scholarly journals Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

2009 ◽  
Vol 20 (24) ◽  
pp. 5096-5105 ◽  
Author(s):  
Vincent Vanoosthuyse ◽  
John C. Meadows ◽  
Sjaak J.A. van der Sar ◽  
Jonathan B.A. Millar ◽  
Kevin G. Hardwick
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Aurora Kinase ◽  
Yeast Cells ◽  
Anaphase Promoting Complex ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Checkpoint Recovery

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3Δ cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.

scholarly journals Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

2003 ◽  
Vol 161 (2) ◽  
pp. 267-280 ◽  
Author(s):  
Claire Ditchfield ◽  
Victoria L. Johnson ◽  
Anthony Tighe ◽  
Rebecca Ellston ◽  
Carolyn Haworth ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Spindle Checkpoint ◽  
Aurora Kinase ◽  
Multiple Roles ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Chromosome Alignment

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

scholarly journals Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation

2009 ◽  
Vol 20 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Quanbin Xu ◽  
Songcheng Zhu ◽  
Wei Wang ◽  
Xiaojuan Zhang ◽  
William Old ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Signaling Cascade ◽  
Checkpoint Activation ◽  
Mitotic Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Anaphase Onset

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.

scholarly journals The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

2018 ◽  
Vol 217 (3) ◽  
pp. 861-876 ◽  
Author(s):  
Eleni Petsalaki ◽  
Maria Dandoulaki ◽  
George Zachos
Keyword(s):  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Metaphase Plate ◽  
Membrane Remodeling ◽  
Mitotic Progression ◽  
Mitotic Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Endosomal Sorting ◽  
Anaphase Onset

The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod–ZW10–Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore–microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport–dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.

scholarly journals Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Atasi Poddar ◽  
P. Todd Stukenberg ◽  
Daniel J. Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
In Cells

ABSTRACT Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

scholarly journals A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhejian Ji ◽  
Haishan Gao ◽  
Luying Jia ◽  
Bing Li ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Phosphorylation Cascade ◽  
Mitotic Checkpoint Complex

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

scholarly journals Spindle Checkpoint Signaling Requires the Mis6 Kinetochore Subcomplex, Which Interacts with Mad2 and Mitotic Spindles

2005 ◽  
Vol 16 (8) ◽  
pp. 3666-3677 ◽  
Author(s):  
Shigeaki Saitoh ◽  
Kojiro Ishii ◽  
Yasuyo Kobayashi ◽  
Kohta Takahashi
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Cycle Progression ◽  
Centromeric Chromatin ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Potential Binding ◽  
Evolutionarily Conserved

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle–kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

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

2004 ◽  
Vol 15 (7) ◽  
pp. 3345-3356 ◽  
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.

scholarly journals MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

2019 ◽  
Vol 218 (4) ◽  
pp. 1108-1117 ◽  
Author(s):  
Tatiana Alfonso-Pérez ◽  
Daniel Hayward ◽  
James Holder ◽  
Ulrike Gruneberg ◽  
Francis A. Barr
Keyword(s):  
Feedback Loop ◽  
Spindle Checkpoint ◽  
Mitotic Exit ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
Mitotic Entry ◽  
Upstream Regulator ◽  
Microtubule Attachment ◽  
Integral Component ◽  
Checkpoint Pathway

Cyclin B–dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

Sign in / Sign up

Export Citation Format

Share Document