The Mal2p Protein Is an Essential Component of the Fission Yeast Centromere

ABSTRACT Precise segregation of chromosomes requires the activity of a specialized chromatin region, the centromere, that assembles the kinetochore complex to mediate the association with spindle microtubules. We show here that Mal2p, previously identified as a protein required for genome stability, is an essential component of the fission yeast centromere. Loss of functional Mal2p leads to extreme missegregation of chromosomes due to nondisjunction of sister chromatids and results in inviable cells. Mal2p associates specifically with the central region of the complex fission yeast centromere, where it is required for the specialized chromatin architecture as well as for transcriptional silencing of this region. Genetic evidence indicates that mal2 + interacts with mis12 +, encoding another component of the inner centromere core complex. In addition, Mal2p is required for correct metaphase spindle length. Our data imply that the Mal2p protein is required to build up a functional fission yeast centromere.

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

The Journal of Cell Biology ◽

10.1083/jcb.201107124 ◽

2012 ◽

Vol 196 (6) ◽

pp. 757-774 ◽

Cited By ~ 43

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.

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Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation

Journal of Cell Science ◽

10.1242/jcs.114.16.2911 ◽

2001 ◽

Vol 114 (16) ◽

pp. 2911-2920 ◽

Cited By ~ 1

Author(s):

Iciar L. Ochotorena ◽

Dai Hirata ◽

Kin-ichiro Kominami ◽

Judith Potashkin ◽

Fikret Sahin ◽

...

Keyword(s):

Fission Yeast ◽

Genome Stability ◽

Large Subunit ◽

Mrna Levels ◽

Mitotic Spindles ◽

Sister Chromatids ◽

Mrna Maturation ◽

Chromosome Separation ◽

Tubulin Mrna ◽

Coupling Protein

Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of α-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of α-tubulin mRNA levels; moreover, transcript levels of genes other than the α-tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

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Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I

Scientific Reports ◽

10.1038/srep25736 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 10

Author(s):

Gheorghe Cojoc ◽

Ana-Maria Florescu ◽

Alexander Krull ◽

Anna H. Klemm ◽

Nenad Pavin ◽

...

Keyword(s):

Cell Division ◽

Fission Yeast ◽

General Feature ◽

Protein Complexes ◽

Spindle Pole ◽

Single Object ◽

Homologous Chromosomes ◽

Angular Movement ◽

Spindle Microtubules ◽

Meiosis I

Abstract Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3–4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

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The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

10.20944/preprints202103.0452.v1 ◽

2021 ◽

Author(s):

Masashi Yukawa ◽

Mitsuki Ohishi ◽

Yusuke Yamada ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Aggregates ◽

Associated Proteins ◽

Spindle Microtubules ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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Monitoring S. pombe genome stress by visualizing end-binding protein Ku

10.1101/2020.06.12.149054 ◽

2020 ◽

Author(s):

Chance Jones ◽

Susan L Forsburg

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Protein Complex ◽

Genome Stability ◽

Binding Protein ◽

Dna Lesions ◽

Live Cells ◽

Dna Damage Checkpoint ◽

Unique Pattern ◽

Ku Protein

AbstractStudies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

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Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast

The Journal of Cell Biology ◽

10.1083/jcb.200902093 ◽

2009 ◽

Vol 187 (3) ◽

pp. 399-412 ◽

Cited By ~ 36

Author(s):

Thibault Courtheoux ◽

Guillaume Gay ◽

Yannick Gachet ◽

Sylvie Tournier

Keyword(s):

Fission Yeast ◽

Mechanical Model ◽

Elongation Rate ◽

Force Balance ◽

Balance Model ◽

Spindle Poles ◽

Sister Chromatids ◽

Spindle Elongation ◽

Dependent Mechanism

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt–MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype.

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Nup132 modulates meiotic spindle attachment in fission yeast by regulating kinetochore assembly

The Journal of Cell Biology ◽

10.1083/jcb.201501035 ◽

2015 ◽

Vol 211 (2) ◽

pp. 295-308 ◽

Cited By ~ 8

Author(s):

Hui-Ju Yang ◽

Haruhiko Asakawa ◽

Tokuko Haraguchi ◽

Yasushi Hiraoka

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Meiotic Prophase ◽

Spindle Assembly Checkpoint ◽

Meiotic Spindle ◽

Spindle Attachment ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Monopolar Spindle ◽

Meiosis I

During meiosis, the kinetochore undergoes substantial reorganization to establish monopolar spindle attachment. In the fission yeast Schizosaccharomyces pombe, the KNL1–Spc7-Mis12-Nuf2 (KMN) complex, which constitutes the outer kinetochore, is disassembled during meiotic prophase and is reassembled before meiosis I. Here, we show that the nucleoporin Nup132 is required for timely assembly of the KMN proteins: In the absence of Nup132, Mis12 and Spc7 are precociously assembled at the centromeres during meiotic prophase. In contrast, Nuf2 shows timely dissociation and reappearance at the meiotic centromeres. We further demonstrate that depletion of Nup132 activates the spindle assembly checkpoint in meiosis I, possibly because of the increased incidence of erroneous spindle attachment at sister chromatids. These results suggest that precocious assembly of the kinetochores leads to the meiosis I defects observed in the nup132-disrupted mutant. Thus, we propose that Nup132 plays an important role in establishing monopolar spindle attachment at meiosis I through outer kinetochore reorganization at meiotic prophase.

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Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015288 ◽

2020 ◽

Vol 295 (40) ◽

pp. 13887-13901

Author(s):

Bhavika Nagareddy ◽

Arafat Khan ◽

Hyungjin Kim

Keyword(s):

Fanconi Anemia ◽

Posttranslational Modifications ◽

Genome Stability ◽

Chromosome Instability ◽

Lysine Residue ◽

Core Complex ◽

Ubiquitin E3 Ligase ◽

Pathway Activation ◽

Anemia Group ◽

Inherited Mutations

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

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Disruption of Astral Microtubule Contact with the Cell Cortex Activates a Bub1, Bub3, and Mad3-dependent Checkpoint in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0256 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3345-3356 ◽

Cited By ~ 37

Author(s):

Sylvie Tournier ◽

Yannick Gachet ◽

Vicky Buck ◽

Jeremy S. Hyams ◽

Jonathan B.A. Millar

Keyword(s):

Actin Cytoskeleton ◽

Fission Yeast ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Yeast Cells ◽

Cell Cortex ◽

Checkpoint Proteins ◽

Sister Chromatids ◽

Astral Microtubule ◽

Spindle Rotation

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

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