scholarly journals Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

2012 ◽  
Vol 198 (4) ◽  
pp. 491-499 ◽  
Author(s):  
Dan Liu ◽  
Olga Davydenko ◽  
Michael A. Lampson
Keyword(s):  
High Activity ◽  
Spindle Checkpoint ◽  
Mitotic Arrest ◽  
Microtubule Dynamics ◽  
Signaling Proteins ◽  
Dynamic Localization ◽  
Checkpoint Signaling ◽  
Microtubule Attachment

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint.

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.

Pharicin a, a Novel Natural Compound That Induces Mitotic Arrest and Catastrophe of Cancer Cells by Perturbing Microtubule Dynamics and the Spindle Checkpoint

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4723-4723
Author(s):  
Guo-Qiang Chen ◽  
Wei Dai ◽  
Han-Zhang Xu ◽  
Dao Li
Keyword(s):  
Functional Characterization ◽  
Folk Medicine ◽  
Spindle Checkpoint ◽  
Mitotic Arrest ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Jurkat Cells ◽  
Cancer Drug

Abstract Microtubule poisons such as taxol represent a potent and effective class of anticancer compounds. In the past decades, great efforts have been directed to identify novel natural products with a mode of action similar to taxol but with a minimal side effect. In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon pharicus leaves, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in Jurkat, Raji and HeLa based on their morphology, DNA content, histone H3 serine-10 phosphorylation, and mitotic marker protein analyses. Pharicin A stabilizes the formation of mitotic spindles, which is coupled with a rapid accumulation of Cdc20 as well as an increased phosphorylation of Cdc27 and BubR1. Pharicin A treatment also results in an enhanced interaction between Cdc20 and spindle checkpoint components including Mad2 and BubR1. Moreover, pharicin A-induced mitotic arrest in HeLa cells is tightly associated with the presence of lagging/missegregated chromosomes at spindle pole regions, which are highly enriched in BubR1, CENP-E and Sgo1. Although pharicin A stabilizes microtubules both in vitro and in vivo, it induces mitotic arrest in taxol-resistant Jurkat cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing microtubule dynamics and the spindle checkpoint in tumor cells, which merits further preclinical and clinical investigations for cancer drug development.

scholarly journals Phosphatase-Regulated Recruitment of the Spindle and Kinetochore Associated (SKA) Complex to Kinetochores

2017 ◽  
Author(s):  
Sushama Sivakumar ◽  
Gary J. Gorbsky
Keyword(s):  
Protein Phosphatase ◽  
Cell Cycle Progression ◽  
Spindle Checkpoint ◽  
Checkpoint Signaling ◽  
Mitotic Kinases ◽  
Feedback Cycle ◽  
Microtubule Attachment ◽  
Phosphatase 2A ◽  
Summary Statement ◽  
And Function

ABSTRACTKinetochores move chromosomes on dynamic spindle microtubules and regulate cell cycle progression by signaling the spindle checkpoint. The Spindle and Kinetochore-Associated (Ska) Complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, participates in both roles. The mitotic kinases, Cdk1, Aurora B, Plk1, Mps1 and Bub1 play key, overlapping tasks in regulating chromosome movement and checkpoint signaling. However, roles for the phosphatases that oppose these kinases are more poorly defined. Recently, we showed that Ska1 is important for recruiting protein phosphatase 1 (PP1) to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A) both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes also seen after depletion of Ska. Tethering of PP1 to the kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and reduces mitotic defects seen after Ska depletion. We propose that kinetochore-associated phosphatases generate a positive feedback cycle to reinforce Ska complex accumulation and function at kinetochores.SUMMARY STATEMENTPhosphatases reinforce recruitment of the Ska complex at kinetochores to stabilize microtubule attachment and oppose spindle checkpoint signaling.

scholarly journals TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes

Open Biology ◽  
2014 ◽  
Vol 4 (6) ◽  
pp. 130108 ◽  
Author(s):  
Roshan L. Shrestha ◽  
Naoka Tamura ◽  
Anna Fries ◽  
Nicolas Levin ◽  
Joanna Clark ◽  
...  
Keyword(s):  
Error Correction ◽  
Chromosomal Instability ◽  
Spindle Checkpoint ◽  
Human Cells ◽  
Microtubule Dynamics ◽  
Regulatory Pathways ◽  
Chromosomal Stability ◽  
Microtubule Attachment ◽  
Drug Treatments

Chromosomal instability can arise from defects in chromosome–microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1's role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome–microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore–microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.

scholarly journals Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

2012 ◽  
Vol 196 (4) ◽  
pp. 469-482 ◽  
Author(s):  
Julien Espeut ◽  
Dhanya K. Cheerambathur ◽  
Lenno Krenning ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Spindle Checkpoint ◽  
Protein Phosphatase 1 ◽  
Checkpoint Activation ◽  
Load Bearing ◽  
Microtubule Binding ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
N Terminus ◽  
Ndc80 Complex ◽  
Checkpoint Signal

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.

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 Kif18A promotes Hec1 dephosphorylation to coordinate chromosome alignment with kinetochore microtubule attachment

2018 ◽  
Author(s):  
Haein Kim ◽  
Jason Stumpff
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Primordial Germ Cells ◽  
Direct Interaction ◽  
Mitotic Arrest ◽  
Microtubule Dynamics ◽  
Mitotic Chromosomes ◽  
Microtubule Attachment ◽  
C Terminus ◽  
Chromosome Alignment

SUMMARYMitotic chromosomes are spatially confined at the spindle equator just prior to chromosome segregation through a process called chromosome alignment. Alignment requires temporal coordination of kinetochore microtubule attachment and dynamics. However, the molecular mechanisms that couple these activities are not understood. Kif18A (kinesin-8) suppresses the dynamics of kinetochore microtubules to promote chromosome alignment during metaphase. Loss of Kif18A function in HeLa and primordial germ cells leads to alignment defects accompanied by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, suggesting the motor also plays a role in regulating kinetochore-microtubule attachments. We show here that Kif18A increases attachment by promoting dephosphorylation of the kinetochore protein Hec1, which provides the primary linkage between kinetochores and microtubules. This function requires a direct interaction between the Kif18A C-terminus and protein phosphatase-1 (PP1). However, the Kif18A-PP1 interaction is not required for chromosome alignment, indicating that regulation of kinetochore microtubule dynamics and attachments are separable Kif18A functions. Mitotic arrest in Kif18A-depleted cells is rescued by expression of a Hec1 variant that mimics a low-phosphorylation state, indicating that Kif18A-dependent Hec1 dephosphorylation is a key step for silencing the checkpoint and promoting mitotic progression. Our data support a model in which Kif18A provides positive feedback for kinetochore microtubule attachment by directly recruiting PP1 to dephosphorylate Hec1. We propose that this function works synergistically with Kif18A’s direct control of kinetochore microtubule dynamics to temporally coordinate chromosome alignment and attachment.

scholarly journals The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1493-1502
Author(s):  
Richard D Gardner ◽  
Atasi Poddar ◽  
Chris Yellman ◽  
Penny A Tavormina ◽  
M Cristina Monteagudo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Spindle Checkpoint ◽  
Essential Genes ◽  
Gene Fusions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Signaling ◽  
One Step ◽  
Diploid Cells ◽  
Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein 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.

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