Ndc80 Complex Research Articles

Contrasting models for kinetochore microtubule attachment in mammalian cells.

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or "kinetochore fibrils" have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules.

Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis

SummaryIn early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules.

Molecular Structures and Interactions in the Yeast Kinetochore

Kinetochores are the elaborate protein assemblies that attach chromosomes to spindle microtubules in mitosis and meiosis. The kinetochores of point-centromere yeast appear to represent an elementary module, which repeats a number of times in kinetochores assembled on regional centromeres. Structural analyses of the discrete protein subcomplexes that make up the budding-yeast kinetochore have begun to reveal principles of kinetochore architecture and to uncover molecular mechanisms underlying functions such as transmission of tension and establishment and maintenance of bipolar attachment. The centromeric DNA is probably wrapped into a compact organization, not only by a conserved, centromeric nucleosome, but also by interactions among various other DNA-bound kinetochore components. The rod-like, heterotetrameric Ndc80 complex, roughly 600 Å long, appears to extend from the DNA-proximal assembly to the plus end of a microtubule, to which one end of the complex is known to bind. Ongoing structural studies will clarify the roles of a number of other well-defined complexes.

Roles for the conserved spc105p/kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint.

BackgroundKinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.Principal FindingsWe show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells.Conclusions/SignificanceSpc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.

SiRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1–KNTC2 and SPC24–SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

Fused sister kinetochores initiate the reductional division in meiosis I

During meiosis I the genome is reduced to the haploid content by a coordinated reductional division event. Homologous chromosomes align, recombine and segregate while the sister chromatids co-orient and move to the same pole. Several data suggest that sister kinetochores co-orient early in metaphase I and that sister chromatid cohesion (which requires Rec8 and Shugoshin) supports monopolar orientation. Nevertheless, it is unclear how the sister kinetochores function as single unit during this period. A gene (monopolin) with a co-orienting role was identified in Saccharomyces cerevisiae; however, it does not have the same function in fission yeast and no similar genes have been found in other species. Here we pursue this issue using knockdown mutants of the core kinetochore protein MIS12 (minichromosome instability 12). MIS12 binds to base of the NDC80 complex, which in turn binds directly to microtubules. In maize plants with systemically reduced levels of MIS12, a visible MIS12-NDC80 bridge between sister kinetochores at meiosis I is broken. Kinetochores separate and orient randomly in metaphase I, causing chromosomes to stall in anaphase due to normal cohesion, marked by Shugoshin, between the chromatids. The data establish that sister kinetochores in meiosis I are fused by a shared microtubule-binding face and that this direct linkage is required for reductional division.

Intrakinetochore localization and essential functional domains of Drosophila Spc105

The kinetochore is assembled during mitotic and meiotic divisions within the centromeric region of chromosomes. It is composed of more than eighty different proteins. Spc105 (also designated as Spc7, KNL-1 or Blinkin in different eukaryotes) is a comparatively large kinetochore protein, which can bind to the Mis12/MIND and Ndc80 complexes and to the spindle assembly checkpoint components Bub1 and BubR1. Our genetic characterization of Drosophila Spc105 shows that a truncated version lacking the rapidly evolving, repetitive central third still provides all essential functions. Moreover, in comparison with Cenp-C that has previously been observed to extend from the inner to the outer kinetochore region, full-length Spc105 is positioned further out and is not similarly extended along the spindle axis. Thus, our results indicate that Spc105 forms neither an extended link connecting inner Cenp-A chromatin with outer kinetochore regions nor a scaffold constraining kinetochore subcomplexes and spindle assembly checkpoint components together into a geometrically rigid supercomplex. Spc105 seems to provide a platform within the outer kinetochore allowing independent assembly of various kinetochore components.

Connecting with Ska, a key complex at the kinetochore–microtubule interface

Connecting with Ska, a key complex at the kinetochore–microtubule interface

Chromosome Segregation: Ndc80 Can Carry the Load

Dynamic attachments between kinetochores and spindle microtubules are required for chromosome bi-orientation in mitosis. A new study provides biophysical insight into how the Ndc80 complex may contribute to the formation of these attachments.

In Vivo Protein Architecture of the Eukaryotic Kinetochore with Nanometer Scale Accuracy

The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus ends of one or more microtubules (MTs) and segregates chromosomes during cell division. Its core structure consists of eight multicomponent protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3D protein architecture of a metaphase kinetochore-microtubule attachment and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is due mainly to a decrease in the length of the Ndc80 complex, which may result either from intramolecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80-Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle-assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres.

CENP-K and CENP-H may form coiled-coils in the kinetochores

Kinetochores are large proteinaceous structure on the surface of chromosomes' primary constriction during mitosis. They link chromosomes to spindle microtubules and also regulate the spindle assembly checkpoint, which is crucial for correct chromosome segregation in all eukaryotes. The better known core networks of kinetochores include the KMN network (K, KNL1; M, Mis12 complex; N, Ndc80 complex)and CCAN (constitutive centromere-associated network). However, the detailed molecular mechanism of the kinetochore protein network remains unclear. This study demonstrates that CENP-H and CENP-K form quite stable subcomplex by TAP (tandem affinity purification) with HEK 293 cells which express TAP-CENP-K, with the ratio of purified CENP-H and CENP-K being close to 1: 1 even with high salt. Bioinformatic analysis suggests that CENP-H and CENP-K are enriched with coiled-coil regions. This implies that CENP-H and CENP-K form heterodimeric coiled-coils. Furthermore, the functional regions which form the complex are respectively located on their N- and C-terminals, but the association between the C-terminals is more complex. It is possible that this is the first identified heterodimeric coiled-coils within the inner kinetochore, which is directly involved in the attachment between kinetochores and the spindle microtubules.

The Ndc80 Kinetochore Complex Forms Load-Bearing Attachments to Dynamic Microtubule Tips via Biased Diffusion

Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical, and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide the combination of plasticity and strength that allows kinetochores to maintain load-bearing tip attachments during both microtubule assembly and disassembly.

Ectopic expression of plasma membrane targeted subunits of the Ndc80-complex as a tool to study kinetochore biochemistry

Genomic stability depends on the normal function of the kinetochore, a multi-protein assemblage, which consists of over 80 molecules including both constitutive and transiently binding components. Information regarding the spatial–temporal assembly of kinetochore subcomplexes is often limited by technical difficulties in their isolation. To study kinetochore subcomplex formation, we targeted separately Hec1 and Spc24, two subunits of the Ndc80 kinetochore compilation, to the plasma membrane by fusing them with the amino-terminal palmitoylation and myristoylation (pm) sequence of the receptor tyrosine kinase Fyn. We found that in early mitotic cells, pm-GFP–Hec1 and pm-GFP–Spc24 fusion proteins localised to the plasma membrane and were able to recruit all subunits of the Ndc80 complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) to these foci. In interphase cells, only Hec1–Nuf2 and Spc24–Spc25 heterodimers accumulated to the plasma membrane foci. The results propose that the assembly of Ndc80 tetramer can take place outside of the kinetochore but requires co-factors that are only present in mitotic cells. These findings provide the first experimental evidence on the successful employment of the plasma membrane targeting technique in the study of kinetochore biochemistry.

Reconstitution Of Microtubule-driven Movement and Force Production by the Ndc80 Kinetochore Complex

The kinetochore-microtubule junction is a central but poorly understood part of the molecular machinery that organizes and separates chromosomes during mitosis. Kinetochores maintain load-bearing attachments between chromosomes and microtubule tips, even as the tips assemble and disassemble under their grip. This end-on coupling is conserved from yeast to humans, suggesting it may be mediated by a common molecular mechanism but the existence of a conserved coupler remains uncertain. Using techniques for manipulating and tracking individual molecules, we discovered that end-on coupling can be reconstituted with the Ndc80 complex, a widely conserved component of the outer kinetochore. Beads decorated with pure Ndc80 complex bind individual microtubules through the N-terminal globular domains of Ndc80p and Nuf2p. Like kinetochores, these Ndc80-based couplers are carried by disassembling tips and they remain tip-bound even under tensile loads up to 2.5 pN. Microtubule-driven movement and force production persist at low surface densities where the number of Ndc80 complexes interacting with the filament is close to the number found at kinetochores in vivo. At the single molecule level, fluorescent-tagged Ndc80 complexes diffuse randomly along microtubules except at disassembling tips where their motion becomes biased in the direction of filament shortening. Our results indicate that Ndc80-based coupling likely depends upon a ‘fluctuating ensemble’ mechanism similar to that proposed by Hill on purely theoretical grounds more than two decades ago*. We also demonstrate that both the yeast and human Ndc80 complexes support microtubule-driven bead movement and force production. This conservation of function, together with the established importance of the Ndc80 complex for kinetochore-microtubule coupling across eukaryotes, strongly suggests that a fluctuating ensemble of Ndc80 complexes represents a core attachment module within the kinetochore.* Hill, T. L. (1985) Proc Natl Acad Sci USA 82:4404-4408.

Systematic Analysis inCaenorhabditis elegansReveals that the Spindle Checkpoint Is Composed of Two Largely Independent Branches

Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1-three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1(MDF-1)/Mad2(MDF-2)-based signal, with a largely independent Mad3(SAN-1)/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2(MDF-2) levels bypasses the requirement for BUB-3 and Mad3(SAN-1) in kinetochore-dependent checkpoint activation. Mad3(SAN-1) does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2(MDF-2). We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1(MDF-1)/Mad2(MDF-2) signal generated at kinetochores is integrated with a separate cytoplasmic Mad3(SAN-1)/BUB-3-based pathway.

Kinetochore Attachment: How the Hec Can a Cell Do It?

Creating stable yet flexible attachments of spindle microtubules to the kinetochore is critical for facilitating chromosome congression, segregation, and checkpoint signaling. Two new studies have elucidated the molecular details of how the Ndc80 complex mediates this dynamic attachment.

Kinetochore-Microtubule Attachment Relies on the Disordered N-Terminal Tail Domain of Hec1

Accurate chromosome segregation is dependent upon stable attachment of kinetochores to spindle microtubules during mitosis. A long-standing question is how kinetochores maintain stable attachment to the plus ends of dynamic microtubules that are continually growing and shortening. The Ndc80 complex is essential for persistent end-on kinetochore-microtubule attachment in cells [1, 2], but how the Ndc80 complex forms functional microtubule-binding sites remains unknown. We show that the 80 amino acid N-terminal unstructured "tail" of Hec1 is required for generating stable kinetochore-microtubule attachments. PtK1 cells depleted of endogenous Hec1 and rescued with Hec1-GFP fusion proteins deleted of the entire N terminus or the disordered N-terminal 80 amino acid tail domain fail to generate stable kinetochore-microtubule attachments. Mutation of nine amino acids within the Hec1 tail to reduce its positive charge also abolishes stable attachment. Furthermore, the mitotic checkpoint remains functional after deletion of the N-terminal 80 amino acid tail, but not after deletion of the N-terminal 207 amino acid region containing both the tail domain and a calponin homology (CH) domain. These results demonstrate that kinetochore-microtubule binding is dependent on electrostatic interactions mediated through the disordered N-terminal 80 amino acid tail domain and mitotic-checkpoint function is dependent on the CH domain of Hec1.

Kinetochore Attachments Require an Interaction between Unstructured Tails on Microtubules and Ndc80Hec1

Kinetochore attachments to microtubules are tight enough to move chromosomes, yet the microtubules' plus ends must remain dynamic and reposition within the attachment pocket during depolymerization-coupled movement. Kinetochores are unable to bind microtubules after any of the four subunits of the Ndc80 complex are knocked down [2, 4]; however, because the Ndc80 complex has important structural roles [1-3], it is unclear whether it directly mediates kinetochore-microtubule attachments. The Ndc80(Hec1) subunit (Hec1) has a microtubule-binding site composed of both an unstructured N-terminal tail and a calponin homology domain [5-7]. Here, we show that, surprisingly, the N-terminal tail is sufficient for microtubule-binding affinity in vitro. The interaction is salt sensitive, and the positively charged Hec1 tail cannot bind microtubules lacking negatively charged tails. We have replaced the endogenous Hec1 subunit with a mutant lacking the N-terminal tail. These cells assemble kinetochores properly but are unable to congress chromosomes, generate tension across sister kinetochores, or establish cold-stable kinetochore-microtubule attachments. Our data argue that the highest affinity interactions between kinetochores and microtubules are ionic attractions between two unstructured domains. We discuss the importance of this finding for models of repositioning of microtubules in the kinetochore during depolymerization.

Hec1 overexpression hyperactivates the mitotic checkpoint and induces tumor formation in vivo

Hec1 (Highly Expressed in Cancer 1) is one of four proteins of the outer kinetochore Ndc80 complex involved in the dynamic interface between centromeres and spindle microtubules. Its overexpression is seen in a variety of human tumors and correlates with tumor grade and prognosis. We show here that the overexpression of Hec1 in an inducible mouse model results in mitotic checkpoint hyperactivation. As previously observed with overexpression of the Mad2 gene, hyperactivation of the mitotic checkpoint leads to aneuploidy in vitro and is sufficient to generate tumors in vivo that harbor significant levels of aneuploidy. These results underscore the role of chromosomal instability as a result of mitotic checkpoint hyperactivation in the initiation of tumorigenesis.

Architecture and Flexibility of the Yeast Ndc80 Kinetochore Complex

Kinetochores mediate microtubule–chromosome attachment and ensure accurate segregation of sister chromatids. The highly conserved Ndc80 kinetochore complex makes direct contacts with the microtubule and is essential for spindle checkpoint signaling. It contains a long coiled-coil region with globular domains at each end involved in kinetochore localization and microtubule binding, respectively. We have directly visualized the architecture of the yeast Ndc80 complex and found a dramatic kink within the 560-Å coiled-coil rod located about 160 Å from the larger globular head. Comparison of our electron microscopy images to the structure of the human Ndc80 complex allowed us to position the kink proximal to the microtubule-binding end and to define the conformational range of the complex. The position of the kink coincides with a coiled-coil breaking region conserved across eukaryotes. We hypothesize that the kink in Ndc80 is essential for correct kinetochore geometry and could be part of a tension-sensing mechanism at the kinetochore.