Conserved and divergent mechanisms of inner kinetochore assembly onto centromeric chromatin

Kinetochores are large protein complexes built on centromeric chromatin that mediate chromosome segregation. The inner kinetochore, or constitutive centromere-associated network (CCAN), assembles onto centromeres defined by centromere protein A (CENP-A) nucleosomes (CENP-A^Nuc), and acts as a platform for the regulated assembly of the microtubule-binding outer kinetochore. Recent cryo-EM work revealed structural conservation of CCAN, from the repeating human regional centromeres to the point centromere of budding yeast. Centromere recognition is determined mainly through engagement of duplex DNA proximal to the CENP-A nucleosome by a DNA-binding CENP-LN channel located at the core of CCAN. Additional DNA interactions formed by other CCAN modules create an enclosed DNA-binding chamber. This configuration explains how kinetochores maintain their tight grip on centromeric DNA to withstand the forces of chromosome segregation. Defining the higher-order architecture of complete kinetochore assemblies with implications for understanding the 3D organisation of regional centromeres and mechanisms of kinetochore dynamics, including how kinetochores sense and respond to tension, are important future directions.     

Where is the right path heading from the centromere to spindle microtubules?

The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.     

CENP-U cooperates with Hec1 to orchestrate kinetochore-microtubule attachment

Mitosis is an orchestration of dynamic interaction between chromosomes and spindle microtubules by which genomic materials are equally distributed into two daughter cells. Previous studies showed that CENP-U is a constitutive centromere component essential for proper chromosome segregation. However, the precise molecular mechanism has remained elusive. Here, we identified CENP-U as a novel interacting partner of Hec1, an evolutionarily conserved kinetochore core component essential for chromosome plasticity. Suppression of CENP-U by shRNA resulted in mitotic defects with an impaired kinetochore-microtubule attachment. Interestingly, CENP-U not only binds microtubules directly but also displays a cooperative microtubule binding activity with Hec1 in vitro. Furthermore, we showed that CENP-U is a substrate of Aurora-B. Importantly, phosphorylation of CENP-U leads to reduced kinetochore-microtubule interaction, which contributes to the error-correcting function of Aurora-B. Taken together, our results indicate that CENP-U is a novel microtubule binding protein and plays an important role in kinetochore-microtubule attachment through its interaction with Hec1.     

The CCAN complex: linking centromere specification to control of kinetochore-microtubule dynamics

For over 70 years, chromosomes have been known to oscillate back-and-forth on the metaphase plate. These movements are directed by kinetochores, the microtubule-attachment complexes on centromeres that regulate the dynamics of bound spindle microtubules. Recent evidence shows that the CCAN (Constitutive Centromere Associated Network) kinetochore network, which directly binds centromeric nucleosomes, plays a crucial role in the control of kinetochore microtubule dynamics. Here we review how this 15-subunit protein network functions within the kinetochore machinery, how it may adapt dynamically both in time and in space to the functional requirements necessary for controlled and faithful chromosome movements during cell division, and how this conserved protein network may have evolved in organisms with different cell division machineries.     

Centromere/kinetochore is assembled through CENP-C oligomerization

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Plc1p is required for proper chromatin structure and activity of the kinetochore in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by facilitating recruitment of the RSC complex

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p’s involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The kinetochore fiber structure in the acentric spindles of the green algaOedogonium

Summary The microtubule (MT) arrangement in three kinetochore fibers in the acentric spindles of the green algaOedogonium cardiacum were reconstructed from serial sections of prometaphase and metaphase cells. The majority of the MTs attached to the kinetochore (kMTs) are relatively short, extending less than a third of the distance to the putative spindle pole region, and none extended the full distance. Fine filaments and a matrix described earlier (Schibler andPickett-Heaps 1980) were associated with the MTs all along the fibers. Live cells ofOedogonium were also studied by time lapse cinematography for correlation with the ultrastructural observations. Late prometaphase and metaphase kinetochore fibers appear to move independently as if unattached at their poleward ends. These observations suggest that kinetochore fibers inOedogonium are not attached to a specific pole structure from late prometaphase until the inception of anaphase. The results are discussed with reference to spindle structure and function in general.     

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

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Maize centromeric chromatin scales with changes in genome size

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.     

The centromere1 (CEN1) region of Arabidopsis thaliana: architecture and functional impact of chromatin

We have analysed the centromere 1 (CEN1) of Arabidopsis thaliana by integration of genetic, sequence and fluorescence in situ hybridisation (FISH) data. CEN1 is considered to include the centromeric core and the flanking left and right pericentromeric regions, which are distinct parts by structural and/or functional properties. CEN1 pericentromeres are composed of different dispersed repetitive elements, sometimes interrupted by functional genes. In contrast the CEN1 core is more uniformly structured harbouring only two different repeats. The presented analysis reveals aspects concerning distribution and effects of the uniformly shaped heterochromatin, which covers all CEN1 regions. A lethal mutation tightly linked to CEN1 enabled us to measure recombination frequencies within the heterochromatin in detail. In the left pericentromere, the change from eu- to heterochromatin is accompanied by a gradual change in sequence composition but by an extreme change in recombination frequency (from normal to 53-fold decrease) which takes place within a small region spanning 15 kb. Generally, heterochromatin is known to suppress recombination. However, the same analysis reveals that left and right pericentromere, though similar in sequence composition, differ markedly in suppression (53-fold versus 10-fold). The centromeric core exhibits at least 200-fold if not complete suppression. We discuss whether differences in (fine) composition reflect quantitative and qualitative differences in binding sites for heterochromatin proteins and in turn render different functional properties. Based on the presented data we estimate the sizes of Arabidopsis centromeres. These are typical for regional centromeres of higher eukaryotes and range from 4.4 Mb (CEN1) to 3.55 Mb (CEN4).     

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly

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Focus on the centre: the role of chromatin on the regulation of centromere identity and function

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere‐specific histone H3 variant CenH3, originally called CENP‐A, which replaces canonical histone H3 at centromeres. CenH3‐chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3‐chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.     

Studies of the Kinetochore Proteins of the Regenerating Liver and the Liver Cells of Rats at Different Stages of Development

The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.     

Adenoassociated virus has a unique chromatin structure

The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2). Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600-2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers. This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.     

Size-dependence of a stable higher-order structure of chromatin

We have recently reported a study of the formation of higher-order structures of chromatin with increasing ionic strength, in which we measured sedimentation coefficients of long nucleosome oligomers. We found that somewhere in the range of 30 to 60mer the sedimentation coefficient developed a jump of about 10% between ionic strengths of 45 mm and 55 mm, which persisted for larger oligomers (Butler & Thomas, 1980).This posed the question of whether the jump observed represented a greater relative compaction at high ionic strength on the part of long polymers, or a relatively lesser resistance to hydrodynamic shear forces at low ionic strength.We now define the dependence of the jump upon oligomer size in detail, the critical size being 50 nucleosomes. We also show that it occurs because the sedimentation of a large oligomer appears “slow” for its size at lower ionic strength, but “normal” at higher ionic strengths. We interpret this as the consequence of insufficient axial interaction to stabilize the helical coiling of long nucleosome filaments at low ionic strength, leading to a more open and slowly sedimenting structure.     

Supranucleosomal structure of chromatin

Rat liver chromatin was moderately digested by micrococcal nuclease and analysed by centrifugation in isokinetic sucrose gradients and electron microscopy. Two classes of particles sedimenting with about 33S and 60S were characterized. Kinetics of their appearance and disappearance during progressive digestion suggests that they represent monomers and dimers cleaved from a higher order (supranucleosomal) structure of chromatin. Biochemical and electron microscopical results suggest that the monomers and dimers contain eight and sixteen nucleosomes, respectively, which are densely packed into 23 nm (monomer) and 29 nm (dimer) globules.