Centromere/kinetochore is assembled through CENP-C oligomerization

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

The structure of a primitive kinetochore

The isolation of yeast centromeres has provided the opportunity to describe the molecular structure of chromosome attachments to the mitotic spindle. Nucleolytic probes of chromatin structure and construction of conditional mutants in centromere function have been used to study the regulation and assembly of centromeres throughout the cell cycle in Saccharomyces cerevisiae.     

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally.     

Rule-based modeling and simulations of the inner kinetochore structure

Background

Combinatorial complexity is a central problem when modeling biochemical reaction networks, since the association of a few components can give rise to a large variation of protein complexes. Available classical modeling approaches are often insufficient for the analysis of very large and complex networks in detail. Recently, we developed a new rule-based modeling approach that facilitates the analysis of spatial and combinatorially complex problems. Here, we explore for the first time how this approach can be applied to a specific biological system, the human kinetochore, which is a multi-protein complex involving over 100 proteins.

Results

Applying our freely available SRSim software to a large data set on kinetochore proteins in human cells, we construct a spatial rule-based simulation model of the human inner kinetochore. The model generates an estimation of the probability distribution of the inner kinetochore 3D architecture and we show how to analyze this distribution using information theory. In our model, the formation of a bridge between CenpA and an H3 containing nucleosome only occurs efficiently for higher protein concentration realized during S-phase but may be not in G1. Above a certain nucleosome distance the protein bridge barely formed pointing towards the importance of chromatin structure for kinetochore complex formation. We define a metric for the distance between structures that allow us to identify structural clusters. Using this modeling technique, we explore different hypothetical chromatin layouts.

Conclusions

Applying a rule-based network analysis to the spatial kinetochore complex geometry allowed us to integrate experimental data on kinetochore proteins, suggesting a 3D model of the human inner kinetochore architecture that is governed by a combinatorial algebraic reaction network. This reaction network can serve as bridge between multiple scales of modeling. Our approach can be applied to other systems beyond kinetochores.     

Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast.

Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.     

Kinetochore structure: electron spectroscopic imaging of the kinetochore

The structure of the kinetochore in thin section has been studied in the Indian muntjac by an electron spectroscopic imaging technique. This procedures allows the analysis of the distribution of phosphorus within the layers of the kinetochore. The results indicate that this element is a major component of both the inner and outer plates whereas it is largely absent in the middle plate and fibrous corona. The majority of the phosphorus is localized to a 30-nm fiber(s) that is woven through the layers of the kinetochore. The presence of phosphorus within this fiber, along with its morphological and biochemical features, indicates that it contains DNA. The fiber(s) occupies a major portion of the inner and outer plate where it forms a series of rows. It is rarely observed in the middle layer except where it passes between the inner and outer layers. The absence of structure in the middle plate suggests that it may represent a space rather than a plate that in turn may be related to the function of this region. The distribution of phosphorus within the kinetochore is neither altered by treatment with colcemid nor by the presence of microtubules at the kinetochore. Analysis of conventional micrographs of the kinetochore together with structural information obtained by electron spectroscopic imaging suggests that most microtubules insert and terminate between the rows of kinetochore fibers in the outer plate. However, some microtubules continue through the middle layer and terminate at the lower plate. The insertion of microtubules at different levels of the kinetochore may reflect the existence of functionally distinct microtubule classes. Electron spectroscopic imaging indicates that the microtubules associated with the kinetochore are phosphorylated.     

Centromere proteins

Human anti-centromere sera from scleroderma patients were used to detect centromere antigens of mouse fibroblast cells. An M_r=59000 centromere protein was localized exclusively on mitotic chromosomes. The association of this protein with the mitotic chromosomes proved to be DNase I sensitive. In interphase nuclei, this centromere antigen was not detectable by immunoblot techniques. The results suggest that the M_r=59000 mitosis specific protein may be necessary for the structural stability of kinetochores during mitosis.     

Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein

Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/ S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.     

Centromere structure and function analysis in wheat–rye translocation lines

1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno‐FISH, chromatin immunoprecipitation (ChIP)‐qPCR and RT‐PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat‐derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere‐specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group‐1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat.     

Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males

Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections. The majority (54%) of the microtubules extended from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole. Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%). We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore. Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position. For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90 degrees to the spindle axis may have microtubules running towards one pole only.     

Centromere dynamics

At the foundation of all eukaryotic kinetochores is a unique histone variant, known as CenH3 (centromere histone H3). We are starting to identify the histone chaperones responsible for CenH3 deposition at centromere DNA, and the mechanisms that restrict CenH3 from chromosome arms. The specialized nucleosome that contains CenH3 in place of canonical histone H3 lies at the interface between microtubules and chromosomes and directs kinetochore protein assembly. By contrast, pericentric chromatin is highly elastic and can stretch or recoil in response to microtubule shortening or growth in mitosis. The variety in histone modification is likely to play a key role in regulating the behavior of these distinct chromatin domains.     

Centromere elasticity

In addition to the role in the spindle apparatus and associated motors, the chromosome themselves play an important role in facilitating chromosome segregation. Sister chromatids are joined at the centromere through a protein complex called cohesin. Chromatids separation requires the degradation by separase of specific proteins acting as a glue to form the cohesin complex. This evolutionally complex is required for the establishment and maintenance of sister chromatids in a ring like structure. It is therefore a key question whether cohesin is indeed a main component of active centromere. Cohesin is insufficient to resist the splitting force exerted by microtubules until anaphase and must be renforced by cohesion provided by flanking DNA. The ring model suggests that cohesine might possess a considerable mobility when associated with chromatin. Observations demonstrate that the interior region of the centromere behaves as an elastic element. Chromosomes display remarkable elasticity, returning to their initial shape after being extended by up to 10 times. For larger deformations the thick filament is converted in thin filament which can be stretched six times before breaking. This article suggests an additional and novel role for the protein titin on chromosome structure and dynamic. Titine was identified as a chromosomal component and it was hypothesised that titin may provide elasticity to chromosome and resistance to chromosome breakages during mitosis. The elastic properties of purified titin correspond well to the elastic properties of chromosome in living cells. The deformability and bending rigidity are consistent with a model developed for titin elasticity. The association of the presence of cohesine ring and the activity of titin could be necessary for segregation.     

Centromere assembly and propagation

Centromere assembly provides a unique example of how elaborate protein structures can be assembled onto DNA, independent of sequence, and then stably propagated through numerous cell divisions. Here, we review the possible epigenetic strategies that organisms ranging from yeast to human use to assemble and propagate active centromeres.     

Chromosome Y Centromere Array Deletion Leads to Impaired Centromere Function

The centromere is an essential chromosomal structure that is required for the faithful distribution of replicated chromosomes to daughter cells. Defects in the centromere can compromise the stability of chromosomes resulting in segregation errors. We have characterised the centromeric structure of the spontaneous mutant mouse strain, BALB/cWt, which exhibits a high rate of Y chromosome instability. The Y centromere DNA array shows a de novo interstitial deletion and a reduction in the level of the foundation centromere protein, CENP-A, when compared to the non-deleted centromere array in the progenitor strain. These results suggest there is a lower threshold limit of centromere size that ensures full kinetochore function during cell division.     

Changes in prometaphase chromosome motion are dependent on experimentally induced changes in kinetochore structure.

Prometaphase PtK₁ cells are treated with low concentrations of sucrose in order to analyze its effects on kinetochore structure, microtubule (MT) associations with the developing kinetochore and chromosome congression. Prometaphase cells treated with 0.15M sucrose slows chromosome congression, yet chromosomes form a metaphase configuration. However, 0.2M sucrose treatment prevents chromosome congression and affects some of the kinetochore MT linkages with the kinetochore, resulting in loss of chromosome congression. We use time lapse video microscopy and ultrastructural analysis to correlate changes in the linkages in the kinetochore MTs and the kinetochore to explain these findings. It appears hyperosmotic shock treatment can produce non-functional linkages between kinetochore MTs and kinetochores such that chromosome congression is affected. When non-functional linkages are formed, the presence of both a corona and matrix-like material is also present, proximal to the kinetochore. The role of this material and its organization at the klnetochore is discussed in its relation to generating mitotic forces.