Protein arms in the kinetochore-microtubule interface of the yeast DASH complex

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface.     

The DASH complex and Klp5/Klp6 kinesin coordinate bipolar chromosome attachment in fission yeast

We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13-117 (cyclin B) and mal3-1 (EB-1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere-associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment.     

The DASH complex component Ask1 is a cell cycle-regulated Cdk substrate in Saccharomyces cerevisiae

The proper timing and fidelity of cell cycle transitions is critical for the survival of organisms. Cyclin-dependent kinases orchestrate many cell cycle transitions in eukaryotes including S phase entry and mitosis. Accurate chromosome segregation during mitosis is one of the key events regulated by the cell cycle and many proteins function together to ensure the fidelity of this process. In S. cerevisiae, the DASH complex is essential for chromosome segregation. The DASH complex binds to microtubules and kinetochores and regulates their association. Here we report that Askl, one component of DASH, is phosphorylated during the cell cycle. This phosphorylation is dependent on Cdks in vivo, and in vitro Cdc28 can phosphorylate Askl. We identify two Cdk phosphorylation sites in Askl and find that the phosphorylation of Askl is important for its full activity in vivo. Thus, the DASH complex is directly regulated by cyclin-dependent kinases to facilitate chromosome segregation.     

The DASH Complex Component Ask1 Is a Cell Cycle-Regulated Cdk Substrate in Saccharomyces cerevisiae

The proper timing and fidelity of cell cycle transitions is critical for the survival of organisms. Cyclin-dependent kinases orchestrate many cell cycle transitions in eukaryotes including S phase entry and mitosis. Accurate chromosome segregation during mitosis is one of the key events regulated by the cell cycle and many proteins function together to ensure the fidelity of this process. In S. cerevisiae, the DASH complex is essential for chromosome segregation. The DASH complex binds to microtubules and kinetochores and regulates their association. Here we report that Ask1, one component of DASH, is phosphorylated during the cell cycle. This phosphorylation is dependent on Cdks in vivo, and in vitro Cdc28 can phosphorylate Ask1. We identify two Cdk phosphorylation sites in Ask1 and find that the phosphorylation of Ask1 is important for its full activity in vivo. Thus, the DASH complex is directly regulated by cyclin-dependent kinases to facilitate chromosome segregation.     

Molecular analysis of kinetochore architecture in fission yeast

Kinetochore composition and structure are critical for understanding how kinetochores of different types perform similar functions in chromosome segregation. We used affinity purification to investigate the kinetochore composition and assembly in Schizosaccharomyces pombe. We identified a conserved DASH complex that functions to ensure precise chromosome segregation. Unlike DASH in budding yeast that is localized onto kinetochores throughout the cell cycle, SpDASH is localized onto kinetochores only in mitosis. We also identified two independent groups of kinetochore components, one of which, the Sim4 complex, contains several novel Fta proteins in addition to known kinetochore components. DASH is likely to be associated with the Sim4 complex via Dad1 protein. The other group, Ndc80-MIND-Spc7 complex, contains the conserved Ndc80 and MIND complexes and Spc7 protein. We propose that fission yeast kinetochore is comprised of at least two major structural motifs that are biochemically separable. Our results suggest a high degree of conservation between the kinetochores of budding yeast and fission yeast even though many individual protein subunits do not have a high degree of sequence similarity.     

Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 ?-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.     

The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment

Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules.     

Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.     

γ‐Tubulin complex in Trypanosoma brucei: molecular composition,subunit interdependence and requirement for axonemal central pair protein assembly

γ‐Tubulin complex constitutes a key component of the microtubule‐organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ‐tubulin small complex (γTuSC) composed of γ‐tubulin, gamma‐tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ‐tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ‐tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ‐tubulin complex in T. brucei is composed of γ‐tubulin and three GCP proteins, GCP2‐GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ‐tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.     

Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and the chromatin is crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. We propose that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.     

Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.     

Actomyosin transports microtubules and microtubules control actomyosin recruitment during Xenopus oocyte wound healing

BACKGROUND: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes. RESULTS: After wounding, microtubules are transported to the wound edge in association with F-actin that is itself recruited to wound borders via actomyosin-powered cortical flow. This transport generates sufficient force to buckle and break microtubules at the wound edge. Transport is complemented by local microtubule assembly around wound borders. The region of microtubule breakage and assembly coincides with a zone of actin assembly, and perturbation of the microtubule cytoskeleton disrupts this zone as well as local recruitment of the Arp2/3 complex and myosin-2. CONCLUSIONS: The results reveal transport of microtubules in association with F-actin that is pulled to wound borders via actomyosin-based contraction. Microtubules, in turn, focus zones of actin assembly and myosin-2 recruitment at the wound border. Thus, wounding triggers the formation of a spatially coordinated feedback loop in which transport and assembly of microtubules maintains actin and myosin-2 in close proximity to the closing contractile array. These results are surprisingly reminiscent of recent findings in locomoting cells, suggesting that similar feedback interactions may be generally employed in a variety of fundamental cell motility processes.     

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

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 ∼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.     

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.     

Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.     

Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding

Rotenone, a widely used insecticide, has been shown to inhibit mammalian cell proliferation and to depolymerize cellular microtubules. In the present study, the effects of rotenone on the assembly of microtubules in relation to its ability to inhibit cell proliferation and mitosis were analyzed. We found that rotenone inhibited the proliferation of HeLa and MCF-7 cells with half maximal inhibitory concentrations of 0.2 +/- 0.1 microm and 0.4 +/- 0.1 microm, respectively. At its effective inhibitory concentration range, rotenone depolymerized spindle microtubules of both cell types. However, it had a much stronger effect on the interphase microtubules of MCF-7 cells compared to that of the HeLa cells. Rotenone suppressed the reassembly of microtubules in living HeLa cells, suggesting that it can suppress microtubule growth rates. Furthermore, it reduced the intercentrosomal distance in HeLa cells at its lower effective concentration range and induced multipolar-spindle formation at a relatively higher concentration range. It also increased the level of checkpoint protein BubR1 at the kinetochore region. Rotenone inhibited both the assembly and the GTP hydrolysis rate of microtubules in vitro. It also inhibited the binding of colchicine to tubulin, perturbed the secondary structure of tubulin, and reduced the intrinsic tryptophan fluorescence of tubulin and the extrinsic fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid complex, suggesting that it binds to tubulin. A dissociation constant of 3 +/- 0.6 microm was estimated for tubulin-rotenone complex. The data presented suggest that rotenone blocks mitosis and inhibits cell proliferation by perturbing microtubule assembly dynamics.