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Mitsuhiro Yanagida

Publications and source records attributed to Mitsuhiro Yanagida.

At least 19 recordsLinked to original sources
PubMed2007

Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1.

The centromere is the chromosomal site that joins to microtubules during mitosis for proper segregation. Determining the location of a centromere-specific histone H3 called CENP-A at the centromere is vital for understanding centromere structure and function. Here, we report the identification of three human proteins essential for centromere/kinetochore structure and function, hMis18alpha, hMis18beta, and M18BP1, the complex of which is accumulated specifically at the telophase-G1 centromere. We provide evidence that such centromeric localization of hMis18 is essential for the subsequent recruitment of de novo-synthesized CENP-A. If any of the three is knocked down by RNAi, centromere recruitment of newly synthesized CENP-A is rapidly abolished, followed by defects such as misaligned chromosomes, anaphase missegregation, and interphase micronuclei. Tricostatin A, an inhibitor to histone deacetylase, suppresses the loss of CENP-A recruitment to centromeres in hMis18alpha RNAi cells. Telophase centromere chromatin may be primed or licensed by the hMis18 complex and RbAp46/48 to recruit CENP-A through regulating the acetylation status in the centromere.

Adaptor Proteins, Signal Transducing↗
PubMed2006

CDC2 phosphorylation of the fission yeast dis1 ensures accurate chromosome segregation.

Shortened kinetochore microtubules take separated chromatids to the opposing spindle poles in anaphase. Fission yeast Dis1 belongs to the Dis1/XMAP215/TOG family that is required for proper microtubule dynamics. Here, we report that Dis1is regulated by Cdc2 phosphorylation and that this mitotic phosphorylation ensures the fidelity of chromosome segregation. Whereas mutants Dis1(6A) and Dis1(6E) that substitute all of the six Cdc2 sites for Ala or Glu, respectively, produce colonies at 22 degrees C-36 degrees C, Dis1(6A) but not Dis1(6E) loses a minichromosome and reveals aberrant chromosome segregation at significant frequencies. Dis1(WT) is recruited to two regions of the mitotic spindle: kinetochores (possibly also kinetochore microtubules) in metaphase and the pole-to-pole microtubule lattice in anaphase. Mutant Dis1(6E) preferentially binds to metaphase kinetochores, whereas Dis1(6A), which is located along microtubules, fails in its accumulation at kinetochores. Dis1(6A) displays synthetic lethality with the mis12-537, which is a mutant that compromises kinetochore function. Dis1(6E) mimics the Cdc2-phosphorylated form of Dis1(WT), whereas Dis1(6A) can partially rescue the phenotype resulting form deletion of Mtc1/Alp14, another XMAP215-like protein. In anaphase, dephosphorylated Dis1 and Dis1(6A), but not Dis1(6E), move to the spindle microtubule lattice near the SPBs. Cdc2 thus directly phosphorylates Dis1, and this phosphorylation regulates Dis1 localization in both metaphase and anaphase and ensures high-fidelity segregation.

CDC2 Protein Kinase↗
PubMed2006

Fission yeast MAP kinase is required for the increased securin-separase interaction that rescues separase mutants under stresses.

Sister chromatid separation requires two steps of proteolysis. Securin, the chaperon and inhibitor of separase, is destructed in anaphase after polyubiquitination, and resulting activated separase cleaves the cohesin subunit Scc1/Rad21. Fission yeast securin/Cut2 and separase/Cut1 that form the complex are essential for viability and a number of temperature-sensitive (ts) mutants have been isolated. We here report that the stresses such as 1.2 M sorbitol, 0.6 M KCl and 0.1 M CaCl(2) in the medium suppress the ts phenotypes of all the cut1 mutants and two of the three cut2 mutants examined. This unexpected finding led us to study how the ts phenotypes of cut1 and cut2 mutants were rescued by the increased stresses. The stresses caused a temporal arrest in the cell number increase, and this arrest was dependent on Spc1/Sty1 but not Rad3 and Mad2. During the 2-3 hr arrested period that occurred prior to the restart of division cycle, the level of securin dramatically increased, apparently accompanying the increased complex formation with mutant separase protein. Securin bound to separase was hyperphosphorylated. The stresses could not rescue the indestructible Cut2 and Rad21 mutants. We postulate that the stresses produce the hyperchaperonic form of Cut2 that can rescue separase mutations.

Calcium Chloride↗
PubMed2006

Cdc48 is required for the stability of Cut1/separase in mitotic anaphase.

Separase, a large protease essential for sister chromatid separation, cleaves the cohesin subunit Scc1/Rad21 during anaphase and leads to dissociation of the link between sister chromatids. Securin, a chaperone and inhibitor of separase, is ubiquitinated by APC/cyclosome, and degraded by 26S proteasome in anaphase. Cdc48/VCP/p97, an AAA ATPase, is involved in a variety of cellular activities, many of which are implicated in the proteasome-mediated degradation. We previously reported that temperature-sensitive (ts) fission yeast Schizosaccharomyces pombe cdc48 mutants were suppressed by multicopy plasmid carrying the cut1(+)/separase gene and that the defective mitotic phenotypes of cut1 and cdc48 were similar. We here describe characterizations of Cdc48 mutant protein and the role of Cdc48 in sister chromatid separation. Mutant residue resides in the conserved D1 domain within the central hole of hexamer, while Cdc48 mutant protein possesses the ATPase activity. Consistent with the phenotypic similarity and the rescue of cdc48 mutant by overproduced Cut1/separase, the levels of Cut1 and also Cut2 are diminished in cdc48 mutant. We show that the stability of Cut1 during anaphase requires Cdc48. Cells lose viability during the traverse of anaphase in cdc48 mutant cells. Cdc48 may protect Cut1/separase and Cut2/securin against the instability during polyubiquitination and degradation in the metaphase-anaphase transition.

Adenosine Triphosphatases↗
PubMed2006

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation.

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

Amino Acid Sequence↗
PubMed2006

Characterization of Schistosoma mansoni Sds homologue, a leucine-rich repeat protein that interacts with protein phosphatase type 1 and interrupts a G2/M cell-cycle checkpoint.

The suppressor of the dis2 mutant (sds22+) has been shown to be an essential regulator in cell division of fission and budding yeast where its deletion causes mitotic arrest. Its role seems to take place through the activation of PP1 (protein phosphatase type 1) in Schizosaccharomyces pombe. In the trematode Schistosoma mansoni, we have identified the Sds22 homologue (SmSds), and the PP1 (SmPP1). We showed by using a GST (glutathione S-transferase) pull-down assay that the SmSds gene product interacts with SmPP1 and that the SmSds-SmPP1 complex is present in parasite extracts. Furthermore, we observed that SmSds inhibited PP1 activity. Functional studies showed that the microinjection of SmSds into Xenopus oocytes interacted with the Xenopus PP1 and disrupted the G2/M cell-cycle checkpoint by promoting progression to GVBD (germinal vesicle breakdown). Similar results showing the appearance of GVBD were observed when oocytes were treated with anti-PP1 antibodies. Taken together, these observations suggest that SmSds can regulate the cell cycle by binding to PP1.

Amino Acid Motifs↗
PubMed2006

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase.

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

Animals↗
PubMed2006

Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance.

Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.

Calcium-Binding Proteins↗
PubMed2006

Distinct modes of DNA damage response in S. pombe G0 and vegetative cells.

Upon nitrogen-starvation, mostly G2 vegetative (VE) fission yeast cells promote two rounds of division and enter the G0 state with 1C DNA via an uncommitted G1. Whilst G0 cells are permanently arrested, they keep viability through recycling the intracellular nitrogen. We here show that, whilst the DNA damages are efficiently repaired in G0 cells, neither Chk1 activation nor Cdc2 implication for Crb2 (53BP1 like) do not occur. ATR-like Rad3 and non-hyperphosphorylated Crb2 participate the repair processes in G0 cells that are more sensitive to UV and gamma-ray than in VE cells. The sensitivity like in VE cells is restored after replication in the nitrogen-replenished medium, suggesting that the damage hyper-sensitive nature of G0 cells is due to the error-prone repair for single DNA duplex chromosome. The double-strand break (DSB) repair in G0 cells required Pku80, one of non-homologous end joining (NHEJ) proteins. S. pombe G0 cells upon DNA damages thus respond distinctively from VE cells in regard with regulation of checkpoint proteins and the mode of repair that is dependent upon the use of NHEJ.

Bacterial Proteins↗
PubMed2006

Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase.

Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation.

Amino Acid Sequence↗
PubMed2005

Regulation of nuclear proteasome by Rhp6/Ubc2 through ubiquitination and destruction of the sensor and anchor Cut8.

While proteasome is central to the degradation of cellular ubiquitinated proteins, the control of its nuclear function is barely understood. Here we show that the fission yeast ubiquitin-conjugating Rhp6/Ubc2/Rad6 and ligating enzymes Ubr1 are responsible for nuclear enrichment of proteasome through the function of Cut8, a nuclear envelope protein. Cut8 is an Rhp6 substrate that physically interacts with and tethers proteasome. Nonubiquitinatable K-all-R Cut8 weakly interacts with proteasome and fails to enrich nuclear proteasome. Consistently, the nuclear enrichment of proteasome also fails in rhp6 and ubr1 null mutants. Further, cut8 null and cut8 K-all-R mutants are hypersensitive to DNA damage, probably due to the paucity of nuclear proteasome. Thus, Rhp6 enhances the retention of nuclear proteasome through regulating Cut8. The short-lived nature of Cut8 is crucial for feedback enrichment of the proteasome within the nucleus. This is likely to be a conserved mechanism as we describe a Cut8 homolog in flies.

Amino Acid Sequence↗
PubMed2005

Basic mechanism of eukaryotic chromosome segregation.

We now have firm evidence that the basic mechanism of chromosome segregation is similar among diverse eukaryotes as the same genes are employed. Even in prokaryotes, the very basic feature of chromosome segregation has similarities to that of eukaryotes. Many aspects of chromosome segregation are closely related to a cell cycle control that includes stage-specific protein modification and proteolysis. Destruction of mitotic cyclin and securin leads to mitotic exit and separase activation, respectively. Key players in chromosome segregation are SMC-containing cohesin and condensin, DNA topoisomerase II, APC/C ubiquitin ligase, securin-separase complex, aurora passengers, and kinetochore microtubule destabilizers or regulators. In addition, the formation of mitotic kinetochore and spindle apparatus is absolutely essential. The roles of principal players in basic chromosome segregation are discussed: most players have interphase as well as mitotic functions. A view on how the centromere/kinetochore is formed is described.

Adenosine Triphosphatases↗
PubMed2004

A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1.

Defects in kinetochore proteins often lead to aneuploidy and cancer. Mis12-Mtw1 is a conserved, essential kinetochore protein family. Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells. Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins. Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12-Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi). Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1alpha and HP1gamma. Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8. Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis.

Base Sequence↗
PubMed2004

Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres.

Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1. Here we report identification of five fission yeast centromere proteins, Mis14-18. Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres. In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway. Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6. Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres. Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively. RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.

Acetylation↗
PubMed2004

Separase-mediated cleavage of cohesin at interphase is required for DNA repair.

Sister chromatids are held together by cohesins. At anaphase, separase is activated by degradation of its inhibitory partner, securin. Separase then cleaves cohesins, thus allowing sister chromatid separation. Fission yeast securin (Cut2) has destruction boxes and a separase (Cut1) interaction site in the amino and carboxyl terminus, respectively. Here we show that securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation. The cut2(EA2) mutant is defective in the repair of ultraviolet damage lesions, although the DNA damage checkpoint is activated normally. In double mutant analysis of ultraviolet sensitivity, checkpoint kinase chk1 (ref. 9) and excision repair rad13 (ref. 10) mutants were additive with cut2(EA2), whereas recombination repair rhp51 (ref. 11) and cohesin subunit rad21 (ref. 12) mutants were not. Cohesin was hyper-modified on ultraviolet irradiation in a Rad3 kinase-dependent way. Experiments using either mutant cohesin that cannot be cleaved by separase or a protease-dead separase provide evidence that this DNA repair function of securin-separase acts through the cleavage of cohesin. We propose that the securin-separase complex might aid DNA repair by removing local cohesin in interphase cells.

Amino Acid Sequence↗
PubMed2004

The nucleolus is involved in mRNA export from the nucleus in fission yeast.

To elucidate the mechanism of mRNA export from the nucleus, we isolated five novel temperature-sensitive mutants (ptr7 to ptr11) that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr11 mutation was found in the top2(+) gene encoding DNA topoisomerase II. In addition to the nuclear accumulation of poly(A)(+) RNA, ptr11 exhibited the cut (cell untimely torn) phenotype at the nonpermissive temperature, like the previously isolated mutant, ptr4. In these two mutants, cytokinesis occurred without prior nuclear division, resulting in cleavage of the undivided nuclei by the septum. To investigate the relationship between mRNA export defects and the cut phenotype observed in ptr4 and ptr11, we analyzed 11 other mutants displaying the cut phenotype and found that all these tested mutants accumulate poly(A)(+) mRNA in the aberrantly cleaved nuclei. Interestingly, nuclear accumulation of poly(A)(+) mRNA was observed only in the anucleolate nuclei produced by aberrant cytokinesis. In addition, nuc1, the S. pombe mutant exhibiting a collapsed nucleolus, trapped poly(A)(+) mRNA in the nucleolar region at the nonpermissive temperature. In ptr11 and nuc1, mRNA transcribed from the intron-containing TBP gene showed nuclear accumulation, but not transcripts from the intron-less TBP cDNA, suggesting that the export pathway differs between the spliced and unspliced TBP mRNAs. These findings support the notion that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus.

Cell Nucleolus↗