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 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.

scholarly journals Divergent polo box domains underpin the unique kinetoplastid kinetochore

Open Biology ◽  
2016 ◽  
Vol 6 (3) ◽  
pp. 150206 ◽  
Author(s):  
Olga O. Nerusheva ◽  
Bungo Akiyoshi
Keyword(s):  
Trypanosoma Brucei ◽  
Chromosome Segregation ◽  
Sequence Similarity ◽  
Rich Region ◽  
Significant Similarity ◽  
Centromeric Dna ◽  
Eukaryotic Chromosome ◽  
Duplication Events ◽  
Spindle Microtubules

Kinetochores are macromolecular machines that drive eukaryotic chromosome segregation by interacting with centromeric DNA and spindle microtubules. While most eukaryotes possess conventional kinetochore proteins, evolutionarily distant kinetoplastid species have unconventional kinetochore proteins, composed of at least 19 proteins (KKT1–19). Polo-like kinase (PLK) is not a structural kinetochore component in either system. Here, we report the identification of an additional kinetochore protein, KKT20, in Trypanosoma brucei . KKT20 has sequence similarity with KKT2 and KKT3 in the Cys-rich region, and all three proteins have weak but significant similarity to the polo box domain (PBD) of PLK. These divergent PBDs of KKT2 and KKT20 are sufficient for kinetochore localization in vivo . We propose that the ancestral PLK acquired a Cys-rich region and then underwent gene duplication events to give rise to three structural kinetochore proteins in kinetoplastids.

scholarly journals Kinetochore-independent chromosome segregation driven by lateral microtubule bundles

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Christina C Muscat ◽  
Keila M Torre-Santiago ◽  
Michael V Tran ◽  
James A Powers ◽  
Sarah M Wignall
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Protein Complex ◽  
Chromosome Movement ◽  
Chromosome Dynamics ◽  
Anaphase Onset ◽  
Independent Segregation ◽  
Main Driver ◽  
Chromosome Associations ◽  
Spindle Microtubules

During cell division, chromosomes attach to spindle microtubules at sites called kinetochores, and force generated at the kinetochore-microtubule interface is the main driver of chromosome movement. Surprisingly, kinetochores are not required for chromosome segregation on acentrosomal spindles in Caenorhabditis elegans oocytes, but the mechanism driving chromosomes apart in their absence is not understood. In this study, we show that lateral microtubule–chromosome associations established during prometaphase remain intact during anaphase to facilitate separation, defining a novel form of kinetochore-independent segregation. Chromosome dynamics during congression and segregation are controlled by opposing forces; plus-end directed forces are mediated by a protein complex that forms a ring around the chromosome center and dynein on chromosome arms provides a minus-end force. At anaphase onset, ring removal shifts the balance between these forces, triggering poleward movement along lateral microtubule bundles. This represents an elegant strategy for controlling chromosomal movements during cell division distinct from the canonical kinetochore-driven mechanism.

scholarly journals Kre28-Spc105 interaction is essential for their recruitment at the kinetochore

2021 ◽  
Author(s):  
Babhrubahan Roy ◽  
Janice Sim ◽  
Simon J. Y. Han ◽  
Ajit P. Joglekar
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
High Fidelity ◽  
Protein Assemblies ◽  
Sister Chromatids ◽  
Exact Function ◽  
Interaction Interface ◽  
Macromolecular Protein ◽  
Spindle Microtubules ◽  
Turn Over

Kinetochores are macromolecular protein assemblies that attach sister chromatids to spindle microtubules and mediate accurate chromosome segregation during mitosis. The outer kinetochore consists of the KMN network, a protein super complex made of Knl1 (yeast Spc105), Mis12 (yeast Mtw1) and Ndc80 (yeast Ndc80), which harbors sites for microtubule binding. Within the KMN network, Spc105 acts as interaction hub of components involved in spindle assembly checkpoint (SAC) signaling. It is known that Spc105 forms a complex with kinetochore component Kre28. However, where Kre28 physically localizes in the budding yeast kinetochore is not clear. The exact function of Kre28 at the kinetochore is also unknown. Here, we reveal how Spc105 and Kre28 interact and how they are organized within bioriented yeast kinetochores using genetics and cell biological experiments. We also identify the interaction interface between the two proteins and show that this interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the kinetochores. We created several truncation mutants of kre28 that do not localize at the kinetochores and so cannot mediate Spc105 loading at the kinetochores. When we over-expressed these mutants, they could sustain the cell viability even though failed to facilitate proper SAC activation and/or error correction. Thus, we inferred that Kre28 indirectly contributes to chromosome biorientation and high-fidelity segregation by regulating Spc105 localization at the kinetochores.

scholarly journals Adaptive Evolution of Cid, a Centromere-Specific Histone in Drosophila

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1293-1298 ◽  
Author(s):  
Harmit S Malik ◽  
Steven Henikoff
Keyword(s):  
Drosophila Melanogaster ◽  
Adaptive Evolution ◽  
Chromosome Segregation ◽  
Rapid Evolution ◽  
Histone H3 ◽  
Closely Related Species ◽  
Centromeric Dna ◽  
Eukaryotic Chromosome ◽  
Histone Fold ◽  
Satellite Repeats

Abstract Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.

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 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 structural basis for Ulp2 recruitment to the kinetochore

2021 ◽  
Author(s):  
Yun Quan ◽  
Stephen M. Hinshaw ◽  
Pang-Che Wang ◽  
Stephen C. Harrison ◽  
Huilin Zhou
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Structural Basis ◽  
Centromeric Dna ◽  
Step Process ◽  
Sumo Protease ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Spindle Microtubules

ABSTRACTThe step-by-step process of chromosome segregation defines the stages of the cell division cycle. In eukaryotes, signaling pathways that control these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to the centromere of each chromosome. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for the SUMO protease, Ulp2. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

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