scholarly journals CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state

2013 ◽  
Vol 201 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Karen E. Gascoigne ◽  
Iain M. Cheeseman
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Exit ◽  
Cyclin Dependent Kinase ◽  
Temporal Regulation ◽  
Regulatory Processes ◽  
Kinetochore Assembly ◽  
Nuclear Exclusion ◽  
Ndc80 Complex ◽  
Mitotic Phosphorylation

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging–based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division.

scholarly journals Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly

2021 ◽  
Vol 118 (48) ◽  
pp. e2104459118
Author(s):  
Qianhua Dong ◽  
Xue-lei Liu ◽  
Xiao-hui Wang ◽  
Yu Zhao ◽  
Yu-hang Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Competitive Exclusion ◽  
Histone H3 ◽  
Cyclin Dependent Kinase ◽  
Null Mutant ◽  
Kinetochore Assembly ◽  
N Terminus ◽  
Ndc80 Complex ◽  
Histone H3 Variant

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

scholarly journals An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

2018 ◽  
Author(s):  
Jackie Lang ◽  
Adrienne Barber ◽  
Sue Biggins
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
De Novo ◽  
Budding Yeast ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Underlying Mechanisms ◽  
Mitotic Phosphorylation

ABSTRACTChromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

scholarly journals An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jackie Lang ◽  
Adrienne Barber ◽  
Sue Biggins
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
De Novo ◽  
Budding Yeast ◽  
Centromeric Dna ◽  
Kinetochore Assembly ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Underlying Mechanisms ◽  
Mitotic Phosphorylation

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

scholarly journals Phosphatase 2A Negatively Regulates Mitotic Exit in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (1) ◽  
pp. 80-89 ◽  
Author(s):  
Yanchang Wang ◽  
Tuen-Yung Ng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Exit ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Catalytic Subunits ◽  
Phosphatase 2A ◽  
Negative Role

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

scholarly journals A mitosis-specific and R loop–driven ATR pathway promotes faithful chromosome segregation

Science ◽  
2017 ◽  
Vol 359 (6371) ◽  
pp. 108-114 ◽  
Author(s):  
Lilian Kabeche ◽  
Hai Dang Nguyen ◽  
Rémi Buisson ◽  
Lee Zou
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Stress Responses ◽  
Chromosome Instability ◽  
Protein A ◽  
Aurora B ◽  
Cyclin Dependent Kinase ◽  
Ataxia Telangiectasia Mutated ◽  
Atr Kinase

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A–regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)–coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop–driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.

scholarly journals CENP-C is a blueprint for constitutive centromere–associated network assembly within human kinetochores

2015 ◽  
Vol 210 (1) ◽  
pp. 11-22 ◽  
Author(s):  
Kerstin Klare ◽  
John R. Weir ◽  
Federica Basilico ◽  
Tomasz Zimniak ◽  
Lucia Massimiliano ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
The Other ◽  
Microtubule Binding ◽  
Kinetochore Assembly ◽  
Binding Interface ◽  
Other Hand ◽  
Spindle Microtubules ◽  
Data Point

Kinetochores are multisubunit complexes that assemble on centromeres to bind spindle microtubules and promote faithful chromosome segregation during cell division. A 16-subunit complex named the constitutive centromere–associated network (CCAN) creates the centromere–kinetochore interface. CENP-C, a CCAN subunit, is crucial for kinetochore assembly because it links centromeres with the microtubule-binding interface of kinetochores. The role of CENP-C in CCAN organization, on the other hand, had been incompletely understood. In this paper, we combined biochemical reconstitution and cellular investigations to unveil how CENP-C promotes kinetochore targeting of other CCAN subunits. The so-called PEST domain in the N-terminal half of CENP-C interacted directly with the four-subunit CCAN subcomplex CENP-HIKM. We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex. When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.

scholarly journals Budding yeast complete DNA replication after chromosome segregation begins

2018 ◽  
Author(s):  
Tsvetomira Ivanova ◽  
Michael Maier ◽  
Alsu Missarova ◽  
Céline Ziegler-Birling ◽  
Lucas B. Carey ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Normal Growth ◽  
Population Level ◽  
Mitotic Exit ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Temporal Separation ◽  
Entire Genome ◽  
Number Variation

AbstractTo faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in a substantial fraction of unperturbed yeast cells, DNA replication finishes during anaphase, late in mitosis. High cyclin-Cdk activity inhibits replication in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA replication to finish at difficult-to-replicate regions. Replication during late mitosis correlates with elevated mutation rates, including copy number variation. Thus, yeast cells temporally overlap replication and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.One Sentence SummaryCompletion of DNA replication is coupled to downregulation of Cyclin-Dependent Kinase during mitotic exit.

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