scholarly journals Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly

2021 ◽  
Author(s):  
Stephen M Hinshaw ◽  
Yun Quan ◽  
Jiaxi Cai ◽  
Ann Zhou ◽  
Huilin Zhou
Keyword(s):  
Chromosome Segregation ◽  
Regulatory Mechanism ◽  
Phosphorylation Sites ◽  
Biochemical System ◽  
Eukaryotic Chromosome ◽  
Kinetochore Assembly ◽  
Spindle Microtubules ◽  
Pest Region ◽  
Microtubule Poisons ◽  
In Cells

Kinetochores control eukaryotic chromosome segregation by connecting chromosomal centromeres to spindle microtubules. Duplication of centromeric DNA necessitates kinetochore disassembly and subsequent reassembly on the nascent sisters. To search for a regulatory mechanism that controls the earliest steps of kinetochore assembly, we studied Mif2/CENP-C, an essential basal component. We found that Polo-like kinase (Cdc5) and Dbf4-dependent kinase (DDK) phosphorylate the conserved PEST region of Mif2/CENP-C and that this phosphorylation directs inner kinetochore assembly. Mif2 phosphorylation promotes kinetochore assembly in a reconstituted biochemical system, and it strengthens Mif2 localization at centromeres in cells. Disrupting one or more phosphorylation sites in the Mif2-PEST region progressively impairs cellular fitness and sensitizes cells to microtubule poisons. The most severe Mif2-PEST mutations are lethal in cells lacking otherwise non-essential Ctf19 complex factors. These data suggest that multi-site phosphorylation of Mif2/CENP-C is a robust switch that controls inner kinetochore assembly, ensuring accurate chromosome segregation.

scholarly journals Phosphorylation of CENP-C by Aurora B facilitates kinetochore attachment error correction in mitosis

2017 ◽  
Vol 114 (50) ◽  
pp. E10667-E10676 ◽  
Author(s):  
Xing Zhou ◽  
Fan Zheng ◽  
Chengliang Wang ◽  
Minhao Wu ◽  
Xiaozhen Zhang ◽  
...  
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Chromosome ◽  
Regulatory Mechanism ◽  
Dynamic Interaction ◽  
Aurora B ◽  
Kinetochore Assembly ◽  
Important Pathway ◽  
Spindle Microtubules

Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12–Ndc80–Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12–Nnf1 complex and showed that N-terminal residues 26–50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C–Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

scholarly journals The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

2006 ◽  
Vol 173 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Susan L. Kline ◽  
Iain M. Cheeseman ◽  
Tetsuya Hori ◽  
Tatsuo Fukagawa ◽  
Arshad Desai
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Essential Role ◽  
Stable Complex ◽  
Wild Type ◽  
Outer Plate ◽  
Checkpoint Protein ◽  
Kinetochore Assembly ◽  
Attachment Sites ◽  
Spindle Microtubules

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.

scholarly journals Decoding the centromeric nucleosome through CENP-N

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Satyakrishna Pentakota ◽  
Keda Zhou ◽  
Charlotte Smith ◽  
Stefano Maffini ◽  
Arsen Petrovic ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Double Helix ◽  
Structural Basis ◽  
Binding Motif ◽  
Kinetochore Assembly ◽  
Nucleosomal Dna ◽  
Chromosome Domains ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Dna Double Helix

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

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 Evolutionary cell biology of chromosome segregation: insights from trypanosomes

Open Biology ◽  
2013 ◽  
Vol 3 (5) ◽  
pp. 130023 ◽  
Author(s):  
Bungo Akiyoshi ◽  
Keith Gull
Keyword(s):  
Chromosome Segregation ◽  
Cell Biology ◽  
Design Principle ◽  
Genetic Material ◽  
Evolutionary Time ◽  
Origin And Evolution ◽  
Eukaryotic Chromosome ◽  
Evolutionary Origins ◽  
Spindle Microtubules ◽  
Kinetoplastid Parasite

Faithful transmission of genetic material is essential for the survival of all organisms. Eukaryotic chromosome segregation is driven by the kinetochore that assembles onto centromeric DNA to capture spindle microtubules and govern the movement of chromosomes. Its molecular mechanism has been actively studied in conventional model eukaryotes, such as yeasts, worms, flies and human. However, these organisms are closely related in the evolutionary time scale and it therefore remains unclear whether all eukaryotes use a similar mechanism. The evolutionary origins of the segregation apparatus also remain enigmatic. To gain insights into these questions, it is critical to perform comparative studies. Here, we review our current understanding of the mitotic mechanism in Trypanosoma brucei , an experimentally tractable kinetoplastid parasite that branched early in eukaryotic history. No canonical kinetochore component has been identified, and the design principle of kinetochores might be fundamentally different in kinetoplastids. Furthermore, these organisms do not appear to possess a functional spindle checkpoint that monitors kinetochore–microtubule attachments. With these unique features and the long evolutionary distance from other eukaryotes, understanding the mechanism of chromosome segregation in T. brucei should reveal fundamental requirements for the eukaryotic segregation machinery, and may also provide hints about the origin and evolution of the segregation apparatus.

scholarly journals The centromere comes into focus: from CENP-A nucleosomes to kinetochore connections with the spindle

Open Biology ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 200051 ◽  
Author(s):  
Kathryn Kixmoeller ◽  
Praveen Kumar Allu ◽  
Ben E. Black
Keyword(s):  
Chromosome Segregation ◽  
Molecular Complexes ◽  
Molecular Models ◽  
Chromosomal Locus ◽  
Centromeric Chromatin ◽  
Eukaryotic Chromosome ◽  
The Core ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Insight Into

Eukaryotic chromosome segregation relies upon specific connections from DNA to the microtubule-based spindle that forms at cell division. The chromosomal locus that directs this process is the centromere, where a structure called the kinetochore forms upon entry into mitosis. Recent crystallography and single-particle electron microscopy have provided unprecedented high-resolution views of the molecular complexes involved in this process. The centromere is epigenetically specified by nucleosomes harbouring a histone H3 variant, CENP-A, and we review recent progress on how it differentiates centromeric chromatin from the rest of the chromosome, the biochemical pathway that mediates its assembly and how two non-histone components of the centromere specifically recognize CENP-A nucleosomes. The core centromeric nucleosome complex (CCNC) is required to recruit a 16-subunit complex termed the constitutive centromere associated network (CCAN), and we highlight recent structures reported of the budding yeast CCAN. Finally, the structures of multiple modular sub-complexes of the kinetochore have been solved at near-atomic resolution, providing insight into how connections are made to the CCAN on one end and to the spindle microtubules on the other. One can now build molecular models from the DNA through to the physical connections to microtubules.

scholarly journals The unconventional kinetoplastid kinetochore: from discovery toward functional understanding

2016 ◽  
Vol 44 (5) ◽  
pp. 1201-1217 ◽  
Author(s):  
Bungo Akiyoshi
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Protein Complex ◽  
Fundamental Function ◽  
Centromeric Dna ◽  
Eukaryotic Chromosome ◽  
Ndc80 Complex ◽  
Functional Understanding ◽  
Macromolecular Protein ◽  
Spindle Microtubules

The kinetochore is the macromolecular protein complex that drives chromosome segregation in eukaryotes. Its most fundamental function is to connect centromeric DNA to dynamic spindle microtubules. Studies in popular model eukaryotes have shown that centromere protein (CENP)-A is critical for DNA-binding, whereas the Ndc80 complex is essential for microtubule-binding. Given their conservation in diverse eukaryotes, it was widely believed that all eukaryotes would utilize these components to make up a core of the kinetochore. However, a recent study identified an unconventional type of kinetochore in evolutionarily distant kinetoplastid species, showing that chromosome segregation can be achieved using a distinct set of proteins. Here, I review the discovery of the two kinetochore systems and discuss how their studies contribute to a better understanding of the eukaryotic chromosome segregation machinery.

scholarly journals Aurora B-dependent Ndc80 Degradation Regulates Kinetochore Composition in Meiosis

2019 ◽  
Author(s):  
Jingxun Chen ◽  
Andrew Liao ◽  
Emily N Powers ◽  
Hanna Liao ◽  
Lori A Kohlstaedt ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Meiotic Prophase ◽  
Aurora B ◽  
Phosphorylation Sites ◽  
Independent Manner ◽  
N Terminus ◽  
Spindle Microtubules ◽  
Necessary And Sufficient ◽  
Single Subunit

ABSTRACTThe kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We have previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule–kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N-terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

scholarly journals Trypanosome outer kinetochore proteins suggest conservation of chromosome segregation machinery across eukaryotes

2016 ◽  
Vol 216 (2) ◽  
pp. 379-391 ◽  
Author(s):  
Simon D’Archivio ◽  
Bill Wickstead
Keyword(s):  
Family Member ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Model Organisms ◽  
Interacting Protein ◽  
Eukaryotic Chromosome ◽  
New Family ◽  
Kinetochore Assembly ◽  
The Family ◽  
Form Part

Kinetochores are multiprotein complexes that couple eukaryotic chromosomes to the mitotic spindle to ensure proper segregation. The model for kinetochore assembly is conserved between humans and yeast, and homologues of several components are widely distributed in eukaryotes, but key components are absent in some lineages. The recent discovery in a lineage of protozoa called kinetoplastids of unconventional kinetochores with no apparent homology to model organisms suggests that more than one system for eukaryotic chromosome segregation may exist. In this study, we report a new family of proteins distantly related to outer kinetochore proteins Ndc80 and Nuf2. The family member in kinetoplastids, KKT-interacting protein 1 (KKIP1), associates with the kinetochore, and its depletion causes severe defects in karyokinesis, loss of individual chromosomes, and gross defects in spindle assembly or stability. Immunopurification of KKIP1 from stabilized kinetochores identifies six further components, which form part of a trypanosome outer kinetochore complex. These findings suggest that kinetochores in organisms such as kinetoplastids are built from a divergent, but not ancestrally distinct, set of components and that Ndc80/Nuf2-like proteins are universal in eukaryotic division.

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