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 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 The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

2018 ◽  
Vol 217 (11) ◽  
pp. 3886-3900 ◽  
Author(s):  
Aida Llauró ◽  
Hanako Hayashi ◽  
Megan E. Bailey ◽  
Alex Wilson ◽  
Patryk Ludzia ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Binding Activity ◽  
Significant Similarity ◽  
Load Bearing ◽  
Kinetochore Protein ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Coupling Protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

scholarly journals Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

Open Biology ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 190236 ◽  
Author(s):  
Olga O. Nerusheva ◽  
Patryk Ludzia ◽  
Bungo Akiyoshi
Keyword(s):  
Trypanosoma Brucei ◽  
Chromosome Segregation ◽  
Rna Binding ◽  
Affinity Purification ◽  
Potential Interaction ◽  
Interacting Proteins ◽  
Interaction Partners ◽  
Spindle Microtubules ◽  
Acetyltransferase Domain ◽  
Mitosis And Meiosis

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, 12 proteins (KKT-interacting proteins 1–12, KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei . KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N -acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

scholarly journals Condensin DC spreads linearly and bidirectionally from recruitment sites to create loop-anchored TADs in C. elegans

2021 ◽  
Author(s):  
David Sebastian Jimenez ◽  
Jun Kim ◽  
Bhavana Ragipani ◽  
Bo Zhang ◽  
Lena Annika Street ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
X Chromosome ◽  
Molecular Motors ◽  
Sequence Motif ◽  
X Chromosomes ◽  
C Elegans ◽  
Eukaryotic Chromosome ◽  
Multiple Copies

AbstractCondensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. Unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads into V and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.

scholarly journals Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

2001 ◽  
Vol 155 (5) ◽  
pp. 763-774 ◽  
Author(s):  
Jung-seog Kang ◽  
Iain M. Cheeseman ◽  
George Kallstrom ◽  
Soundarapandian Velmurugan ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Related Protein ◽  
Centromeric Dna ◽  
Centromere Protein ◽  
Kinetochore Function

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

scholarly journals SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation

2021 ◽  
Author(s):  
Manjuan Zhang ◽  
Fengrui Yang ◽  
Wenwen Wang ◽  
Xiwei Wang ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Spindle Microtubule ◽  
Dynamic Interactions ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
And Behavior

Abstract Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

scholarly journals Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

2018 ◽  
Vol 218 (2) ◽  
pp. 455-473 ◽  
Author(s):  
Cai Tong Ng ◽  
Li Deng ◽  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Depolymerization ◽  
Yeast Chromosome ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Electron Cryotomography ◽  
Binding Position

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

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 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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