Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins.

Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

Disorder in CENP-ACse4 tail-chaperone interaction facilitates binding with Ame1/Okp1 at the kinetochore.

The centromere is epigenetically marked by a histone H3 variant-CENP-A. The budding yeast CENP-A called Cse4, consists of an unusually long N-terminus that is known to be involved in kinetochore assembly. Its disordered chaperone, Scm3 is responsible for the centromeric deposition of Cse4 as well as in the maintenance of a segregation-competent kinetochore. In this study, we show that the Cse4 N-terminus is intrinsically disordered and interacts with Scm3 at multiple sites, and the complex does not gain any substantial structure. Additionally, the complex forms a synergistic association with an essential inner kinetochore component (Ctf19-Mcm21-Okp1-Ame1), and a model has been suggested to this effect. Thus, our study provides mechanistic insights into the Cse4 N-terminus-chaperone interaction and also illustrates how intrinsically disordered proteins mediate assembly of complex multiprotein networks, in general.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4.

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463-645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1.

Budding uninhibited by benzimidazole 1-related protein 1 (BUBR1) is a mitotic checkpoint (better known as the spindle assembly checkpoint) protein that forms part of an inhibitory complex required to delay mitosis when errors occur in the attachment between chromosomes and the mitotic spindle. If these errors remain uncorrected, it could result in unequal distribution of genetic material to each of the nascent daughter cells, leading to potentially disastrous consequences at both the cellular and organismal level. In some higher eukaryotes including vertebrates, BUBR1 has a C-terminal kinase fold that is largely thought to be inactive, whereas in many species this domain has been lost through evolution and the truncated protein is known as mitotic arrest deficient 3 (MAD3). Here we present advice and practical considerations for the design of experiments, their analysis and interpretation to study the functions of the vertebrate BUBR1 during mitosis with emphasis on analysis implicating the pseudokinase domain.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome.

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

Kinetochore-bound Mps1 regulates kinetochore-microtubule attachments via Ndc80 phosphorylation.

Dividing cells detect and correct erroneous kinetochore-microtubule attachments during mitosis, thereby avoiding chromosome missegregation. The Aurora B kinase phosphorylates microtubule-binding elements specifically at incorrectly attached kinetochores, promoting their release and providing another chance for proper attachments to form. However, growing evidence suggests that the Mps1 kinase is also required for error correction. Here we directly examine how Mps1 activity affects kinetochore-microtubule attachments using a reconstitution-based approach that allows us to separate its effects from Aurora B activity. When endogenous Mps1 that copurifies with kinetochores is activated in vitro, it weakens their attachments to microtubules via phosphorylation of Ndc80, a major microtubule-binding protein. This phosphorylation contributes to error correction because phospho-deficient Ndc80 mutants exhibit genetic interactions and segregation defects when combined with mutants in other error correction pathways. In addition, Mps1 phosphorylation of Ndc80 is stimulated on kinetochores lacking tension. These data suggest that Mps1 provides an additional mechanism for correcting erroneous kinetochore-microtubule attachments, complementing the well-known activity of Aurora B.

Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase.

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein.

The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

Structural and dynamic mechanisms of CBF3-guided centromeric nucleosome formation.

Accurate chromosome segregation relies on the specific centromeric nucleosome-kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.

Emerging roles of centromeric RNAs in centromere formation and function.

BACKGROUND: Centromeres are specialized chromosomal domains involved in kinetochore formation and faithful chromosome segregation. Despite a high level of functional conservation, centromeres are not identified by DNA sequences, but by epigenetic means. Universally, centromeres are typically formed on highly repetitive DNA, which were previously considered to be silent. However, recent studies have shown that transcription occurs in this region, known as centromeric-derived RNAs (cenRNAs). CenRNAs that contribute to fundamental aspects of centromere function have been recently investigated in detail. However, the distribution, behavior and contributions of centromeric transcripts are still poorly understood. OBJECTIVE: The aim of this article is to provide an overview of the roles of cenRNAs in centromere formation and function. METHODS: We describe the structure and DNA sequence of centromere from yeast to human. In addition, we briefly introduce the roles of cenRNAs in centromere formation and function, kinetochore structure, accurate chromosome segregation, and pericentromeric heterochromatin assembly. Centromeric circular RNAs (circRNAs) and R-loops are rising stars in centromere function. CircRNAs have been successfully identified in various species with the assistance of high-throughput sequencing and novel computational approaches for non-polyadenylated RNA transcripts. Centromeric R-loops can be identified by the single-strand DNA ligation-based library preparation technique. But the molecular features and function of these centromeric R-loops and circRNAs are still being investigated. CONCLUSION: In this review, we summarize recent findings on the epigenetic regulation of cenRNAs across species, which would provide useful information about cenRNAs and interesting hints for further studies.

The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans.

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans.

Biochemical evidence for diverse strategies in the inner kinetochore.

The kinetochore is a complex structure whose function is absolutely essential. Unlike the centromere, the kinetochore at first appeared remarkably well conserved from yeast to humans, especially the microtubule-binding outer kinetochore. However, recent efforts towards biochemical reconstitution of diverse kinetochores challenge the notion of a similarly conserved architecture for the constitutively centromere-associated network of the inner kinetochore. This review briefly summarizes the evidence from comparative genomics for interspecific variability in inner kinetochore composition and focuses on novel biochemical evidence indicating that even homologous inner kinetochore protein complexes are put to different uses in different organisms.

Crystal structure of the Cenp-HIKHead-TW sub-module of the inner kinetochore CCAN complex.

Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14-16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead-Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN-Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.

A dCas9-Based System Identifies a Central Role for Ctf19 in Kinetochore-Derived Suppression of Meiotic Recombination.

In meiosis, crossover (CO) formation between homologous chromosomes is essential for faithful segregation. However, misplaced meiotic recombination can have catastrophic consequences on genome stability. Within pericentromeres, COs are associated with meiotic chromosome missegregation. In organisms ranging from yeast to humans, pericentromeric COs are repressed. We previously identified a role for the kinetochore-associated Ctf19 complex (Ctf19c) in pericentromeric CO suppression. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits. Using this approach, we query sufficiency in meiotic CO suppression, and identify Ctf19 as a mediator of kinetochore-associated CO control. The effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues important for the recruitment of the Scc2-Scc4 cohesin regulator. This work provides insight into kinetochore-derived control of meiotic recombination. We establish an experimental platform to investigate and manipulate meiotic CO control. This platform can easily be adapted in order to investigate other aspects of chromosome biology.

The whole is greater than the sum of its parts: at the intersection of order, disorder, and kinetochore function.

The kinetochore (KT) field has matured tremendously since Earnshaw first identified CENP-A, CENP-B, and CENP-C [1,2]. In the past 35 years, the accumulation of knowledge has included: defining the parts list, identifying epistatic networks of interdependence within the parts list, understanding the spatial organization of subcomplexes into a massive structure - hundreds of megadaltons in size, and dissecting the functions of the KT in its entirety as well as of its individual parts. Like nearly all cell and molecular biology fields, the structure-function paradigm has been foundational to advances in the KT field. A point nicely highlighted by the fact that we are at the precipice of the in vitro reconstitution of a functional KT holo complex. Yet conventional notions of structure cannot provide a complete picture of the KT especially since it contains an abundance of unstructured or intrinsically disordered constituents. The combination of structured and disordered proteins within the KT results in an assembled system that is functionally greater than the sum of its parts.

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness of the spindle assembly checkpoint.

During mitosis, the Spindle Assembly Checkpoint (SAC) maintains genome stability while also ensuring timely anaphase onset. To maintain genome stability, the SAC must be strong to delay anaphase even if just one chromosome is unattached, but for timely anaphase onset, it must promptly respond to silencing mechanisms. How the SAC meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of 'MELT' motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Many strong MELT motifs per Spc105/KNL1 minimize chromosome missegregation, but too many delay anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for much more accurate chromosome segregation. We propose that the necessity of balancing SAC strength and responsiveness drives the dual evolutionary trend of the amplification of MELT motif number, but degeneration of their functionally optimal amino acid sequence.

Artificial generation of centromeres and kinetochores to understand their structure and function.

The centromere is an essential genomic region that provides the surface to form the kinetochore, which binds to the spindle microtubes to mediate chromosome segregation during mitosis and meiosis. Centromeres of most organisms possess highly repetitive sequences, making it difficult to study these loci. However, an unusual centromere called a "neocentromere," which does not contain repetitive sequences, was discovered in a patient and can be generated experimentally. Recent advances in genome biology techniques allow us to analyze centromeric chromatin using neocentromeres. In addition to neocentromeres, artificial kinetochores have been generated on non-centromeric loci, using protein tethering systems. These are powerful tools to understand the mechanism of the centromere specification and kinetochore assembly. In this review, we introduce recent studies utilizing the neocentromeres and artificial kinetochores and discuss current problems in centromere biology.

Dynamics of kinetochore structure and its regulations during mitotic progression.

Faithful chromosome segregation during mitosis in eukaryotes requires attachment of the kinetochore, a large protein complex assembled on the centromere of each chromosome, to the spindle microtubules. The kinetochore is a structural interface for the microtubule attachment and provides molecular surveillance mechanisms that monitor and ensure the precise microtubule attachment as well, including error correction and spindle assembly checkpoint. During mitotic progression, the kinetochore undergoes dynamic morphological changes that are observable through electron microscopy as well as through fluorescence microscopy. These structural changes might be associated with the kinetochore function. In this review, we summarize how the dynamics of kinetochore morphology are associated with its functions and discuss recent findings on the switching of protein interaction networks in the kinetochore during cell cycle progression.

CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.