scholarly journals Unrestrained Spindle Elongation during Recovery from Spindle Checkpoint Activation in cdc15-2 Cells Results in Mis-Segregation of Chromosomes

2010 ◽  
Vol 21 (14) ◽  
pp. 2384-2398 ◽  
Author(s):  
Chuan Chung Chai ◽  
Ee Mei Teh ◽  
Foong May Yeong
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Checkpoint Activation ◽  
Sister Chromatids ◽  
Spindle Pole Bodies ◽  
Balance Of Forces ◽  
Spindle Elongation ◽  
Pulling Force

During normal metaphase in Saccharomyces cerevisiae, chromosomes are captured at the kinetochores by microtubules emanating from the spindle pole bodies at opposite poles of the dividing cell. The balance of forces between the cohesins holding the replicated chromosomes together and the pulling force from the microtubules at the kinetochores result in the biorientation of the sister chromatids before chromosome segregation. The absence of kinetochore–microtubule interactions or loss of cohesion between the sister chromatids triggers the spindle checkpoint which arrests cells in metaphase. We report here that an MEN mutant, cdc15-2, though competent in activating the spindle assembly checkpoint when exposed to Noc, mis-segregated chromosomes during recovery from spindle checkpoint activation. cdc15-2 cells arrested in Noc, although their Pds1p levels did not accumulate as well as in wild-type cells. Genetic analysis indicated that Pds1p levels are lower in a mad2Δ cdc15-2 and bub2Δ cdc15-2 double mutants compared with the single mutants. Chromosome mis-segregation in the mutant was due to premature spindle elongation in the presence of unattached chromosomes, likely through loss of proper control on spindle midzone protein Slk19p and kinesin protein, Cin8p. Our data indicate that a slower rate of transition through the cell division cycle can result in an inadequate level of Pds1p accumulation that can compromise recovery from spindle assembly checkpoint activation.

scholarly journals Histone H3 Exerts a Key Function in Mitotic Checkpoint Control

2009 ◽  
Vol 30 (2) ◽  
pp. 537-549 ◽  
Author(s):  
Jianjun Luo ◽  
Xinjing Xu ◽  
Hana Hall ◽  
Edel M. Hyland ◽  
Jef D. Boeke ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Histone H3 ◽  
Spindle Assembly ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Yeast Cells ◽  
Checkpoint Control ◽  
Spindle Pole Bodies ◽  
Key Factor ◽  
Pulling Force

ABSTRACT It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.

scholarly journals Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

2016 ◽  
Vol 27 (11) ◽  
pp. 1753-1763 ◽  
Author(s):  
Hirohisa Masuda ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Synthetic Lethality ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Spindle Pole Bodies ◽  
Mitotic Spindle Pole

In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

scholarly journals Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Heather E Arsenault ◽  
Julie M Ghizzoni ◽  
Cassandra M Leech ◽  
Anne R Diers ◽  
Stephane Gesta ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Cycle Arrest ◽  
Microtubule Poisons

Abstract The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase-anaphase transition. Here we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging and half-life. Additionally, stabilization of Ubc1 prevented its downregulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that downregulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe

1998 ◽  
Vol 111 (12) ◽  
pp. 1635-1647 ◽  
Author(s):  
X. He ◽  
M.H. Jones ◽  
M. Winey ◽  
S. Sazer
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Sequence Similarity ◽  
Spindle Pole Body ◽  
Spindle Checkpoint ◽  
Spindle Pole ◽  
Metaphase Arrest ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Activation ◽  
Checkpoint Pathway ◽  
Dual Specificity

The spindle assembly checkpoint pathway is not essential for normal mitosis but ensures accurate nuclear division by blocking the metaphase to anaphase transition in response to a defective spindle. Here, we report the isolation of a new spindle checkpoint gene, mph1 (Mps1p-like pombe homolog), in the fission yeast Schizosaccharomyces pombe, that is required for checkpoint activation in response to spindle defects. mph1 functions upstream of mad2, a previously characterized component of the spindle checkpoint. Overexpression of mph1, like overexpression of mad2, mimics activation of the checkpoint and imposes a metaphase arrest. mph1 protein shares sequence similarity with Mps1p, a dual specificity kinase that functions in the spindle checkpoint of the budding yeast Saccharomyces cerevisiae. Complementation analysis demonstrates that mph1 and Mps1p are functionally related. They differ in that Mps1p, but not mph1, has an additional essential role in spindle pole body duplication. We propose that mph1 is the MPS1 equivalent in the spindle checkpoint pathway but not in the SPB duplication pathway. Overexpression of mad2 does not require mph1 to impose a metaphase arrest, which indicates a mechanism of spindle checkpoint activation other than mph1/Mps1p kinase-dependent phosphorylation. In the same screen which led to the isolation of mad2 and mph1, we also isolated dph1, a cDNA that encodes a protein 46% identical to an S. cerevisiae SPB duplication protein, Dsk2p. Our initial characterization indicates that S.p. dph1 and S.c. DSK2 are functionally similar. Together these results suggest that the budding and fission yeasts share common elements for SPB duplication, despite differences in SPB structure and the timing of SPB duplication relative to mitotic entry.

scholarly journals Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast

2009 ◽  
Vol 20 (10) ◽  
pp. 2626-2637 ◽  
Author(s):  
Elena Chiroli ◽  
Giulia Rancati ◽  
Ilaria Catusi ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Sister Chromatid ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Centrosome Separation ◽  
Spindle Pole Bodies ◽  
Early Anaphase ◽  
Sister Chromatid Separation

The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.

scholarly journals Loss of Kif18A Results in Spindle Assembly Checkpoint Activation at Microtubule-Attached Kinetochores

2018 ◽  
Vol 28 (17) ◽  
pp. 2685-2696.e4 ◽  
Author(s):  
Louise M.E. Janssen ◽  
Tessa V. Averink ◽  
Vincent A. Blomen ◽  
Thijn R. Brummelkamp ◽  
René H. Medema ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Checkpoint Activation

The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

2014 ◽  
Vol 307 (5) ◽  
pp. C466-C478 ◽  
Author(s):  
Shao-Chih Chiu ◽  
Jo-Mei Maureen Chen ◽  
Tong-You Wade Wei ◽  
Tai-Shan Cheng ◽  
Ya-Hui Candice Wang ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Spindle Assembly Checkpoint ◽  
Morphological Changes ◽  
Spindle Assembly ◽  
Aurora A ◽  
Protein Protein Interaction ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Complicated Process ◽  
Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

scholarly journals Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Irsa Shoukat ◽  
Corey Frazer ◽  
John S. Allingham
Keyword(s):  
Candida Albicans ◽  
Mitotic Spindle ◽  
Fungal Pathogens ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Content Type ◽  
Bipolar Spindle ◽  
Spindle Elongation ◽  
Simultaneous Loss ◽  
Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

scholarly journals Mitosis in the cellular slime mold Polysphondylium violaceum.

1975 ◽  
Vol 64 (2) ◽  
pp. 480-491 ◽  
Author(s):  
U P Roos
Keyword(s):  
Spindle Pole ◽  
Equatorial Region ◽  
Cellular Slime Mold ◽  
Central Spindle ◽  
Spindle Pole Bodies ◽  
Cellular Slime ◽  
Late Telophase ◽  
Spindle Elongation ◽  
Well Differentiated ◽  
Opaque Body

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.

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