Chromosome Segregation In Yeast Research Articles

Fission yeast spindle dynamics and chromosome segregation fidelity show distinct thermosensitivity.

Cellular processes rely on proteins with temperature-dependent stability and activity. While thermosensitivity in biological networks is well-explored, the effect of temperature on complex mechanochemical assemblies, like the spindle, is rarely studied. We examined fission yeast spindle dynamics and chromosome segregation from 15⁰C to 40⁰C. Our findings reveal that these parameters follow U-shaped temperature-dependent curves but reach their minima at different temperatures. Specifically, spindle dynamics peak around 35⁰C, whereas chromosome segregation defects are minimized at 25⁰C. This suggests a scenario in which mitotic errors are tolerated to expedite rapid cell cycle progression.

Structural characterisation of the Chaetomium thermophilum Chl1 helicase.

Chl1 is a member of the XPD family of 5'-3' DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Shake It Off: The Elimination of Erroneous Kinetochore-Microtubule Attachments and Chromosome Oscillation.

Cell proliferation and sexual reproduction require the faithful segregation of chromosomes. Chromosome segregation is driven by the interaction of chromosomes with the spindle, and the attachment of chromosomes to the proper spindle poles is essential. Initial attachments are frequently erroneous due to the random nature of the attachment process; however, erroneous attachments are selectively eliminated. Proper attachment generates greater tension at the kinetochore than erroneous attachments, and it is thought that attachment selection is dependent on this tension. However, studies of meiotic chromosome segregation suggest that attachment elimination cannot be solely attributed to tension, and the precise mechanism of selective elimination of erroneous attachments remains unclear. During attachment elimination, chromosomes oscillate between the spindle poles. A recent study on meiotic chromosome segregation in fission yeast has suggested that attachment elimination is coupled to chromosome oscillation. In this review, the possible contribution of chromosome oscillation in the elimination of erroneous attachment is discussed in light of the recent finding.

The role of CSE1L expression in cervical lymph node metastasis of larynx tumors

IntroductionAccording to international reports, 30–40% of all head and neck cancers are larynx cancers, comprising 1–2.5% of all cancer types. Cervical nodal involvement has been reported to be 40% and 65% in T3 and T4 cases, respectively. Five-year survival in patients with cervical lymph node metastasis has been demonstrated to be 50% lower compared to patients with no metastasis. Chromosome segregation like 1 protein; is a DNA fragment isolated by Brinkmann et al. in 1995 that corresponds to yeast chromosome segregation protein. Studies on the effect of chromosome segregation like 1 protein expression in head and neck tumors are rare and it has been shown that nuclear chromosome segregation like 1 protein is over-expressed in these studies where gastrointestinal and breast tumors over-expressed cytoplasmic chromosome segregation like 1 protein. ObjectiveChromosome segregation like 1 protein may regulate the proliferation and metastasis of T3–T4 glottic larynx cancer. The aim of this study is to show the relationship between chromosome segregation like 1 protein expression and cervical lymph node metastasis of T3–T4 glottic larynx cancer. MethodsA total of 57 male patients who were operated for T3–T4 glottic cancer in a tertiary referral hospital was included in this study. There were 28 patients with cervical lymph node metastasis and 29 patients without lymph node metastasis. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded archival glottic larynx tumour tissue. According to the percentage of immunoreactive cells, chromosome segregation like 1 protein status was analyzed. ResultsAmong the patients, who had no cervical lymph node metastasis, 15 patients showed weak nuclear staining, 12 patients showed moderate nuclear staining and only 2 patients showed high nuclear staining for chromosome segregation like 1 protein. Among the patients who had cervical lymph node metastasis, 18 patients showed high nuclear staining, 9 patients showed moderate staining and only one patient showed weak staining for chromosome segregation like 1 protein. None of the metastatic patients showed cytoplasmic staining and only one patient in the non-metastatic group showed cytoplasmic staining for chromosome segregation like 1 protein. There was a positive correlation between nuclear chromosome segregation like 1 protein expression and cervical lymph node metastasis (r = 0,668) and it was statistically significant (p < 0,001). ConclusionChromosome segregation like 1 protein expression is correlated with lymph node metastasis in T3–T4 glottic cancers. This may change the approach to cervical node treatment in patients with glottic cancers in future.

Chromosome detachment from the nuclear envelope is required for genomic stability in closed mitosis.

Mitosis in metazoans involves detachment of chromosomes from the nuclear envelope (NE) and NE breakdown, whereas yeasts maintain the nuclear structure throughout mitosis. It remains unknown how chromosome attachment to the NE might affect chromosome movement in yeast. By using a rapamycin-induced dimerization system to tether a specific locus of the chromosome to the NE, I found that the tethering delays the separation and causes missegregation of the region distal to the tethered site. The phenotypes are exacerbated by mutations in kinetochore components and Aurora B kinase Ipl1. The chromosome region proximal to the centromere is less affected by the tether, but it exhibits excessive oscillation before segregation. Furthermore, the tether impacts full extension of the mitotic spindle, causing abrupt shrinkage or bending of the spindle in shortened anaphase. The study supports detachment of chromosomes from the NE being required for faithful chromosome segregation in yeast and segregation of tethered chromosomes being dependent on a fully functional mitotic apparatus.

The Agrobacterium VirD5 protein hyperactivates the mitotic Aurora kinase in host cells

Summary Aided by translocated virulence proteins, Agrobacterium tumefaciens transforms plant cells with oncogenic T‐DNA. In the host cells the virulence protein VirD5 moves to the nucleus, where it becomes localized at the kinetochores, and disturbs faithful chromosome segregation, but the molecular mechanism underlying this remains unknown.To gain more insight, we screened amongst the kinetochore proteins for VirD5 interactors using bimolecular fluorescence complementation assays, and tested chromosome segregation in yeast cells.We found that VirD5 interacts with the conserved mitotic Aurora kinase Ipl1 in yeast and likewise with plant Aurora kinases. In vitro VirD5 was found to stimulate the activity of Ipl1. Phosphorylation of substrates by Ipl1 in vivo is known to result in the detachment between kinetochore and spindle microtubule. This is necessary for error correction, but increased Ipl1/Aurora kinase activity is known to cause spindle instability, explaining enhanced chromosome mis‐segregation seen in the presence of VirD5. That activation of the Ipl1/Aurora kinase at least partially underlies the toxicity of VirD5 became apparent by artificial boosting the activity of the specific counteracting phosphatase Glc7 in vivo, which relieved the toxicity.These findings reveal a novel mechanism by which a pathogenic bacterium manipulates host cells.

Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

A Genome-Wide Screen with Nicotinamide to Identify Sirtuin-Dependent Pathways in Saccharomyces cerevisiae.

Sirtuins are evolutionarily conserved NAD-dependent deacetylases that catalyze the cleavage of NAD+ into nicotinamide (NAM), which can act as a pan-sirtuin inhibitor in unicellular and multicellular organisms. Sirtuins regulate processes such as transcription, DNA damage repair, chromosome segregation, and longevity extension in yeast and metazoans. The founding member of the evolutionarily conserved sirtuin family, SIR2, was first identified in budding yeast. Subsequent studies led to the identification of four yeast SIR2 homologs HST1, HST2, HST3, and HST4. Understanding the downstream physiological consequences of inhibiting sirtuins can be challenging since most studies focus on single or double deletions of sirtuins, and mating defects in SIR2 deletions hamper genome-wide screens. This represents an important gap in our knowledge of how sirtuins function in highly complex biological processes such as aging, metabolism, and chromosome segregation. In this report, we used a genome-wide screen to explore sirtuin-dependent processes in Saccharomyces cerevisiae by identifying deletion mutants that are sensitive to NAM. We identified 55 genes in total, 36 of which have not been previously reported to be dependent on sirtuins. We find that genome stability pathways are particularly vulnerable to loss of sirtuin activity. Here, we provide evidence that defects in sister chromatid cohesion renders cells sensitive to growth in the presence of NAM. The results of our screen provide a broad view of the biological pathways sensitive to inhibition of sirtuins, and advance our understanding of the function of sirtuins and NAD+ biology.

Building an Accurate Chromosome Segregation Machine in Fission Yeast

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B which destabilizes kinetochore microtubules (ktMT) in the absence of tension between sister chromatids. We previously described a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of kinetochore microtubules. This model accurately reproduces the timing of correct chromosome bi-orientation and segregation seen in fission yeast. This force-balance model of mitosis describes global spindle dynamics and predicts chromosome segregation defects (Courtheoux J.Cell Biol 2009; Gay, J.Cell Biol 2012).We recently implemented new features to our model that describe kinetochore alignment during metaphase. In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. We provide a quantitative analysis of kinetochore oscillations and positioning in S. pombe, a model lacking chromokinesins. In wild type cells, chromosomes align during prophase and while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. We propose that Kinesin-8 aligns chromosomes by controlling pulling forces in a length dependent manner. Our chromosome segregation model implemented with a length-dependent process that controls the force at kinetochores is sufficient to mimic congression and to prevent aneuploidy. These model illustrate how a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes.Altogether, our work illustrates how a simplified force balance model, with stochastic attachment and detachment events, correction mechanisms (Aurora B, and kinetochore orientation effect) and a length dependent process for kinetochore alignment, can explain the segregation of chromosomes with a timing and accuracy similar to chromosome segregation in living wild-type fission yeast cells.

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.

Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast.

Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2+ could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-Acnp1-1 was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words]

A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B-like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy.

A Homolog of the Cell Apoptosis Susceptibility Gene Involved in Ovary Development of Chinese Shrimp Fenneropenaeus chinensis1

The cell apoptosis susceptibility (CAS) gene is a homolog of the yeast chromosome segregation (CSE1) gene, which functions in cell proliferation and apoptosis. In the present study, a homolog of CAS was cloned from Chinese shrimp Fenneropenaeus chinensis (FcCAS). The full-length FcCAS cDNA is 3534 bp and contains an open reading frame encoding 968 amino acids. The predicted tertiary FcCAS structure is highly similar to that of CSE1 from the yeast Saccharomyces cerevisiae. RT-PCR analysis showed that the FcCAS gene is expressed mainly in testis, ovary, stomach, lymphoid organs, gills, and hemocytes. RNA in situ hybridization showed that FcCAS transcripts were distributed mainly in the cytoplasm of oocytes. Western blot analysis showed that FcCAS could be detected only in testis and ovary, and its expression levels differed at different developmental stages of ovaries. Immunohistochemical analysis showed that FcCAS existed in both the cytoplasm and the nucleus, which suggested that FcCAS might function as a nuclear protein. No transcript was detected in the abnormally developed ovaries of triploid shrimp. Therefore, we inferred that the FcCAS gene might be one of the key genes that is closely related to ovary development in shrimp.

Female gametophytic cell specification and seed development require the function of the putativeArabidopsisINCENP orthologWYRD

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

CTF18 plays critical roles in female gametogenesis and ovarian folliculogenesis in mammals

OBJECTIVE: Infertility is a major medical concern affecting 1 of every 10 individuals of childbearing age worldwide. In order to explore possible genetic causes of infertility, we are studying CTF18, an evolutionarily conserved protein that is crucial for germline development in the fruitfly and essential for accurate chromosome segregation in yeast. We recently demonstrated that CTF18, termed Chtf18 in mice, is expressed throughout the female germline of the mouse. The objective of our study is to determine the roles of CTF18 /Chtf18[/italic] in mammalian female gametogenesis.DESIGN: We employed gene targeting to derive mice that lack Chtf18 function.MATERIALS AND METHODS: Follicle counting techniques were used to examine hemotoxylin and eosin stained sections of ovaries from age-matched Chtf18-null and wild-type mice at postnatal and adult ages. Surface spread nuclei were prepared and immunofluorescence microscopy was performed to evaluate meiotic prophase I in oocytes of Chtf18-null compared to wild-type females. Experiments to assess developmental competency of oocytes by culture and in vitro maturation are currently underway.RESULTS: Ovaries from Chtf18-null females were smaller and morphologically abnormal, and contained follicles that were aberrant and appeared to be degenerating, compared to wild-type females. Chtf18-null follicles were significantly decreased in number throughout all stages of folliculogenesis. While chromosomal synapsis was complete, the diplotene stage of meiotic prophase I revealed the presence of possible univalent chromosomes in Chtf18-null oocytes. Results regarding developmental competency of Chtf18-null oocytes compared to wild-type oocytes will be presented.CONCLUSION: CTF18 plays crucial roles in female gametogenesis and ovarian folliculogenesis in mammals. Preliminary results reveal that homologous chromosomes separate prematurely in Chtf18-null oocytes during prophase I. CTF18 likely plays important roles in human female gametogenesis and fertility. OBJECTIVE: Infertility is a major medical concern affecting 1 of every 10 individuals of childbearing age worldwide. In order to explore possible genetic causes of infertility, we are studying CTF18, an evolutionarily conserved protein that is crucial for germline development in the fruitfly and essential for accurate chromosome segregation in yeast. We recently demonstrated that CTF18, termed Chtf18 in mice, is expressed throughout the female germline of the mouse. The objective of our study is to determine the roles of CTF18 /Chtf18[/italic] in mammalian female gametogenesis. DESIGN: We employed gene targeting to derive mice that lack Chtf18 function. MATERIALS AND METHODS: Follicle counting techniques were used to examine hemotoxylin and eosin stained sections of ovaries from age-matched Chtf18-null and wild-type mice at postnatal and adult ages. Surface spread nuclei were prepared and immunofluorescence microscopy was performed to evaluate meiotic prophase I in oocytes of Chtf18-null compared to wild-type females. Experiments to assess developmental competency of oocytes by culture and in vitro maturation are currently underway. RESULTS: Ovaries from Chtf18-null females were smaller and morphologically abnormal, and contained follicles that were aberrant and appeared to be degenerating, compared to wild-type females. Chtf18-null follicles were significantly decreased in number throughout all stages of folliculogenesis. While chromosomal synapsis was complete, the diplotene stage of meiotic prophase I revealed the presence of possible univalent chromosomes in Chtf18-null oocytes. Results regarding developmental competency of Chtf18-null oocytes compared to wild-type oocytes will be presented. CONCLUSION: CTF18 plays crucial roles in female gametogenesis and ovarian folliculogenesis in mammals. Preliminary results reveal that homologous chromosomes separate prematurely in Chtf18-null oocytes during prophase I. CTF18 likely plays important roles in human female gametogenesis and fertility.

Physical Linkages between Sister Chromatids and Their Removal during Yeast Chromosome Segregation

The fidelity of chromosome inheritance is of paramount importance to all living organisms. In eukaryotic cells, the strategy to ensure physical segregation of chromosomes to daughter cells relies on two basic steps ordered in time: an initial linkage, or cohesion, of sister chromatids and its timely and complete dissolution during anaphase. The current view is that these two basic steps are accomplished around the regulation of a protein complex called cohesin that serves as "clamp brackets" distributed at intervals throughout the genome. However, many of the DNA metabolic activities during interphase also produce physical linking of chromatids. For example, during replication, intertwines between sister chromatids are formed. Here, we review our understanding of the processes that generate physical linkages between chromatids and discuss potential mechanisms that are involved in the removal of such obstacles to the complete physical separation of chromatids at anaphase.

Histone Variant H2A.Z Regulates Centromere Silencing and Chromosome Segregation in Fission Yeast

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.

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.

Phosphorylation of HsMis13 by Aurora B Kinase Is Essential for Assembly of Functional Kinetochore

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilising drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.