Kinetochore Assembly Research Articles

TMOD-01. EGFR AMPLIFICATION ACCOMPANIES ADAPTATION TO MITOGEN-INDUCED DEFECTIVE MITOSIS IN PDGFA

Abstract Using a newly described in vitro murine model of GBM [Bohm et al., Neuro-Oncol 2020 and Omairi et al., Neuro-Oncol 2023] in which P53 null neural progenitor cells (NPCs) divide abnormally and evolve a GBM-like genome during exposure to Platelet-Derived Growth Factor-AA (PDGFA), we asked how a brain-abundant mitogen could transform NPCs. By analyzing gene and protein expression over time, we found that PDGFA fails to induce the transcription of kinetochore and spindle assembly checkpoint genes, while simultaneously driving NPCs to enter mitosis. These dual effects caused chromosome miss-segregation in continuously dividing NPCs; moreover, they occurred in both WT and null NPCs, although only null cells survived defective mitosis. These surviving cells gradually expanded in PDGFA accumulating both random and clonal chromosomal re-arrangements. Transcriptome analysis of NPCs in PDGFA revealed significant under-expression of Foxm1, the major regulator of kinetochore transcription, together with over-expression and phosphorylation of the immediate early response gene, FOS. Analysis of signalling downstream of PDGFRα identified the Ras-MAPK pathway, especially ERK, as responsible for FOS activation. As surviving null cells gradually expanded, they accumulated random and recurrent chromosomal rearrangements. Expansion and subsequent PDGFA-independent proliferation and tumorigenicity were associated with re-expression of Foxm1 and kinetochore proteins, and accompanied by over-expression of Egfr, an RTK-signature that defines human GBM. By stimulating proliferation without setting the stage for error-free mitosis, exposure to PDGFA transforms p53 null NPCs and generates Egfr amplified GBM-like cancer cells.

Binding Site Maturation Modulated by Molecular Density Underlies Ndc80 Binding to Kinetochore Receptor CENP-T.

A distinctive mechanism of protein-protein interaction underpins the assembly of kinetochores, which is critical for human cell division. During mitosis, the Ndc80 complex must bind tightly to the unstructured N-terminus of its receptor, CENP-T, which is densely clustered at kinetochores. Using single-molecule in vitro assays, we show that Ndc80 binding is mediated by an initially unstable yet tunable interface. The high molecular density of CENP-T at the kinetochores accelerates the maturation of this binding interface, favoring the formation of stable complexes within the kinetochore structure, rather than in the soluble phase. This environment-driven modulation of binding site maturation may represent a key regulatory mechanism for ensuring strong and specific interactions during the assembly of macromolecular complexes such as kinetochores.

Argonaute protein CSR-1 restricts localization of holocentromere protein HCP-3, the C. elegans CENP-A homolog.

Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos.

Regulation of outer kinetochore assembly during meiosis I and II by CENP-A and KNL-2/M18BP1 in C. elegans oocytes

During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C.elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28ELYS. Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28ELYS. Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2M18BP1 (or MEL-28ELYS) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2M18BP1 and MEL-28ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2M18BP1 recruits MEL-28ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C.elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2M18BP1 and MEL-28ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin.

ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.

Zeste white 10 (ZW10) was first identified as a centromere/kinetochore protein encoded by the ZW10 gene in Drosophila. ZW10 guides the spindle assembly checkpoint signaling during mitotic chromosome segregation in metazoans. Recent studies have shown that ZW10 is also involved in membranous organelle interactions during interphase and plays a vital role in membrane transport between the endoplasmic reticulum and Golgi apparatus. Despite these findings, the precise molecular mechanisms by which ZW10 regulates interactions between membranous organelles in interphase and the assembly of membraneless organelle kinetochore in mitosis remain elusive. Here, we highlight how ZW10 forms context-dependent protein complexes during the cell cycle. These complexes are essential for mediating membrane trafficking in interphase and ensuring the accurate segregation of chromosomes in mitosis.

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.

Unveiling the distinctive traits of functional rye centromeres: minisatellites, retrotransposons, and R-loop formation.

Centromeres play a vital role in cellular division by facilitating kinetochore assembly and spindle attachments. Despite their conserved functionality, centromeric DNA sequences exhibit rapid evolution, presenting diverse sizes and compositions across species. The functional significance of rye centromeric DNA sequences, particularly in centromere identity, remains unclear. In this study, we comprehensively characterized the sequence composition and organization of rye centromeres. Our findings revealed that these centromeres are primarily composed of long terminal repeat retrotransposons (LTR-RTs) and interspersed minisatellites. We systematically classified LTR-RTs into five categories, highlighting the prevalence of younger CRS1, CRS2, and CRS3 of CRSs (centromeric retrotransposons of Secale cereale) were primarily located in the core centromeres and exhibited a higher association with CENH3 nucleosomes. The minisatellites, mainly derived from retrotransposons, along with CRSs, played a pivotal role in establishing functional centromeres in rye. Additionally, we observed the formation of R-loops at specific regions of CRS1, CRS2, and CRS3, with both rye pericentromeres and centromeres exhibiting enrichment in R-loops. Notably, these R-loops selectively formed at binding regions of the CENH3 nucleosome in rye centromeres, suggesting a potential role in mediating the precise loading of CENH3 to centromeres and contributing to centromere specification. Our work provides insights into the DNA sequence composition, distribution, and potential function of R-loops in rye centromeres. This knowledge contributes valuable information to understanding the genetics and epigenetics of rye centromeres, offering implications for the development of synthetic centromeres in future plant modifications and beyond.

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.

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.

Structure of the human outer kinetochore KMN network complex

Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

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.

Dicentric chromosomes are resolved through breakage and repair at their centromeres

Chromosomes with two centromeres provide a unique opportunity to study chromosome breakage and DNA repair using completely endogenous cellular machinery. Using a conditional transcriptional promoter to control the second centromere, we are able to activate the dicentric chromosome and follow the appearance of DNA repair products. We find that the rate of appearance of DNA repair products resulting from homology-based mechanisms exceeds the expected rate based on their limited centromere homology (340 bp) and distance from one another (up to 46.3 kb). In order to identify whether DNA breaks originate in the centromere, we introduced 12 single-nucleotide polymorphisms (SNPs) into one of the centromeres. Analysis of the distribution of SNPs in the recombinant centromeres reveals that recombination was initiated with about equal frequency within the conserved centromere DNA elements CDEII and CDEIII of the two centromeres. The conversion tracts range from about 50 bp to the full length of the homology between the two centromeres (340 bp). Breakage and repair events within and between the centromeres can account for the efficiency and distribution of DNA repair products. We propose that in addition to providing a site for kinetochore assembly, the centromere may be a point of stress relief in the face of genomic perturbations.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

A dynamic population of prophase CENP-C is required for meiotic chromosome segregation.

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.

Humanization reveals pervasive incompatibility of yeast and human kinetochore components.

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

A role for β-1,6- and β-1,3-glucans in kinetochore function in Saccharomyces cerevisiae.

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/ transglycosidase required for β-1,6-glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a constitutive centromere-associated network protein. Similarly, the absence of Fks1, a β-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular β-1,6- and β-1,3-glucans, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.

Centromere structure and function: lessons from Drosophila.

The fruit fly Drosophila melanogaster serves as a powerful model organism for advancing our understanding of biological processes, not just by studying its similarities with other organisms including ourselves but also by investigating its differences to unravel the underlying strategies that evolved to achieve a common goal. This is particularly true for centromeres, specialized genomic regions present on all eukaryotic chromosomes that function as the platform for the assembly of kinetochores. These multiprotein structures play an essential role during cell division by connecting chromosomes to spindle microtubules in mitosis and meiosis to mediate accurate chromosome segregation. Here, we will take a historical perspective on the study of fly centromeres, aiming to highlight not only the important similarities but also the differences identified that contributed to advancing centromere biology. We will discuss the current knowledge on the sequence and chromatin organization of fly centromeres together with advances for identification of centromeric proteins. Then, we will describe both the factors and processes involved in centromere organization and how they work together to provide an epigenetic identity to the centromeric locus. Lastly, we will take an evolutionary point of view of centromeres and briefly discuss current views on centromere drive.

The role of centromeric repeats and transcripts in kinetochore assembly and function.

Centromeres are the chromosomal domains, where the kinetochore protein complex is formed, mediating proper segregation of chromosomes during cell division. Although the function of centromeres has remained conserved during evolution, centromeric DNA is highly variable, even in closely related species. In addition, the composition of the kinetochore complexes varies among organisms. Therefore, it is assumed that the centromeric position is determined epigenetically, and the centromeric histone H3 (CENH3) serves as an epigenetic marker. The loading of CENH3 onto centromeres depends on centromere-licensing factors, chaperones, and transcription of centromeric repeats. Several proteins that regulate CENH3 loading and kinetochore assembly interact with the centromeric transcripts and DNA in a sequence-independent manner. However, the functional aspects of these interactions are not fully understood. This review discusses the variability of centromeric sequences in different organisms and the regulation of their transcription through the RNA Pol II and RNAi machinery. The data suggest that the interaction of proteins involved in CENH3 loading and kinetochore assembly with centromeric DNA and transcripts plays a role in centromere, and possibly neocentromere, formation in a sequence-independent manner.

Evidence of kinesin motors involved in stable kinetochore assembly during early meiosis.

During mitosis, the budding yeast, kinetochores remain attached to microtubules, except for a brief period during S phase. Sister-kinetochores separate into two clusters (bilobed organization) upon stable end-on attachment to microtubules emanating from opposite spindle poles. However, in meiosis, the outer kinetochore protein (Ndc80) reassembles at the centromeres much later after prophase I, establishing new kinetochore-microtubule attachments. Perhaps due to this, despite homolog bi-orientation, we observed that the Ndc80 are linearly dispersed between spindle poles during metaphase I of meiosis. The presence of end-on attachment marker Dam1 as a cluster near each pole suggests one of the other possibilities that the pole-proximal and pole-distal kinetochores are attached end-on and laterally to the microtubules, respectively. Colocalization studies of kinetochores and kinesin motors suggest that budding yeast kinesin 5, Cin8, and Kip1 perhaps localize to the end-on attached kinetochores while kinesin 8 and Kip3 resides at all the kinetochores. Our findings, including kinesin 5 and Ndc80 coappearance after prophase I and reduced Ndc80 levels in cin8 null mutant, suggest that kinesin motors are crucial for kinetochore reassembly and stability during early meiosis. Thus, this work reports yet another meiosis specific function of kinesin motors.