Roles for the conserved spc105p/kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint.
ABSTRACT: BACKGROUND:Kinetochores 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 approximately 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 FINDINGS:We 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/SIGNIFICANCE:Spc105p/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.
Project description:The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (?14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes). Each CENP-T molecule recruits ?2 Ndc80 complexes; one as part of a KMN network. In contrast, ?40% of CENP-C recruits only a KMN network. Replacing the CENP-C domain that binds KMN with the CENP-T domain that recruits both an Ndc80 complex and KMN network yielded functional kinetochores. These results provide a quantitative picture of the linkages between centromeric chromatin and the microtubule-binding Ndc80 complex at the human kinetochore.
Project description:The microtubule-based mitotic spindle segregates chromosomes during cell division. During chromosome segregation, the centromeric regions of chromosomes build kinetochores that establish end-coupled attachments to spindle microtubules. Here, we used the Caenorhabditis elegans embryo as a model system to examine the crosstalk between two kinetochore protein complexes implicated in temporally distinct stages of attachment formation. The kinetochore dynein module, which mediates initial lateral microtubule capture, inhibited microtubule binding by the Ndc80 complex, which ultimately forms the end-coupled attachments that segregate chromosomes. The kinetochore dynein module directly regulated Ndc80, independently of phosphorylation by Aurora B kinase, and this regulation was required for accurate segregation. Thus, the conversion from initial dynein-mediated, lateral attachments to correctly oriented, Ndc80-mediated end-coupled attachments is actively controlled.
Project description:The kinetochore is a macromolecular protein machine  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.
Project description: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.
Project description:Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.
Project description:The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochore-microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex--microtubule interface.
Project description:How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.
Project description:Kinetochores (KTs) assemble on centromeric DNA, bi-orient paired sister chromatids on spindle microtubules (MTs) and control cell-cycle progression via the spindle assembly checkpoint. Genetic and biochemical studies in budding yeast have established that three 'linker' complexes, MIND, COMA and NDC80, play essential but distinct roles in KT assembly and chromosome segregation. To determine whether similar linker activities are present at human KTs, we have compared the functions of Nnf1R and Mcm21R, recently identified MIND and COMA subunits, and Nuf2R, a well-characterized NDC80 subunit. We find that the three proteins bind to KTs independent of each other and with distinct cell-cycle profiles. MT-KT attachment is aberrant in Nnf1R- and Mcm21R-depleted cells, whereas it is lost in the absence of Nuf2R. Defective attachments in Nnf1R-depleted cells prevent chromosome congression, whereas those in Mcm21R-depleted cells interfere with spindle assembly. All three human KT proteins are necessary for correct binding of spindle checkpoint proteins to KTs. The differing functions and KT-binding properties of Nnf1R, Mcm21R and Nuf2R suggest that, like their yeast counterparts, the proteins act independent of each other in KT assembly, but that their combined activities are required for checkpoint signaling.
Project description:During metaphase, sister chromatids are connected to microtubules extending from the opposite spindle poles via kinetochores to protein complexes on the chromosome. Kinetochores congress to the equatorial plane of the spindle and oscillate around it, with kinesin-8 motors restricting these movements. Yet, the physical mechanism underlying kinetochore movements is unclear. We show that kinetochore movements in the fission yeast Schizosaccharomyces pombe are regulated by kinesin-8-promoted microtubule catastrophe, force-induced rescue, and microtubule dynamic instability. A candidate screen showed that among the selected motors only kinesin-8 motors Klp5/Klp6 are required for kinetochore centering. Kinesin-8 accumulates at the end of microtubules, where it promotes catastrophe. Laser ablation of the spindle resulted in kinetochore movement toward the intact spindle pole in wild-type and klp5? cells, suggesting that kinetochore movement is driven by pulling forces. Our theoretical model with Langevin description of microtubule dynamic instability shows that kinesin-8 motors are required for kinetochore centering, whereas sensitivity of rescue to force is necessary for the generation of oscillations. We found that irregular kinetochore movements occur for a broader range of parameters than regular oscillations. Thus, our work provides an explanation for how regulation of microtubule dynamic instability contributes to kinetochore congression and the accompanying movements around the spindle center.
Project description:BACKGROUND:Kinetochores are large multiprotein complexes indispensable for proper chromosome segregation. Although Drosophila is a classical model organism for studies of chromosome segregation, little is known about the organization of its kinetochores. METHODOLOGY/PRINCIPAL FINDINGS:We employed bioinformatics, proteomics and cell biology methods to identify and analyze the interaction network of Drosophila kinetochore proteins. We have shown that three Drosophila proteins highly diverged from human and yeast Ndc80, Nuf2 and Mis12 are indeed their orthologues. Affinity purification of these proteins from cultured Drosophila cells identified a further five interacting proteins with weak similarity to subunits of the SPC105/KNL-1, MIND/MIS12 and NDC80 kinetochore complexes together with known kinetochore associated proteins such as dynein/dynactin, spindle assembly checkpoint components and heterochromatin proteins. All eight kinetochore complex proteins were present at the kinetochore during mitosis and MIND/MIS12 complex proteins were also centromeric during interphase. Their down-regulation led to dramatic defects in chromosome congression/segregation frequently accompanied by mitotic spindle elongation. The systematic depletion of each individual protein allowed us to establish dependency relationships for their recruitment onto the kinetochore. This revealed the sequential recruitment of individual members of first, the MIND/MIS12 and then, NDC80 complex. CONCLUSIONS/SIGNIFICANCE:The Drosophila MIND/MIS12 and NDC80 complexes and the Spc105 protein, like their counterparts from other eukaryotic species, are essential for chromosome congression and segregation, but are highly diverged in sequence. Hierarchical dependence relationships of individual proteins regulate the assembly of Drosophila kinetochore complexes in a manner similar, but not identical, to other organisms.