scholarly journals Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore

2016 ◽  
Vol 212 (7) ◽  
pp. 767-776 ◽  
Author(s):  
Gheorghe Cojoc ◽  
Emanuele Roscioli ◽  
Lijuan Zhang ◽  
Alfonso García-Ulloa ◽  
Jagesh V. Shah ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Mechanical Response ◽  
Viscoelastic Behavior ◽  
Length Change ◽  
Live Cells ◽  
Microtubule Severing ◽  
Microtubule Attachment ◽  
Pulling Forces

Accurate chromosome segregation depends on proper kinetochore–microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore–centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

scholarly journals Kinetochore Targeting of Fission Yeast Mad and Bub Proteins Is Essential for Spindle Checkpoint Function but Not for All Chromosome Segregation Roles of Bub1p

2004 ◽  
Vol 24 (22) ◽  
pp. 9786-9801 ◽  
Author(s):  
Vincent Vanoosthuyse ◽  
Rebekka Valsdottir ◽  
Jean-Paul Javerzat ◽  
Kevin G. Hardwick
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Spindle Checkpoint ◽  
Central Domain ◽  
Checkpoint Response ◽  
Terminal Domain ◽  
Microtubule Attachment ◽  
Lines Of Evidence

ABSTRACT Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a “wait anaphase” signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

scholarly journals Dicer promotes genome stability via the bromodomain transcriptional co-activator Brd4.

2021 ◽  
Author(s):  
Michael Gutbrod ◽  
Benjamin Roche ◽  
Joshua Steinberg ◽  
Asad Lakhani ◽  
Kenneth Chang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Genome Instability ◽  
Embryonic Stem ◽  
Major Satellite ◽  
Suppressor Mutations ◽  
Satellite Repeats ◽  
Transcription And Replication

RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.

scholarly journals Convergent genes shape budding yeast pericentromeres

2019 ◽  
Author(s):  
Flora Paldi ◽  
Bonnie Alver ◽  
Daniel Robertson ◽  
Stephanie A. Schalbetter ◽  
Alastair Kerr ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Budding Yeast ◽  
3D Structure ◽  
Linear Arrangement ◽  
3D Genome ◽  
Microtubule Attachment ◽  
Pulling Forces ◽  
3D Architecture ◽  
Transcriptional Units ◽  
And Function

AbstractThe 3D architecture of the genome governs its maintenance, expression and transmission. The conserved ring-shaped cohesin complex organises the genome by topologically linking distant loci on either a single DNA molecule or, after DNA replication, on separate sister chromatids to provide the cohesion that resists the pulling forces of spindle microtubules during mitosis1,2. Cohesin is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres3-7. However, the structural organisation of pericentromeres and implications for chromosome segregation are unknown. Here we report the 3D structure of budding yeast pericentromeres and establish the relationship between genome organisation and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organised into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Re-orienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation in mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading at centromeres shapes pericentromeres into a structure competent for chromosome segregation during mitosis. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded and the re-structuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between 3D genome organization of a specific chromosomal domain and cellular function.

scholarly journals Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase.

1995 ◽  
Vol 129 (5) ◽  
pp. 1195-1204 ◽  
Author(s):  
M S Campbell ◽  
G J Gorbsky
Keyword(s):  
Monoclonal Antibody ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Metaphase Plate ◽  
Chromosome Movement ◽  
Normal Cells ◽  
Microtubule Attachment ◽  
Checkpoint Pathway ◽  
Chromosome Congression ◽  
Asymmetric Expression

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

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 Aurora B and Condensin are dispensable for chromosome arm and telomere separation during meiosis II

2020 ◽  
Author(s):  
Julien Berthezene ◽  
Céline Reyes ◽  
Tong Li ◽  
Stéphane Coulon ◽  
Pascal Bernard ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Microtubule Attachment ◽  
Chromosome Arm ◽  
Mitosis And Meiosis ◽  
Chromatin Bridges ◽  
Meiosis I

ABSTRACTIn mitosis, while the importance of kinetochore-microtubule attachment has been known for many years, increasing evidence suggests that telomere dysfunctions also perturb chromosome segregation by contributing to the formation of chromatin bridges at anaphase. Recent evidence suggests that Aurora B ensures proper chromosome segregation during mitosis not only by controlling kinetochore-microtubule attachment but also by regulating telomere and chromosome arm separation. However, whether and how Aurora-B governs telomere separation during meiosis has remained unknown. Here, we show that fission yeast Aurora B localizes at telomeres during meiosis I and promotes telomere separation independently of the meiotic cohesin Rec8. In meiosis II, Aurora-B controls kinetochore-microtubule attachment but appears dispensable for telomere and chromosome arm separation. Likewise, condensin activity is nonessential in meiosis II for telomere and chromosome arm separation. Thus, in meiosis, the requirements for Aurora-B are distinct at centromeres and telomeres, illustrating the critical differences in the control of chromosome segregation between mitosis and meiosis II.

scholarly journals Tetraploidy causes chromosomal instability in acentriolar mouse embryos

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lia Mara Gomes Paim ◽  
Greg FitzHarris
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Chromosomal Instability ◽  
Current Model ◽  
Cell Stage ◽  
Multipolar Spindle ◽  
Genome Doubling ◽  
Microtubule Attachment ◽  
Multipolar Spindles ◽  
Bipolar Spindles

Abstract Tetraploidisation is considered a common event in the evolution of chromosomal instability (CIN) in cancer cells. The current model for how tetraploidy drives CIN in mammalian cells is that a doubling of the number of centrioles that accompany the genome doubling event leads to multipolar spindle formation and chromosome segregation errors. By exploiting the unusual scenario of mouse blastomeres, which lack centrioles until the ~64-cell stage, we show that tetraploidy can drive CIN by an entirely distinct mechanism. Tetraploid blastomeres assemble bipolar spindles dictated by microtubule organising centres, and multipolar spindles are rare. Rather, kinetochore-microtubule turnover is altered, leading to microtubule attachment defects and anaphase chromosome segregation errors. The resulting blastomeres become chromosomally unstable and exhibit a dramatic increase in whole chromosome aneuploidies. Our results thus reveal an unexpected mechanism by which tetraploidy drives CIN, in which the acquisition of chromosomally-unstable microtubule dynamics contributes to chromosome segregation errors following tetraploidisation.

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