scholarly journals Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics

2019 ◽  
Vol 12 (6) ◽  
pp. 462-476 ◽  
Author(s):  
Ruoying Yu ◽  
Huihui Wu ◽  
Hazrat Ismail ◽  
Shihao Du ◽  
Jun Cao ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Kinase Activity ◽  
Genomic Stability ◽  
Lysine Methylation ◽  
Mitotic Chromosomes ◽  
Methyltransferase Activity ◽  
Sister Chromatids ◽  
Spindle Microtubules ◽  
Chemical Inhibition ◽  
Early Mitosis

Abstract Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.

scholarly journals The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

2003 ◽  
Vol 160 (3) ◽  
pp. 329-339 ◽  
Author(s):  
Stéphanie Buvelot ◽  
Sean Y. Tatsutani ◽  
Danielle Vermaak ◽  
Sue Biggins
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Genomic Stability ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Spindle Disassembly ◽  
Spindle Midzone ◽  
Spindle Microtubules

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

scholarly journals A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

2012 ◽  
Vol 196 (6) ◽  
pp. 757-774 ◽  
Author(s):  
Guillaume Gay ◽  
Thibault Courtheoux ◽  
Céline Reyes ◽  
Sylvie Tournier ◽  
Yannick Gachet
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Segregation Behavior ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Yeast Chromosome ◽  
Spindle Microtubules ◽  
Early Mitosis

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B–like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B–like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy.

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

scholarly journals SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A

2021 ◽  
Author(s):  
Bin Yu ◽  
Qiaoyu Lin ◽  
Chao Huang ◽  
Boyan Zhang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
In Vitro Assays ◽  
Aurora A ◽  
Sumo Proteases ◽  
Spatiotemporal Control ◽  
Early Mitosis

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator, Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupted the deSUMOylation of AurA, leading to an increased kinase activity and abnormalities in spindle assembly and chromosomes segregation which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render a spatiotemporal control on its kinase activity in mitosis.

Driving chromosome segregation: lessons from the human and Drosophila centromere–kinetochore machinery

2010 ◽  
Vol 38 (6) ◽  
pp. 1667-1675 ◽  
Author(s):  
Bernardo Orr ◽  
Olga Afonso ◽  
Tália Feijão ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Genomic Stability ◽  
Mitotic Progression ◽  
Chromosomal Locus ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
And Function ◽  
Drosophila Cells

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

scholarly journals Dynamic bonds and polar ejection force distribution explain kinetochore oscillations in PtK1 cells

2013 ◽  
Vol 201 (4) ◽  
pp. 577-593 ◽  
Author(s):  
Gul Civelekoglu-Scholey ◽  
Bin He ◽  
Muyao Shen ◽  
Xiaohu Wan ◽  
Emanuele Roscioli ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Quantitative Model ◽  
Force Distribution ◽  
Mitotic Chromosomes ◽  
Chromosome Behavior ◽  
Sister Chromatids ◽  
Ejection Force ◽  
Sister Kinetochore ◽  
Spindle Microtubules ◽  
Key Features

Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments to spindle microtubules via their kinetochores, and multiple motor and nonmotor proteins cooperate to regulate their behavior. Depending on the system, sister chromatids may display either of two distinct behaviors, namely (1) the presence or (2) the absence of oscillations about the metaphase plate. Significantly, in PtK1 cells, in which chromosome behavior appears to be dependent on the position along the metaphase plate, both types of behavior are observed within the same spindle, but how and why these distinct behaviors are manifested is unclear. Here, we developed a new quantitative model to describe metaphase chromosome dynamics via kinetochore–microtubule interactions mediated by nonmotor viscoelastic linkages. Our model reproduces all the key features of metaphase sister kinetochore dynamics in PtK1 cells and suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed dichotomy of chromosome behavior.

scholarly journals Aurora B Kinase Exists in a Complex with Survivin and INCENP and Its Kinase Activity Is Stimulated by Survivin Binding and Phosphorylation

2002 ◽  
Vol 13 (9) ◽  
pp. 3064-3077 ◽  
Author(s):  
Margaret A. Bolton ◽  
Weijie Lan ◽  
Shannon E. Powers ◽  
Mark L. McCleland ◽  
Jian Kuang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Kinase Activity ◽  
Genetic Interactions ◽  
Aurora B ◽  
Hydrodynamic Properties ◽  
Aurora B Kinase ◽  
Survivin Gene ◽  
Spindle Microtubules ◽  
Cycle Dependent

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned theXenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopusearly embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.

Proteins of the inner and outer centromere of mitotic chromosomes

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 541-552 ◽  
Author(s):  
William C. Earnshaw ◽  
Carol A. Cooke
Keyword(s):  
Negative Charge ◽  
Dna Binding Protein ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Region I ◽  
Spindle Microtubules ◽  
Region Ii ◽  
Chromosome Scaffold

We have used immunocytochemistry and molecular cloning methods to identify and characterize structural polypeptides of the centromere. These studies permit us to resolve two distinct regions: the inner and outer centromere, (i) Components of the outer centromere: autoantibodies from certain patients with rheumatic disease identify a family of three immunologically related polypeptides that we have designated CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa). CENP-B has been cloned and sequenced. DNA sequence analysis indicates that this polypeptide possesses two large regions with extraordinary concentrations of acidic residues (region I: 61 residues with 79% glu + asp; region II: 31 residues with 87% glu + asp). Despite this concentration of negative charge, immunocytochemical experiments suggest that CENP-B may be a DNA binding protein. In these experiments, the levels of CENP-B are seen to vary reproducibly from chromosome to chromosome. The role of CENP-B in vivo is unknown. However, it is unlikely to bind directly to the spindle microtubules since it is found at an inactive centromere that apparently does not attach to the spindle. (ii) Components of the inner centromere: we have injected mice with the whole chromosome scaffold fraction to elicit production of monoclonal antibodies. One such antibody identifies two structurally related polypeptides (the INCENP antigens, 135 and 155 kDa) that are preferentially located between the sister chromatids at the centromere. The INCENP antigens undergo dramatic movements from the chromosomes to the central spindle during mitosis. They are ultimately sequestered in the midbody and discarded. Several lines of evidence suggest that the INCENP polypeptides may be involved in the regulation of sister chromatid separation at the metaphase–anaphase transition.Key words: mitosis, centromere, CENP antigens, INCENP antigens, kinetochore, disjunction.

scholarly journals Kre28-Spc105 interaction is essential for their recruitment at the kinetochore

2021 ◽  
Author(s):  
Babhrubahan Roy ◽  
Janice Sim ◽  
Simon J. Y. Han ◽  
Ajit P. Joglekar
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
High Fidelity ◽  
Protein Assemblies ◽  
Sister Chromatids ◽  
Exact Function ◽  
Interaction Interface ◽  
Macromolecular Protein ◽  
Spindle Microtubules ◽  
Turn Over

Kinetochores are macromolecular protein assemblies that attach sister chromatids to spindle microtubules and mediate accurate chromosome segregation during mitosis. The outer kinetochore consists of the KMN network, a protein super complex made of Knl1 (yeast Spc105), Mis12 (yeast Mtw1) and Ndc80 (yeast Ndc80), which harbors sites for microtubule binding. Within the KMN network, Spc105 acts as interaction hub of components involved in spindle assembly checkpoint (SAC) signaling. It is known that Spc105 forms a complex with kinetochore component Kre28. However, where Kre28 physically localizes in the budding yeast kinetochore is not clear. The exact function of Kre28 at the kinetochore is also unknown. Here, we reveal how Spc105 and Kre28 interact and how they are organized within bioriented yeast kinetochores using genetics and cell biological experiments. We also identify the interaction interface between the two proteins and show that this interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the kinetochores. We created several truncation mutants of kre28 that do not localize at the kinetochores and so cannot mediate Spc105 loading at the kinetochores. When we over-expressed these mutants, they could sustain the cell viability even though failed to facilitate proper SAC activation and/or error correction. Thus, we inferred that Kre28 indirectly contributes to chromosome biorientation and high-fidelity segregation by regulating Spc105 localization at the kinetochores.

scholarly journals Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes

2021 ◽  
Author(s):  
Elvira Nikalayevich ◽  
Safia El Jailani ◽  
Damien Cladiere ◽  
Yulia Gryaznova ◽  
Celia Fosse ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Kinase Activity ◽  
Model Systems ◽  
Aurora B ◽  
Dependent Manner ◽  
Centromere Region ◽  
C Elegans ◽  
Sister Chromatids ◽  
Turn Over ◽  
Meiosis I

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II, to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease Separase[1, 2]. In yeast, Rec8 is kept in a non-phosphorylated state by the action of PP2A-B56, which is localised to the centromere region, thereby preventing cohesin removal from this region in meiosis I[3-5]. However, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and whether centromeric cohesin protection is indeed brought about by dephosphorylation of Rec8 preventing cleavage, in mammalian meiosis. The identity of one or several potential Rec8-specific kinase(s) is also unknown. This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals, and additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phospho mutant analysis of functional Rec8. To address how Rec8 cleavage is brought about in mammals, we adapted a biosensor for Separase to study Rec8 cleavage in single mouse oocytes by live imaging, and identified phosphorylation sites promoting cleavage. We found that Rec8 cleavage by Separase depends on Aurora B/C kinase activity, and identified a residue promoting cleavage and being phosphorylated in an Aurora B/C kinase-dependent manner. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

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