The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules

The Dam1 complex attaches the kinetochore to spindle microtubules and is a processivity factor in vitro. In Saccharomyces cerevisiae, which has point centromeres that attach to a single microtubule, deletion of any Dam1 complex member results in chromosome segregation failures and cell death. In Schizosaccharomyces pombe, which has epigenetically defined regional centromeres that each attach to 3-5 kinetochore microtubules, Dam1 complex homologs are not essential. To determine why the complex is essential in some organisms and not in others, we used Candida albicans, a multimorphic yeast with regional centromeres that attach to a single microtubule. Interestingly, the Dam1 complex was essential in C. albicans, suggesting that the number of microtubules per centromere is critical for its requirement. Importantly, by increasing CENP-A expression levels, more kinetochore proteins and microtubules were recruited to the centromeres, which remained fully functional. Furthermore, Dam1 complex members became less crucial for growth in cells with extra kinetochore proteins and microtubules. Thus, the requirement for the Dam1 complex is not due to the DNA-specific nature of point centromeres. Rather, the Dam1 complex is less critical when chromosomes have multiple kinetochore complexes and microtubules per centromere, implying that it functions as a processivity factor in vivo as well as in vitro.     

A coordinated interdependent protein circuitry stabilizes the kinetochore ensemble to protect CENP-A in the human pathogenic yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3-5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.     

HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres

The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.     

Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.     

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.     

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole)

The kinetochore is a multi‐protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock‐down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan‐specific motif with a putative role in nuclear localization.     

Kinetochore recruitment of two nucleolar proteins is required for homolog segregation in meiosis I

Halving of the chromosome number during meiosis I depends on the segregation of maternal and paternal centromeres. This process relies on the attachment of sister centromeres to microtubules emanating from the same spindle pole. We describe here the identification of a protein complex, Csm1/Lrs4, that is essential for monoorientation of sister kinetochores in Saccharomyces cerevisiae. Both proteins are present in vegetative cells, where they reside in the nucleolus. Only shortly before meiosis I do they leave the nucleolus and form a "monopolin" complex with the meiosis-specific Mam1 protein, which binds to kinetochores. Surprisingly, Csm1's homolog in Schizosaccharomyces pombe, Pcs1, is essential for accurate chromosome segregation during mitosis and meiosis II. Csm1 and Pcs1 might clamp together microtubule binding sites on the same (Pcs1) or sister (Csm1) kinetochores.     

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.     

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.     

Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.     

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.     

Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.     

CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans

Proper assembly of the kinetochore, a multi-protein complex that mediates attachment of centromere DNA to spindle microtubules on each chromosome, is required for faithful chromosome segregation. Each previously characterized member of the Mis12/Mtw1 protein family is part of an essential subcomplex in the kinetochore. In this work, we identify and characterize CaMTW1, which encodes the homologue of the human Mis12 protein in the pathogenic budding yeast Candida albicans. Subcellular localization and chromatin immunoprecipitation assays confirmed CaMtw1 is a kinetochore protein. CaMtw1 is essential for viability. CaMtw1-depleted cells and cells in which CaMtw1 was inactivated with a temperature-sensitive mutation had reduced viability, accumulated at the G2/M stage of the cell cycle, and exhibited increased chromosome missegregation. CaMtw1 depletion also affected spindle length and alignment. Interestingly, in C. albicans, CaMtw1 and the centromeric histone, CaCse4, influence each other for kinetochore localization. In addition, CaMtw1 is required for efficient kinetochore recruitment of another inner kinetochore protein, the CENP-C homologue, CaMif2. Mis12/Mtw1 proteins have well-established roles in the recruitment and maintenance of outer kinetochore proteins. We propose that Mis12/Mtw1 proteins also have important co-dependent interactions with inner kinetochore proteins and that these interactions may increase the fidelity of kinetochore formation.     

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.     

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.     

Dissection of CENP-C–directed Centromere and Kinetochore Assembly

Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.     

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.     

Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore

The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.