Yeast Kinetochore Research Articles

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.

Splitting the yeast centromere by recombination.

Although fusions between the centromeres of different human chromosomes have been observed cytologically in cancer cells, since the centromeres are long arrays of satellite sequences, the details of these fusions have been difficult to investigate. We developed methods of detecting recombination within the centromeres of the yeast Saccharomyces cerevisiae (intercentromere recombination). These events occur at similar rates (about 10-8/cell division) between two active or two inactive centromeres. We mapped the breakpoints of most of the recombination events to a region of 43 base pairs of uninterrupted homology between the two centromeres. By whole-genome DNA sequencing, we showed that most (>90%) of the events occur by non-reciprocal recombination (gene conversion/break-induced replication). We also found that intercentromere recombination can involve non-homologous chromosome, generating whole-arm translocations. In addition, intercentromere recombination is associated with very frequent chromosome missegregation. These observations support the conclusion that intercentromere recombination generally has negative genetic consequences.

Sequence-dependent heterochromatin formation in the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum represses transcription of the gene encoding AP2-G, which is the master regulator of germ cell differentiation, via heterochromatin condensation following histone H3 lysine 9 trimethylation (H3K9me3). Although H3K9me3-marked heterochromatin is typically constitutive and its establishment depends on the RNA interference (RNAi) pathway in fission yeast centromeres, malaria parasites lack molecular members essential for RNAi. We developed a strategy to assess heterochromatin establishment on artificial chromosomes introduced into P. falciparum. We show that a particular DNA sequence in the AP2-G promoter is able to induce de novo H3K9me3 nucleosome deposition. In addition, we also found that the AP2-G promoter contains a distinct element required in maintenance of the repression memory. Thus, we speculate that malaria parasites have evolutionarily acquired a sequence-dependent establishment system of non-constitutive, i.e. facultative, H3K9me3-marked heterochromatin.

Direct observation of coordinated assembly of individual native centromeric nucleosomes

Eukaryotic chromosome segregation requires the kinetochore, a megadalton‐sized machine that forms on specialized centromeric chromatin containing CENP‐A, a histone H3 variant. CENP‐A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP‐A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP‐A can arrive at centromeres without its dedicated centromere‐specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA‐binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP‐A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

Working strokes produced by curling protofilaments at disassembling microtubule tips: Their role in kinetochore motility and microtubule dynamics.

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, for example, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the popular conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. However, there are other possible mechanisms and rigorously testing the conformational wave idea will require modifying the mechanical and energetic properties of curling protofilaments. By direct measurement of working strokes generated in vitro by curling protofilaments, we previously showed that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. These increases in work output occurred independently of any change in the bending stiffness or intrinsic curvature of the protofilament curls, and were largely attributable to increases in contour length alone. Here we investigate the role of protofilament curls in the motility of the yeast kinetochore and explore the relationship between tubulin bending stiffness and microtubule dynamics.

Nanoscale structural organization and stoichiometry of the budding yeast kinetochore.

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multiprotein complex that physically links the DNA to spindle microtubules and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions and how they ensure an error-free outcome of mitosis is still limited, partly because we lack a complete understanding of the kinetochore structure in the cell. In this study, we use single-molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For major kinetochore proteins, we measured their abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging, our results present a unifying nanoscale model of the kinetochore in budding yeast.

The fission yeast kinetochore complex Mhf1-Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S-CENP-X is involved in regulating the SAC and kinetochore-microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1-Mhf2, the CENP-S-CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1-Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments. This article has an associated First Person interview with the first author of the paper.

First person – Yanze Jian

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Yanze Jian is first author on ‘ The fission yeast kinetochore complex Mhf1–Mhf2 regulates the spindle assembly checkpoint and faithful chromosome segregation’, published in JCS. Yanze is a PhD student in the lab of Chuanhai Fu at the University of Science and Technology of China, Hefei, China, investigating checkpoint signalling in mitosis and meiosis.

Structural insights into human CCAN complex assembled onto DNA

In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

Establishment of centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

Centromere Identity and the Regulation of Chromosome Segregation.

Eukaryotes segregate their chromosomes during mitosis and meiosis by attaching chromosomes to the microtubules of the spindle so that they can be distributed into daughter cells. The complexity of centromeres ranges from the point centromeres of yeast that attach to a single microtubule to the more complex regional centromeres found in many metazoans or holocentric centromeres of some nematodes, arthropods and plants, that bind to dozens of microtubules per kinetochore. In vertebrates, the centromere is defined by a centromere specific histone variant termed Centromere Protein A (CENP-A) that replaces histone H3 in a subset of centromeric nucleosomes. These CENP-A nucleosomes are distributed on long stretches of highly repetitive DNA and interspersed with histone H3 containing nucleosomes. The mechanisms by which cells control the number and position of CENP-A nucleosomes is unknown but likely important for the organization of centromeric chromatin in mitosis so that the kinetochore is properly oriented for microtubule capture. CENP-A chromatin is epigenetically determined thus cells must correct errors in CENP-A organization to prevent centromere dysfunction and chromosome loss. Recent improvements in sequencing complex centromeres have paved the way for defining the organization of CENP-A nucleosomes in centromeres. Here we discuss the importance and challenges in understanding CENP-A organization and highlight new discoveries and advances enabled by recent improvements in the human genome assembly.

A transcriptional roadblock protects yeast centromeres.

Centromeres are the chromosomal loci essential for faithful chromosome segregation during cell division. Although centromeres are transcribed and produce non-coding RNAs (cenRNAs) that affect centromere function, we still lack a mechanistic understanding of how centromere transcription is regulated. Here, using a targeted RNA isoform sequencing approach, we identified the transcriptional landscape at and surrounding all centromeres in budding yeast. Overall, cenRNAs are derived from transcription readthrough of pericentromeric regions but rarely span the entire centromere and are a complex mixture of molecules that are heterogeneous in abundance, orientation, and sequence. While most pericentromeres are transcribed throughout the cell cycle, centromere accessibility to the transcription machinery is restricted to S-phase. This temporal restriction is dependent on Cbf1, a centromere-binding transcription factor, that we demonstrate acts locally as a transcriptional roadblock. Cbf1 deletion leads to an accumulation of cenRNAs at all phases of the cell cycle which correlates with increased chromosome mis-segregation that is partially rescued when the roadblock activity is restored. We propose that a Cbf1-mediated transcriptional roadblock protects yeast centromeres from untimely transcription to ensure genomic stability.

Shaping centromeres to resist mitotic spindle forces.

The centromere serves as the binding site for the kinetochore and is essential for the faithful segregation of chromosomes throughout cell division. The point centromere in yeast is encoded by a ∼115 bp specific DNA sequence, whereas regional centromeres range from 6-10 kbp in fission yeast to 5-10 Mbp in humans. Understanding the physical structure of centromere chromatin (pericentromere in yeast), defined as the chromatin between sister kinetochores, will provide fundamental insights into how centromere DNA is woven into a stiff spring that is able to resist microtubule pulling forces during mitosis. One hallmark of the pericentromere is the enrichment of the structural maintenance of chromosome (SMC) proteins cohesin and condensin. Based on studies from population approaches (ChIP-seq and Hi-C) and experimentally obtained images of fluorescent probes of pericentromeric structure, as well as quantitative comparisons between simulations and experimental results, we suggest a mechanism for building tension between sister kinetochores. We propose that the centromere is a chromatin bottlebrush that is organized by the loop-extruding proteins condensin and cohesin. The bottlebrush arrangement provides a biophysical means to transform pericentromeric chromatin into a spring due to the steric repulsion between radial loops. We argue that the bottlebrush is an organizing principle for chromosome organization that has emerged from multiple approaches in the field.

Genome editing with removable TALEN vectors harboring a yeast centromere and\xa0autonomous replication sequence in\xa0oleaginous microalga

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

Kre28-Spc105 interaction is essential for Spc105 loading at the kinetochore.

Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.

Correction for Carlsten et al., "Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres".

Volume 32, no. 19, p. 4035–4043, 2012, https://doi.org/10.1128/MCB.00374-12. Page 4037, Fig. 1F, right panel: The bottom left image duplicates the bottom center image by mistake. The dg row should appear as shown below.

Aurora B switches relative strength of kinetochore-microtubule attachment modes for error correction.

To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.

A case of convergent-gene interference in the budding yeast knockout library causing chromosome instability.

To maintain genome stability, organisms depend on faithful chromosome segregation, a process affected by diverse genetic pathways, some of which are not directly linked to mitosis. In this study, we set out to explore one such pathway represented by an undercharacterized gene, SNO1, identified previously in screens of the yeast knockout (YKO) library for mitotic fidelity genes. We found that the causative factor increasing mitotic error rate in the sno1Δ mutant is not loss of the Sno1 protein, but rather perturbation to the mRNA of the neighboring convergent gene, CTF13, encoding an essential component for forming the yeast kinetochore. This is caused by a combination of the Kanamycin resistance gene and the transcriptional terminator used in the YKO library affecting the CTF13 mRNA level and quality . We further provide a list of gene pairs potentially subjected to this artifact, which may be useful for accurate phenotypic interpretation of YKO mutants.

Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8.

Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the double strand break-forming topoisomerase Spo11 and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells lacking the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly1; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodeling factors mediate centromere dismantlement by Spo11 and Rec8. Remarkably, ectopic expression of either protein in proliferating cells leads to mitotic kinetochore loss in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability, and may jeopardize kinetochores in the increasing number of cancers known to express meiotic proteins.

Structural basis of Stu2 recruitment to yeast kinetochores.

Chromosome segregation during cell division requires engagement of kinetochores of sister chromatids with microtubules emanating from opposite poles. As the corresponding microtubules shorten, these 'bioriented' sister kinetochores experience tension-dependent stabilization of microtubule attachments. The yeast XMAP215 family member and microtubule polymerase, Stu2, associates with kinetochores and contributes to tension-dependent stabilization in vitro. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function in vivo. These findings show that the role of Stu2 in tension-sensing depends on its association with kinetochores by binding with Ndc80c.