: 10.7554/eLife.59999 -
BioRxiv : the Preprint Server For... Sep 2023Many Lamin A-associated proteins (LAAP's) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and...
Many Lamin A-associated proteins (LAAP's) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and mitotic exit. However, the identity and function of the chromosomal core domains remain ill-defined. Here, we show that a distinct section of the core domain overlaps with the centromeres/kinetochores of chromosomes during mitotic telophase. The core domain can thus be demarcated into a kinetochore proximal core (KPC) on one side of the segregated chromosomes and the kinetochore distal core (KDC) on the opposite side, close to the central spindle. We next tested if centromere assembly is connected to NE re-formation. We find that centromere assembly is markedly perturbed after inhibiting the function of LMNA and the core-localized LAAPs, BANF1 and Emerin. We also find that the LAAPs exhibit multiple biochemical interactions with the centromere and inner kinetochore proteins. Consistent with this, normal mitotic progression and chromosome segregation was severely impeded after inhibiting LAAP function. Intriguingly, the inhibition of centromere function also interferes with the assembly of LAAP components at the core domain, suggesting a mutual dependence of LAAP and centromeres for their assembly at the core domains. Finally, we find that the localization of key proteins involved in the centromeric loading of CENP-A, including the Mis18 complex and HJURP were markedly affected in LAAP-inhibited cells. Our evidence points to a model where LAAP assembly at the core domain serves a key function in loading new copies of centromeric proteins during or immediately after mitotic exit.
PubMed: 37808683
DOI: 10.1101/2023.09.25.559341 -
The Journal of Cell Biology May 2023Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the...
Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity, and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C knockout cells, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C knockout cells. In regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control and thus the fidelity of cell division.
Topics: Cell Cycle Proteins; Kinetochores; Microtubules; Mitosis; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Spindle Apparatus; Cytoskeletal Proteins; Aurora Kinase A; Microtubule-Associated Proteins
PubMed: 36897279
DOI: 10.1083/jcb.202205117 -
Genes & Development Jul 2020During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the...
During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division.
Topics: Animals; Cell Cycle Checkpoints; Chromatin; Chromosomes; Enhancer Elements, Genetic; Genome; Histone Code; Histones; Humans; Mitosis; Promoter Regions, Genetic; Protein Binding; Protein Processing, Post-Translational; Time Factors; Transcriptional Activation
PubMed: 32499403
DOI: 10.1101/gad.335794.119 -
Cells Jul 2020The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which...
The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.
Topics: Animals; Cell Nucleus; Chromatin; Chromatin Assembly and Disassembly; Conserved Sequence; Down-Regulation; HeLa Cells; Histones; Humans; Mitosis; Protein Domains; Repressor Proteins; Telophase; Xenopus
PubMed: 32708675
DOI: 10.3390/cells9071702 -
Archives of Biochemistry and Biophysics Jun 2011At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during... (Review)
Review
At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.
Topics: Animals; Cytokinesis; Humans; Mitosis; Myosin Type II; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase
PubMed: 21396909
DOI: 10.1016/j.abb.2011.03.002 -
Journal of Cell Science Sep 2021Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory...
Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory mechanism that allow timely lamin A dephosphorylation remain unclear. Here, we report that RepoMan (also known as CDCA2), a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, SUMOylated RepoMan contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of the mitotic lamin A phosphorylation. Expression of a SUMO-2 mutant that has a defective interaction with the SUMO-interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggest that RepoMan recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatiotemporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis.
Topics: Lamin Type A; Mitosis; Nuclear Proteins; Sumoylation; Telophase
PubMed: 34387316
DOI: 10.1242/jcs.247171