Ndc80复合体在外层动粒中的作用

动粒是参与有丝分裂过程中染色体分离的蛋白的附着支架。结构保守的Ndc80复合体位于动粒的外层,连接动粒和微管,与动粒-微管连接的稳定性有关。Aurora B/Ipl1激酶参与纠正动粒-微管的错误连接。Ndc80复合体对纺锤体组装检查点的功能非常重要。本文主要介绍了Ndc80复合体的研究进展。     

CENP‐T provides a structural platform for outer kinetochore assembly

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule‐binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high‐resolution structural analysis, we demonstrate that the N‐terminal region of vertebrate CENP‐T interacts with the ‘RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP‐T strengthens a cryptic hydrophobic interaction between CENP‐T and Spc25 resulting in a phospho‐regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP‐T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho‐regulated manner.     

Perturbation of Spc25 expression affects meiotic spindle organization,chromosome alignment and spindle assembly checkpoint in mouse oocytes.

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment and the spindle assembly checkpoint in mitosis. The roles of Spc25 in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 by mRNA injection resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi by siRNA injection resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and proper spindle checkpoint signaling during meiosis.     

Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes

Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects. The spindle assembly checkpoint (SAC) guarantees proper cell cycle progression. Highly Expressed in Cancer protein 1 (Hec1, also called Ndc80) is the core component of the Ndc80 complex and is involved in regulating both kinetochore-microtubule interactions and the SAC during mitosis in multiple cell types. However, its involvement in pig oocyte meiotic maturation remains uncertain. Thus, we investigated Hec1 expression, localization, and possible functions during porcine oocyte meiosis. Immunofluorescent staining showed that Hec1 was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages. Disrupting Hec1 function with its inhibitor INH1 resulted in polar body extrusion defects in porcine oocytes. Moreover, inhibiting Hec1 activity also resulted in severe chromosome misalignments and aberrant spindle morphology. Our results showed a unique localization pattern for Hec1 in porcine oocytes and suggested that Hec1 was required for chromosome alignment and spindle organization. Thus, Hec1 might regulate spindle checkpoint activity during mammalian oocyte meiosis.     

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.     

The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation

We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.     

The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end

Proper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment). The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface, and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex, in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.     

The Ndc80 complex: hub of kinetochore activity

Kinetochores are protein scaffolds coordinating the process of chromosome segregation in mitosis. Kinetochore components are organized in functionally and topologically distinct domains that are designed to connect the sister chromatids to the mitotic spindle. The inner kinetochore proteins are in direct contact with the centromeric DNA, whilst the outer kinetochore proteins are responsible for binding to spindle microtubules. The conserved Ndc80 complex is implicated in several essential outer kinetochore functions, including microtubule binding and control of a safety device known as the spindle assembly checkpoint. Here, we describe how current work is contributing to unravel the complex endeavors of this essential kinetochore complex.     

Looping in on Ndc80 – How does a protein loop at the kinetochore control chromosome segregation?

Segregation of chromosomes during mitosis requires the interaction of dynamic microtubules with the kinetochore, a large protein structure established on the centromere region of sister chromatids. The core microtubule‐binding activity of the kinetochore resides in the KMN network, an outer kinetochore complex. As part of the KMN network, the Ndc80 complex, which is composed of Ndc80, Nuf2, Spc24, and Spc25, is able to bind directly to microtubules and has the ability to track with depolymerizing microtubules to produce chromosome movement. The Ndc80 complex binds directly to microtubules through a calponin homology domain and an unstructured tail in the N terminus of the Ndc80 protein. A recent flurry of papers has highlighted the importance of an internal loop region in Ndc80 in establishing end‐on attachment to microtubules. Here I discuss these recent findings that suggest that the Ndc80 internal loop functions as a binding site for proteins required for kinetochore‐microtubule interactions.     

Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.     

CENP-T proteins are conserved centromere receptors of the Ndc80 complex

Centromeres direct the assembly of kinetochores, microtubule-attachment sites that allow chromosome segregation on the mitotic spindle. Fundamental differences in size and organization between evolutionarily distant eukaryotic centromeres have in many cases obscured general principles of their function. Here we demonstrate that centromere-binding proteins are highly conserved between budding yeast and humans. We identify the histone-fold protein Cnn1(CENP-T) as a direct centromere receptor of the microtubule-binding Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24-25 domain of the Ndc80 complex. Consistent with the critical role of this interaction, artificial tethering of the Ndc80 complex through Cnn1 allows mini-chromosomes to segregate in the absence of a natural centromere. Our results reveal the molecular function of CENP-T proteins and demonstrate how the Ndc80 complex is anchored to centromeres in a manner that couples chromosome movement to spindle dynamics.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

Resonance assignments of the microtubule-binding domain of the C. elegans spindle and kinetochore-associated protein 1

During mitosis, kinetochores coordinate the attachment of centromeric DNA to the dynamic plus ends of microtubules, which is hypothesized to pull sister chromatids toward opposing poles of the mitotic spindle. The outer kinetochore Ndc80 complex acts synergistically with the Ska (spindle and kinetochore-associated) complex to harness the energy of depolymerizing microtubules and power chromosome movement. The Ska complex is a hexamer consisting of two copies of the proteins Ska1, Ska2 and Ska3, respectively. The C-terminal domain of the spindle and kinetochore-associated protein 1 (Ska1) is the microtubule-binding domain of the Ska complex. We solved the solution structure of the C. elegans microtubule-binding domain (MTBD) of the protein Ska1 using NMR spectroscopy. Here, we report the resonance assignments of the MTBD of C. elegans Ska1.     

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore–microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore–microtubule attachment is regulated by conserved signals for error correction.     

Insights into the kinetochore

The Ndc80 complex is a core component of the kinetochore, which links chromosomes to microtubules. Recently, Ciferri et al. (2008) published an atomic-level structure of the complex with implications for kinetochore architecture and for the generation and control of chromosome movements during mitosis.     

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.     

Orientation and structure of the Ndc80 complex on the microtubule lattice

The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential α/β-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.     

Spc24 and Stu2 promote spindle integrity when DNA replication is stalled

The kinetochore, a protein complex that links chromosomes to microtubules (MTs), is required to prevent spindle expansion during S phase in budding yeast, but the mechanism of how the kinetochore maintains integrity of the bipolar spindle before mitosis is not well understood. Here, we demonstrate that a mutation of Spc24, a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. We propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants. Identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle.     

Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.