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.     

Polo-like kinase-activating kinases

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

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.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

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.     

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.     

MAD2蛋白在蚕豆细胞周期中的同源定位

细胞周期是一个高度有序的过程,其运行过程受到多个检验点(checkpoint)的严格监控.MAD2是动物细胞纺锤体检验点(spindle checkpoint)中一种重要的蛋白。我们利用免疫印记法从蚕豆组织中检测到一种MAD2的同源蛋白,该蛋白的分子量在26KD左右,其亚细胞分布方式与动物细胞大致相同,都随细胞周期的进行而变化:在间期细胞中,MAD2分布于整个细胞中,并且在核膜外侧有优先聚集;在有丝分裂前期细胞中,MAD2在核膜破裂后开始在动粒处聚集;到早中期(prometaphase),MAD2定位于动粒;随着微管的延伸,MAD2逐渐减少直至消失,在中期,晚中期及后期细胞中,动粒处已无MAD2的分布。     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada)

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.     

Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori

The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species.