Chromosome segregation: Mps1 and Dam1 battle to bind a shared interaction site at the kinetochore.

The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the kinetochore that integrates kinetochore attachment state with spindle checkpoint activity and kinetochore assembly.

Native and tagged CENP-A histones are functionally inequivalent.

BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

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.

Chromosome size-dependent polar ejection force impairs mammalian mitotic error correction.

Accurate chromosome segregation requires sister kinetochores to biorient, attaching to opposite spindle poles. To this end, the mammalian kinetochore destabilizes incorrect attachments and stabilizes correct ones, but how it discriminates between these is not yet clear. Here, we test the model that kinetochore tension is the stabilizing cue and ask how chromosome size impacts that model. We live image PtK2 cells, with just 14 chromosomes, widely ranging in size, and find that long chromosomes align at the metaphase plate later than short chromosomes. Enriching for errors and imaging error correction live, we show that long chromosomes exhibit a specific delay in correcting attachments. Using chromokinesin overexpression and laser ablation to perturb polar ejection forces, we find that chromosome size and force on arms determine alignment order. Thus, we propose a model where increased force on long chromosomes can falsely stabilize incorrect attachments, delaying their biorientation. As such, long chromosomes may require compensatory mechanisms for correcting errors to avoid chromosomal instability.

A conserved site on Ndc80 complex facilitates dynamic recruitment of Mps1 to yeast kinetochores to promote accurate chromosome segregation.

Accurate chromosome segregation relies on kinetochores carrying out multiple functions, including establishing and maintaining microtubule attachments, forming precise bi-oriented attachments between sister chromatids, and activating the spindle assembly checkpoint. Central to these processes is the highly conserved Ndc80 complex. This kinetochore subcomplex interacts directly with microtubules but also serves as a critical platform for recruiting kinetochore-associated factors and as a key substrate for error correction kinases. The precise manner in which these kinetochore factors interact and regulate each other's function remains unknown, considerably hindering our understanding of how Ndc80 complex-dependent processes function together to orchestrate accurate chromosome segregation. Here, we aimed to uncover the role of Nuf2's CH domain, a component of the Ndc80 complex, in ensuring these processes. Through extensive mutational analysis, we identified a conserved interaction domain composed of two segments in Nuf2's CH domain that form the binding site for Mps1 within the yeast Ndc80 complex. Interestingly, this site also associates with the Dam1 complex, suggesting Mps1 recruitment may be subject to regulation by competitive binding with other factors. Mutants disrupting this "interaction hub" exhibit defects in spindle assembly checkpoint function and severe chromosome segregation errors. Significantly, specifically restoring Mps1-Ndc80 complex association rescues these defects. Our findings shed light on the intricate regulation of Ndc80 complex-dependent functions and highlight the essential role of Mps1 in kinetochore bi-orientation and accurate chromosome segregation.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor.

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

A communication hub for phosphoregulation of kinetochore-microtubule attachment.

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin.

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.

Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis.

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

The variation and evolution of complete human centromeres.

Human centromeres have been traditionally very difficult to sequence and assemble owing to their repetitive nature and large size1. As a result, patterns of human centromeric variation and models for their evolution and function remain incomplete, despite centromeres being among the most rapidly mutating regions2,3. Here, using long-read sequencing, we completely sequenced and assembled all centromeres from a second human genome and compared it to the finished reference genome4,5. We find that the two sets of centromeres show at least a 4.1-fold increase in single-nucleotide variation when compared with their unique flanks and vary up to 3-fold in size. Moreover, we find that 45.8% of centromeric sequence cannot be reliably aligned using standard methods owing to the emergence of new α-satellite higher-order repeats (HORs). DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by >500 kb. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan and macaque genomes. Comparative analyses reveal a nearly complete turnover of α-satellite HORs, with characteristic idiosyncratic changes in α-satellite HORs for each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the short (p) and long (q) arms across centromeres and reveals that novel α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

The outer kinetochore components KNL-1 and Ndc80 complex regulate axon and neuronal cell body positioning in the C. elegans nervous system.

The KMN (Knl1/Mis12/Ndc80) network at the kinetochore, primarily known for its role in chromosome segregation, has been shown to be repurposed during neurodevelopment. Here, we investigate the underlying neuronal mechanism and show that the KMN network promotes the proper axonal organization within the C. elegans head nervous system. Postmitotic degradation of KNL-1, which acts as a scaffold for signaling and has microtubule-binding activities at the kinetochore, led to disorganized ganglia and aberrant placement and organization of axons in the nerve ring - an interconnected axonal network. Through gene-replacement approaches, we demonstrate that the signaling motifs within KNL-1, responsible for recruiting protein phosphatase 1, and activating the spindle assembly checkpoint are required for neurodevelopment. Interestingly, while the microtubule-binding activity is crucial to KMN's neuronal function, microtubule dynamics and organization were unaffected in the absence of KNL-1. Instead, the NDC-80 microtubule-binding mutant displayed notable defects in axon bundling during nerve ring formation, indicating its role in facilitating axon-axon contacts. Overall, these findings provide evidence for a noncanonical role for the KMN network in shaping the structure and connectivity of the nervous system in C. elegans during brain development.

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.

Non-canonical role for the BAF complex subunit DPF3 in mitosis and ciliogenesis.

DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.

Kinetochore scaffold 1 downregulation suppressed the development of non-small cell lung cancer by inactivating the phosphatidylinositol 3 kinase/protein kinase B (AKT)/nuclear factor-kappa B pathway.

Kinetochore scaffold 1 (KNL1) is indispensable for generating motile micro-tubule attachments and isolating chromosomes. KNL1 is highly expressed in multiple middle-route tissues and promotes tumor development. However, how it functions in non-small cell lung cancer (NSCLC) is unclear. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to determine KNL1 expression in NSCLC tissues and cells. The sh-KNL1 or oe-KNL1 was transfected into NSCLC cells. The colony formation assay, cell counting kit-8 (CCK-8) assay, and flow cytometry were used to evaluate cell proliferation and apoptosis. A transwell assay was used to monitor invasion and migration. The CCK-8 assay was used to measure NSCLC cell sensitivity to chemotherapy drugs. WB confirmed the protein levels of apoptosis-related proteins, cell cycle-associated proteins, and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappaB (NF-κB) pathway. A PI3K/AKT/NF-κB pathway inhibitor was used to intervene in NSCLC cell transfection along with oe-KNL1, thus revealing the function of the pathway in carcinogenicity mediated by KNL1. In result KNL1 expression was substantially increased in NSCLC tissues and cells. High-level KNL1 expression is related to the poor prognosis of NSCLC patients. KNL1 silencing bolstered promoted NSCLC cell apoptosis and inhibited proliferation, cell cycle progression, invasion, and EMT, whereas KNL1 silencing had the opposite effect. KNL1 knockdown increased NSCLC cell sensitivity to chemical drugs. KNL1 promoted PI3K/AKT/NF-κB pathway activation, while PI3K/AKT/NF-κB pathway inhibition weakened the procancer effect mediated by KNL1 overexpression but had little influence on KNL1 levels. We conclude that KNL1 activates the PI3K/AKT/NF-κB pathway to increase NSCLC progression and attenuate NSCLC sensitivity to chemotherapy drugs.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

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.

Set2 regulates Ccp1 and Swc2 to ensure centromeric stability by retargeting CENP-A.

Precise positioning of the histone-H3 variant, CENP-A, ensures centromere stability and faithful chromosomal segregation. Mislocalization of CENP-A to extra-centromeric loci results in aneuploidy and compromised cell viability associated with formation of ectopic kinetochores. The mechanism that retargets mislocalized CENP-A back to the centromere is unclarified. We show here that the downregulation of the histone H3 lysine 36 (H3K36) methyltransferase Set2 can preserve centromere localization of a temperature-sensitive mutant cnp1-1 Schizosaccharomyces pombe CENP-A (SpCENP-A) protein and reverse aneuploidy by redirecting mislocalized SpCENP-A back to centromere from ribosomal DNA (rDNA) loci, which serves as a sink for the delocalized SpCENP-A. Downregulation of set2 augments Swc2 (SWR1 complex DNA-binding module) expression and releases histone chaperone Ccp1 from the centromeric reservoir. Swc2 and Ccp1 are directed to the rDNA locus to excavate the SpCENP-Acnp1-1, which is relocalized to the centromere in a manner dependent on canonical SpCENP-A loaders, including Mis16, Mis17 and Mis18, thereby conferring cell survival and safeguarding chromosome segregation fidelity. Chromosome missegregation is a severe genetic instability event that compromises cell viability. This mechanism thus promotes CENP-A presence at the centromere to maintain genomic stability.

The Arabidopsis BUB1/MAD3 family protein BMF3 requires BUB3.3 to recruit CDC20 to kinetochores in spindle assembly checkpoint signaling.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

CKAP5 stabilizes CENP-E at kinetochores by regulating microtubule-chromosome attachments.

Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.