Updated on 2022/01/19

写真a

 
SATO, Masamitsu
 
Affiliation
Faculty of Science and Engineering, School of Advanced Science and Engineering
Job title
Professor
Profile
細胞や組織において微小管などの細胞骨格はどのように形態を作り、どのような機能を担っているのかについて研究を展開しています。
特に、微小管形成の分子メカニズム、組織における微小管の形態と機能の制御、さらに細胞周期における遺伝子発現と細胞の運命決定の解析を研究しています。

Concurrent Post

  • Affiliated organization   Global Education Center

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

Research Institute

  • 2020
    -
    2022

    理工学術院総合研究所   兼任研究員

Education

  •  
    -
    2001

    University of Tokyo   Graduate School of Science   Department of Biophysics and Biochemistry  

  •  
    -
    1998

    University of Tokyo   Graduate School of Science   Department of Biophysics and Biochemistry  

  •  
    -
    1996

    University of Tokyo   Faculty of Science   Department of Biophysics and Biochemistry  

Degree

  • University of Tokyo   Ph.D.

Research Experience

  • 2020.10
    -
    Now

    大隅基礎科学創成財団 酵母コンソーシアムフェロー

  • 2018.04
    -
    Now

    Waseda University   Department of Life Science and Medical Bioscience   Professor

  • 2013.04
    -
    2018.03

    Waseda University   Department of Life Science and Medical Bioscience   Associate Professor

  • 2009.09
    -
     

    ~2012.3: PRESTO researcher, JST

  • 2006.09
    -
     

    ~2013.3: Assistant Professor, Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo

  • 2006.04
    -
     

    ~2006.9: JSPS Postdoctoral Fellow for Research Abroad

  • 2002.04
    -
     

    ~2006.9: Postdoctoral Fellow, Cancer Research UK, London Research Institute

  • 2001.04
    -
     

    ~2002.3: Research Fellow, University of Tokyo

▼display all

Professional Memberships

  •  
     
     

    The Molecular Biology Society of Japan

  •  
     
     

    Japan Society for Cell Biology

 

Research Areas

  • Genetics

  • Molecular biology

  • Cell biology

Research Interests

  • gene expression

  • oocyte

  • Single Cell Analysis

  • cell division

  • mitosis

  • meiosis

  • Chemical Genetics

  • Transcriptome (Gene Expression Profile)

  • Fission Yeast

  • Nucleosome

  • Centromere

  • Nuclear Dynamics

  • Mitosis and Meiosis

  • Epithelial Tissue

  • Microtyubule

  • Cell Cycle

  • Cytoskeleton

▼display all

Papers

  • Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating.

    Hiroko Saito, Fumiko Matsukawa-Usami, Toshihiko Fujimori, Toshiya Kimura, Takahiro Ide, Takaki Yamamoto, Tatsuo Shibata, Kenta Onoue, Satoko Okayama, Shigenobu Yonemura, Kazuyo Misaki, Yurina Soba, Yasutaka Kakui, Masamitsu Sato, Mika Toya, Masatoshi Takeichi

    Molecular biology of the cell   32 ( 20 ) ar12  2021.10  [Refereed]  [International journal]

     View Summary

    Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

    DOI PubMed

  • Wake-up alarm: virtual time-lapse gene expression landscape illuminates mechanisms underlying dormancy breaking of germinating spores

    Hayato Tsuyuzaki, Ryosuke Ujiie, Masamitsu Sato

    Current Genetics   67 ( 4 ) 519 - 534  2021.08  [Refereed]  [Invited]

    Authorship:Last author, Corresponding author

    DOI

  • Simplification of nutritional conditions in transformation procedures for genome editing with the CRISPR/Cas9 system for fission yeast.

    Seibun Li, Mika Toya, Masamitsu Sato

    Gene   784   145595 - 145595  2021.06  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    CRISPR/Cas9 is a powerful tool for genome editing. Several studies have been conducted to take the benefit of the versatile tool in the fission yeast Schizosaccharomyces pombe. However, the protocols for the CRISPR/Cas9 system proposed in previous studies are complicated in culture conditions compared to traditional genome editing methods. In this study, we introduced vectors for expression of sgRNA as well as Cas9, which employ natMX6 and bsdMX6 dominant selection markers. Using these materials, we examined nutritional conditions of cell cultures and found that nitrogen depletion introduced in previous methods does not affect the efficiency of genome editing. We found that bsdMX6-based plasmids enable us to skip any recovery steps before plating onto medium containing blasticidin S, unlike other antibiotic resistance selection markers. We thus propose easier transformation procedures with natMX6 and particularly bsdMX6 markers. We also simulate prescreening of mutants by genotyping with DNA endonucleases or proofreading PCR instead of relying on existing knowledge of mutant phenotypes. These materials and methods assist easy construction of S. pombe strains using CRISPR/Cas9, thereby accelerating seamless introduction of CRISPR/Cas9 to S. pombe researchers.

    DOI PubMed

  • Dual Impact of a Benzimidazole Resistant β-Tubulin on Microtubule Behavior in Fission Yeast

    Mamika Minagawa, Minamo Shirato, Mika Toya, Masamitsu Sato

    Cells   10 ( 5 ) 1042 - 1042  2021.04  [Refereed]

    Authorship:Last author, Corresponding author

    DOI

  • Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

    Yuto Mitsuhata, Takaya Abe, Kazuyo Misaki, Yuna Nakajima, Keita Kiriya, Miwa Kawasaki, Hiroshi Kiyonari, Masatoshi Takeichi, Mika Toya, Masamitsu Sato

    Scientific reports   11 ( 1 ) 5857 - 5857  2021.03  [Refereed]  [International journal]

    Authorship:Last author

     View Summary

    Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

    DOI PubMed

  • Tell the Difference Between Mitosis and Meiosis: Interplay Between Chromosomes, Cytoskeleton, and Cell Cycle Regulation.

    Masamitsu Sato, Yasutaka Kakui, Mika Toya

    Frontiers in cell and developmental biology   9   660322 - 660322  2021  [Refereed]  [International journal]

    Authorship:Lead author, Corresponding author

     View Summary

    Meiosis is a specialized style of cell division conserved in eukaryotes, particularly designed for the production of gametes. A huge number of studies to date have demonstrated how chromosomes behave and how meiotic events are controlled. Yeast substantially contributed to the understanding of the molecular mechanisms of meiosis in the past decades. Recently, evidence began to accumulate to draw a perspective landscape showing that chromosomes and microtubules are mutually influenced: microtubules regulate chromosomes, whereas chromosomes also regulate microtubule behaviors. Here we focus on lessons from recent advancement in genetical and cytological studies of the fission yeast Schizosaccharomyces pombe, revealing how chromosomes, cytoskeleton, and cell cycle progression are organized and particularly how these are differentiated in mitosis and meiosis. These studies illuminate that meiosis is strategically designed to fulfill two missions: faithful segregation of genetic materials and production of genetic diversity in descendants through elaboration by meiosis-specific factors in collaboration with general factors.

    DOI PubMed

  • Mechanisms for Cellular Wake-up in Fission Yeast Revealed by Single-cell RNA-seq

    Hayato Tsuyuzaki, Masahito Hosokawa, Haruko Takeyama, Masamitsu Sato

    Bioscience and Industry (B&I)   78 ( 6 ) 507 - 509  2020.11  [Invited]

    Authorship:Last author, Corresponding author

  • Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking in fission yeast.

    Hayato Tsuyuzaki, Masahito Hosokawa, Koji Arikawa, Takuya Yoda, Naoyuki Okada, Haruko Takeyama, Masamitsu Sato

    Nature communications   11 ( 1 ) 1265 - 1265  2020.03  [Refereed]  [International journal]

    Authorship:Last author, Corresponding author

     View Summary

    How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.

    DOI PubMed

  • Kinetochore-mediated outward force promotes spindle pole separation in fission yeast.

    Shirasugi Y, Sato M

    Molecular biology of the cell   30 ( 22 ) 2802 - 2813  2019.10  [Refereed]

    Authorship:Last author, Corresponding author

    DOI PubMed

  • Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    Yasutaka Kakui, Tomonari Sunaga, Kunio Arai, James Dodgson, Liang Ji, Attila Csikasz-Nagy, Rafael Carazo-Salas, Masamitsu Sato

    OPEN BIOLOGY   5 ( 6 )  2015.06  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy-and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a 'module', can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.

    DOI PubMed

  • The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

    Hayato Hirai, Kunio Arai, Ryo Kariyazono, Masayuki Yamamoto, Masamitsu Sato

    PLOS ONE   9 ( 11 ) e111905  2014.11  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.

    DOI

  • CDK-dependent phosphorylation of Alp7-Alp14 (TACC-TOG) promotes its nuclear accumulation and spindle microtubule assembly

    Naoyuki Okada, Takashi Toda, Masayuki Yamamoto, Masamitsu Sato

    MOLECULAR BIOLOGY OF THE CELL   25 ( 13 ) 1969 - 1982  2014.07  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7-Alp14 (transforming acidic coiled-coil-tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7-Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7-Alp14 complex to localize it to the nucleus.

    DOI

  • Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis

    Yuki Aoi, Shigehiro A. Kawashima, Viesturs Simanis, Masayuki Yamamoto, Masamitsu Sato

    OPEN BIOLOGY   4 ( 7 ) 140063  2014.07  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.

    DOI

  • Dissecting the first and the second meiotic divisions using a marker-less drug-hypersensitive fission yeast

    Yuki Aoi, Masamitsu Sato, Takashi Sutani, Katsuhiko Shirahige, Tarun M. Kapoor, Shigehiro A. Kawashima

    CELL CYCLE   13 ( 8 ) 1327 - 1334  2014.04  [Refereed]

     View Summary

    Faithful chromosome segregation during meiosis is indispensable to prevent birth defects and infertility. Canonical genetic manipulations have not been very useful for studying meiosis II, since mutations of genes involved in cell cycle regulation or chromosome segregation may affect meiosis I, making interpretations of any defects observed in meiosis II complicated. Here we present a powerful strategy to dissect meiosis I and meiosis II, using chemical inhibitors in genetically tractable model organism fission yeast (Schizosaccharomyces pombe). As various chemical probes are not active in fission yeast, mainly due to an effective multidrug resistance (MDR) response, we have recently developed a drug-hypersensitive MDR-sup strain by suppression of the key genes responsible for MDR response. We further developed the MDR-supML (marker-less) strain by deleting 7 MDR genes without commonly used antibiotic markers. The new strain makes fluorescent tagging and gene deletion much simpler, which enables effective protein visualization in varied genetic backgrounds. Using the MDR-supML strain with chemical inhibitors and live cell fluorescence microscopy, we established cell cycle arrest at meiosis I and meiosis II and examined Aurora-dependent spindle assembly checkpoint (SAC) regulation during meiosis. We found that Aurora B/Ark1 kinase activity is required for recruitment of Bub1, an essential SAC kinase, to unattached kinetochore in prometaphase I and prometaphase II as in mitosis. Thus, Aurora's role in SAC activation is likely conserved in mitosis, meiosis I, and meiosis II. Together, our MDR-supML strain will be useful to dissect complex molecular mechanisms in mitosis and 2 successive meiotic divisions.

    DOI

  • Microtubules and Alp7-Alp14 (TACC-TOG) reposition chromosomes before meiotic segregation

    Yasutaka Kakui, Masamitsu Sato, Naoyuki Okada, Takashi Toda, Masayuki Yamamoto

    Nature Cell Biology   15 ( 7 ) 786 - 796  2013.07  [Refereed]

    Authorship:Corresponding author

     View Summary

    Tethering kinetochores at spindle poles facilitates their efficient capture and segregation by microtubules at mitotic onset in yeast. During meiotic prophase of fission yeast, however, kinetochores are detached from the poles, which facilitates meiotic recombination but may cause a risk of chromosome mis-segregation during meiosis. How cells circumvent this dilemma remains unclear. Here we show that an extensive microtubule array assembles from the poles at meiosis I onset and retrieves scattered kinetochores towards the poles to prevent chromosome drift. Moreover, the microtubule-associated protein complex Alp7-Alp14 (the fission yeast orthologues of mammalian TACC-TOG) is phosphorylated by Polo kinase, which promotes its meiosis-specific association to the outer kinetochore complex Nuf2-Ndc80 of scattered kinetochores, thereby assisting in capturing remote kinetochores. Although TOG was recently characterized as a microtubule polymerase, Dis1 (the other TOG orthologue in fission yeast), together with the Dam1 complex, plays a role in microtubule shortening to pull kinetochores polewards. Thus, microtubules and their binding proteins uniquely reconstitute chromosome configuration during meiosis. © 2013 Macmillan Publishers Limited. All rights reserved.

    DOI PubMed

  • Spatial segregation of polarity factors into distinct cortical clusters is required for cell polarity control

    James Dodgson, Anatole Chessel, Miki Yamamoto, Federico Vaggi, Susan Cox, Edward Rosten, David Albrecht, Marco Geymonat, Attila Csikasz-Nagy, Masamitsu Sato, Rafael E. Carazo-Salas

    NATURE COMMUNICATIONS   4  2013.05  [Refereed]

     View Summary

    Cell polarity is regulated by evolutionarily conserved polarity factors whose precise higher-order organization at the cell cortex is largely unknown. Here we image frontally the cortex of live fission yeast cells using time-lapse and super-resolution microscopy. Interestingly, we find that polarity factors are organized in discrete cortical clusters resolvable to similar to 50-100 nm in size, which can form and become cortically enriched by oligomerization. We show that forced co-localization of the polarity factors Tea1 and Tea3 results in polarity defects, suggesting that the maintenance of both factors in distinct clusters is required for polarity. However, during mitosis, their co-localization increases, and Tea3 helps to retain the cortical localization of the Tea1 growth landmark in preparation for growth reactivation following mitosis. Thus, regulated spatial segregation of polarity factor clusters provides a means to spatio-temporally control cell polarity at the cell cortex. We observe similar clusters in Saccharomyces cerevisiae and Caenorhabditis elegans cells, indicating this could be a universal regulatory feature.

    DOI

  • Spindle pole body components are reorganized during fission yeast meiosis

    Midori Ohta, Masamitsu Sato, Masayuki Yamamoto

    MOLECULAR BIOLOGY OF THE CELL   23 ( 10 ) 1799 - 1811  2012.05  [Refereed]

    Authorship:Corresponding author

     View Summary

    During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.

    DOI

  • Interpolar microtubules are dispensable in fission yeast meiosis II

    Takashi Akera, Masamitsu Sato, Masayuki Yamamoto

    NATURE COMMUNICATIONS   3   695  2012.02  [Refereed]

     View Summary

    The mitotic spindle consists of two types of microtubules. Dynamic kinetochore microtubules capture kinetochores, whereas stable interpolar microtubules serve as the structural backbone that connects the two spindle poles. Both have been believed to be indispensable for cell division in eukaryotes. Here we demonstrate that interpolar microtubules are dispensable for the second division of meiosis in fission yeast. Even when interpolar microtubules are disrupted by a microtubule-depolymerizing drug, spindle poles separate and chromosomes segregate poleward in second division of meiosis in most zygotes, producing viable spores. The forespore membrane, which encapsulates the nucleus in second division of meiosis and is guided by septins and the leading-edge proteins, is responsible for carrying out meiotic events in the absence of interpolar microtubules. Furthermore, during physiological second division of meiosis without microtubule perturbation, the forespore membrane assembly contributes structurally to spindle pole separation and nuclear division, generating sufficient force for spindle pole separation and subsequent events independently of interpolar microtubules.

    DOI

  • SCF Ensures Meiotic Chromosome Segregation Through a Resolution of Meiotic Recombination Intermediates

    Shin-ya Okamoto, Masamitsu Sato, Takashi Toda, Masayuki Yamamoto

    PLOS ONE   7 ( 1 ) e0030622  2012.01  [Refereed]

     View Summary

    The SCF (Skp1-Cul1-F-box) complex contributes to a variety of cellular events including meiotic cell cycle control, but its function during meiosis is not understood well. Here we describe a novel function of SCF/Skp1 in meiotic recombination and subsequent chromosome segregation. The skp1 temperature-sensitive mutant exhibited abnormal distribution of spindle microtubules in meiosis II, which turned out to originate from abnormal bending of the spindle in meiosis I. Bent spindles were reported in mitosis of this mutant, but it remained unknown how SCF could affect spindle morphology. We found that the meiotic bent spindle in skp1 cells was due to a hypertension generated by chromosome entanglement. The spindle bending was suppressed by inhibiting double strand break (DSB) formation, indicating that the entanglement was generated by the meiotic recombination machinery. Consistently, Rhp51/Rad51-Rad22/Rad52 foci persisted until meiosis I in skp1 cells, proving accumulation of recombination intermediates. Intriguingly bent spindles were also observed in the mutant of Fbh1, an F-box protein containing the DNA helicase domain, which is involved in meiotic recombination. Genetic evidence suggested its cooperation with SCF/Skp1. Thus, SCF/Skp1 together with Fbh1 is likely to function in the resolution of meiotic recombination intermediates, thereby ensuring proper chromosome segregation.

    DOI

  • Nuclear Compartmentalization Is Abolished during Fission Yeast Meiosis

    Kunio Arai, Masamitsu Sato, Kayoko Tanaka, Masayuki Yamamoto

    CURRENT BIOLOGY   20 ( 21 ) 1913 - 1918  2010.11  [Refereed]

     View Summary

    In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells [1]. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle [2]. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.

    DOI

  • Visualization of fluorescence-tagged proteins in fission yeast: The analysis of mitotic spindle dynamics using GFP-tubulin under the native promoter

    Masamitsu Sato, Mika Toya, Takashi Toda

    Methods in Molecular Biology   545   185 - 203  2009

     View Summary

    Mitotic spindle microtubules pull chromosomes toward each pole to generate two daughter cells. Proper spindle formation and function are required to prevent tumorigenesis and cell death. The fission yeast Schizosaccharomyces pombe has been widely used as a model organism to understand the molecular mechanism of mitosis due to its convenience in genetics, molecular biology, and cell biology. The development of fluorescent protein systems and microscopy enables us to investigate the "true" behavior of proteins in living fission yeast cells using a strain with a fluorescence-tagged gene under its native promoter. In this way the level of expression of tagged protein is similar to the level of wild-type nontagged protein. In this chapter we illustrate standard methods to generate strains expressing fluorescently tagged proteins and to observe them under the microscope. Specifically, we introduce a GFP-tubulin strain to analyze the dynamic behavior of spindle microtubules. Observation of GFP-tubulin under its native promoter has illuminated the process of kinetochore-microtubule attachment process in fission yeast. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

    DOI PubMed

  • gamma-Tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast

    Mika Toya, Masamitsu Sato, Uta Haselmann, Kazuhide Asakawa, Damian Brunner, Claude Antony, Takashi Toda

    NATURE CELL BIOLOGY   9 ( 6 ) 646 - U55  2007.06  [Refereed]

     View Summary

    The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail(1-4), it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the gamma-tubulin complex(5) (gamma-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with gamma-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.

    DOI

  • New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe

    M Sato, S Dhut, T Toda

    YEAST   22 ( 7 ) 583 - 591  2005.05  [Refereed]

     View Summary

    We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (Bahler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of proyein functions in fission yeast. Copyright (c) 2005 John Wiley & Sons, Ltd.

    DOI

  • Interdependency of fission yeast Alp14/T0G and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation

    M Sato, L Vardy, MA Garcia, N Koonrugsa, T Toda

    MOLECULAR BIOLOGY OF THE CELL   15 ( 4 ) 1609 - 1622  2004.04  [Refereed]

     View Summary

    The Dis1/TOG family plays a pivotal role in microtubule organization. In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation. Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles. Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles. Alp14 localization to these sites is fully dependent upon Alp7. Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles. Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7. Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization. Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14. Alp7 may be a functional homologue of animal TACC. Our results shed light on an interdependent relationship between Alp14/TOG and Alp7. We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules.

    DOI

  • Deletion of Mia1/Alp7 activates Mad2-dependent spindle assembly checkpoint in fission yeast

    M Sato, N Koonrugsa, T Toda, L Vardy, S Tournier, JBA Millar

    NATURE CELL BIOLOGY   5 ( 9 ) 764 - 766  2003.09  [Refereed]

    DOI

  • 14-3-3 protein interferes with the binding of RNA to the phosphorylated form of fission yeast meiotic regulator Mei2p

    M Sato, Y Watanabe, Y Akiyoshi, M Yamamoto

    CURRENT BIOLOGY   12 ( 2 ) 141 - 145  2002.01  [Refereed]

     View Summary

    The switch from mitosis to meiosis is controlled by the Pat1(Ran1) kinase-Mei2p system in Schizosaccharomyces pombe [1]. Mei2p promotes both premeiotic DNA synthesis and meiosis 1, and its RNA binding ability is essential for these two processes [2,3]. Mei2p forms a dot structure in the nucleus prior to meiosis 1, aided by a specific RNA species named "meiRNA" [3-5]. Pat1 kinase phosphorylates Mei2p on two positions and downregulates its activity [4]. Pat1 kinase undergoes inactivation under meiotic conditions, as a result of the production of a tethering pseudosubstrate Mei3p [6], and accumulation of the unphosphorylated form of Mei2p commits cells to meiosis [4]. However, the mechanism of how phosphorylation of Mei2p suppresses its activity to induce meiosis remains largely unknown. Here we show that S. pombe Rad24p, a 14-3-3 protein, functions as a negative factor for meiosis by antagonizing the function of meiRNA to promote the formation of a nuclear Mei2p dot. Rad24p binds preferentially to Mei2p phosphorylated by Pat1 kinase. It inhibits association of meiRNA to the phosphorylated form of Mei2p but not to the unphosphorylated form in vitro. We speculate that Rad24p, bound tightly to the residues phosphorylated by Pat1 kinase, may mask the RNA recognition motifs on Mei2p. This model will explain, at least partly, why phosphorylation by Pat1 kinase inhibits the meiosis-inducing activity of Mei2p.

    DOI

  • CLASP promotes microtubule bundling in metaphase spindle independently of Ase1/PRC1 in fission yeast.

    Ebina H, Ji L, Sato M

    Biology open    2019.10  [Refereed]

    Authorship:Last author, Corresponding author

    DOI PubMed

  • Module-based systematic construction of plasmids for episomal gene expression in fission yeast

    Keita Kiriya, Hayato Tsuyuzaki, Masamitsu Sato

    GENE   637   14 - 24  2017.12  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    The fission yeast Schizosaccharomyces pombe is a powerful model organism for cell biology and molecular biology, as genetic manipulation is easily achieved. Introduction of exogenous genes cloned in episomal plasmids into yeast cells can be done through well-established transformation methods. For expression of genes in S. pombe cells, the multi-copy plasmid pREP1 and its derivatives, including pREP41 and pREP81, have been widely used as vectors. Although recent advancement of technology brought a number of useful genetic elements such as new promoters, selection marker genes and fluorescent protein tags, introduction of those elements into conventional pREP1 requires a large commitment of both time and effort because cloning procedures need to be repeated until the final products are constructed. Here, we introduce materials and methods to construct many pREP1-type plasmids easily and systematically using the Golden Gate shuffling method, which enables one-step ligation of many DNA fragments into a plasmid. These materials and methods support creation of expression plasmids employing a variety of novel genetic elements, which will further facilitate genetic studies using S. pombe.

    DOI

  • Differentiating the roles of microtubule-associated proteins at meiotic kinetochores during chromosome segregation

    Yasutaka Kakui, Masamitsu Sato

    CHROMOSOMA   125 ( 2 ) 309 - 320  2016.06  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Meiosis is a specialised cell division process for generating gametes. In contrast to mitosis, meiosis involves recombination followed by two consecutive rounds of cell division, meiosis I and II. A vast field of research has been devoted to understanding the differences between mitotic and meiotic cell divisions from the viewpoint of chromosome behaviour. For faithful inheritance of paternal and maternal genetic information to offspring, two events are indispensable: meiotic recombination, which generates a physical link between homologous chromosomes, and reductional segregation, in which homologous chromosomes move towards opposite poles, thereby halving the ploidy. The cytoskeleton and its regulators play specialised roles in meiosis to accomplish these divisions. Recent studies have shown that microtubule-associated proteins (MAPs), including tumour overexpressed gene (TOG), play unique roles during meiosis. Furthermore, the conserved mitotic protein kinase Polo modulates MAP localisation in meiosis I. As Polo is a well-known regulator of reductional segregation in meiosis, the evidence suggests that Polo constitutes a plausible link between meiosis-specific MAP functions and reductional segregation. Here, we review the latest findings on how the localisation and regulation of MAPs in meiosis differ from those in mitosis, and we discuss conservation of the system between yeast and higher eukaryotes.

    DOI

  • Systems level understanding of cell polarity regulation

    Attila Csikasz-Nagy, Federico Vaggi, James Dodgson, Anatole Chessel, Marco Geymonat, Marco Giordan, Kunio Arai, Masamitsu Sato, Rafael Edgardo Carazo Salas

    YEAST   32   S247 - S247  2015.09  [Refereed]

  • Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

    Naoyuki Okada, Masamitsu Sato

    CELLS   4 ( 3 ) 406 - 426  2015.09  [Refereed]

    Authorship:Last author, Corresponding author

     View Summary

    Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells.

    DOI PubMed

  • Mad1 promotes chromosome congression by anchoring a kinesin motor to the kinetochore

    Takashi Akera, Yuhei Goto, Masamitsu Sato, Masayuki Yamamoto, Yoshinori Watanabe

    NATURE CELL BIOLOGY   17 ( 9 ) 1124 - +  2015.09  [Refereed]

     View Summary

    For proper partitioning of genomes in mitosis, all chromosomes must be aligned at the spindle equator before the onset of anaphase. The spindle assembly checkpoint (SAC) monitors this process, generating a 'wait anaphase' signal at unattached kinetochores of misaligned chromosomes. However, the link between SAC activation and chromosome alignment is poorly understood. Here we show that Mad1, a core SAC component, plays a hitherto concealed role in chromosome alignment. Protein-protein interaction screening revealed that fission yeast Mad1 binds the plus-end-directed kinesin-5 motor protein Cut7 (Eg5 homologue), which is generally thought to promote spindle bipolarity. We demonstrate that Mad1 recruits Cut7 to kinetochores of misaligned chromosomes and promotes chromosome gliding towards the spindle equator. Similarly, human Mad1 recruits another kinetochore motor CENP-E, revealing that Mad1 is the conserved dual-function protein acting in SAC activation and chromosome gliding. Our results suggest that the mitotic checkpoint has co-evolved with a mechanism to drive chromosome congression.

    DOI PubMed

  • Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast

    Imene B. Bouhlel, Midori Ohta, Adeline Mayeux, Nicole Bordes, Florent Dingli, Jerome Boulanger, Guilhem Velve Casquillas, Damarys Loew, Phong T. Tran, Masamitsu Sato, Anne Paoletti

    JOURNAL OF CELL SCIENCE   128 ( 8 ) 1481 - 1493  2015.04  [Refereed]

     View Summary

    Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.

    DOI

  • Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

    Ngang Heok Tang, Naoyuki Okada, Chii Shyang Fong, Kunio Arai, Masamitsu Sato, Takashi Toda

    FEBS Letters   588 ( 17 ) 2814 - 2821  2014.08  [Refereed]

     View Summary

    The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI PubMed

  • Functional significance of nuclear export and mRNA binding of meiotic regulator Spo5 in fission yeast

    Naoyuki Togashi, Akira Yamashita, Masamitsu Sato, Masayuki Yamamoto

    BMC MICROBIOLOGY   14 ( 1 ) 188  2014.07  [Refereed]

     View Summary

    Background: Meiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not fully understood. The fission yeast spo5(+) gene encodes a meiosis-specific RNA-binding protein, which is required for the progression of meiosis II and spore formation. However, the target RNA molecules of Spo5 are yet to be identified. Characterization of meiosis-specific RNA-binding proteins will provide insight into how post-transcriptional regulation influence gene expression during sexual differentiation.
    Results: To assess the functional significance of RNA-recognition motifs (RRMs) of Spo5, we constructed a series of new spo5 truncated mutants and previously reported spo5 missense mutants. In addition, we isolated novel spo5 missense mutants. The phenotypic characteristics of these mutants indicated that the RRMs are essential for both the localization and function of the protein. Interestingly, Spo5 is exported from the nucleus to the cytoplasm via the Rae1-dependent mRNA export pathway, but is unlikely to be involved in global mRNA export. Furthermore, cytoplasmic localization of Spo5 is important for its function, which suggests the involvement of Spo5 in post-transcriptional regulation. We identified pcr1(+) mRNA as one of the critical targets of Spo5. The pcr1(+) gene encodes an activating transcription factor/cAMP response element binding (ATF/CREB) transcription factor family. Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.
    Conclusions: Spo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway. RRMs are necessary for this process and also for the function of Spo5 after the nuclear export. Spo5 appears to influence the activity of pcr1(+) mRNA, and the mechanism of how Spo5 stimulates the mRNA to promote the progression of meiosis II and spore formation remains an intriguing question for future research.

    DOI

  • The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast

    Mayumi Arata, Masamitsu Sato, Akira Yamashita, Masayuki Yamamoto

    GENES TO CELLS   19 ( 3 ) 225 - 238  2014.03  [Refereed]

     View Summary

    Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II.

    DOI

  • A network approach to mixing delegates at meetings

    Federico Vaggi, Tommaso Schiavinotto, Jonathan L. D. Lawson, Anatole Chessel, James Dodgson, Marco Geymonat, Masamitsu Sato, Rafael Edgardo Carazo Salas, Attila Csikasz-Nagy

    ELIFE   3   e02273  2014.02  [Refereed]

    DOI

  • Projecting cell polarity into the next decade Introduction

    Attila Csikasz-Nagy, Masamitsu Sato, Rafael E. Carazo Salas

    PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES   368 ( 1629 ) 20130001  2013.11

    DOI

  • Dynamics of SIN Asymmetry Establishment

    Archana Bajpai, Anna Feoktistova, Jun-Song Chen, Dannel McCollum, Masamitsu Sato, Rafael E. Carazo-Salas, Kathleen L. Gould, Attila Csikasz-Nagy

    PLOS COMPUTATIONAL BIOLOGY   9 ( 7 ) e1003147  2013.07  [Refereed]

     View Summary

    Timing of cell division is coordinated by the Septation Initiation Network (SIN) in fission yeast. SIN activation is initiated at the two spindle pole bodies (SPB) of the cell in metaphase, but only one of these SPBs contains an active SIN in anaphase, while SIN is inactivated in the other by the Cdc16-Byr4 GAP complex. Most of the factors that are needed for such asymmetry establishment have been already characterized, but we lack the molecular details that drive such quick asymmetric distribution of molecules at the two SPBs. Here we investigate the problem by computational modeling and, after establishing a minimal system with two antagonists that can drive reliable asymmetry establishment, we incorporate the current knowledge on the basic SIN regulators into an extended model with molecular details of the key regulators. The model can capture several peculiar earlier experimental findings and also predicts the behavior of double and triple SIN mutants. We experimentally tested one prediction, that phosphorylation of the scaffold protein Cdc11 by a SIN kinase and the core cell cycle regulatory Cyclin dependent kinase (Cdk) can compensate for mutations in the SIN inhibitor Cdc16 with different efficiencies. One aspect of the prediction failed, highlighting a potential hole in our current knowledge. Further experimental tests revealed that SIN induced Cdc11 phosphorylation might have two separate effects. We conclude that SIN asymmetry is established by the antagonistic interactions between SIN and its inhibitor Cdc16-Byr4, partially through the regulation of Cdc11 phosphorylation states.

    DOI

  • Cuf2 boosts the transcription of APC/C activator Fzr1 to terminate the meiotic division cycle

    Yuki Aoi, Kunio Arai, Masaya Miyamoto, Yuji Katsutaw, Akira Yamashita, Masamitsu Sato, Masayuki Yamamoto

    EMBO REPORTS   14 ( 6 ) 553 - 560  2013.06  [Refereed]

    Authorship:Corresponding author

     View Summary

    The number of nuclear divisions in meiosis is strictly limited to two. Although the precise mechanism remains unknown, this seems to be achieved by adjusting the anaphase-promoting complex/cyclosome (APC/C) activity to degrade cyclin. Here, we describe a fission yeast cuf2 mutant that enters into a third nuclear division cycle, represented by ectopic spindle assembly and abnormal chromosome segregation. Cuf2 is a meiotic transcription factor, and its critical target is fzr1(+)/mfr1(+), which encodes a meiotic APC/C activator. fzr1 Delta also enters a third nuclear division. Thus, Cuf2 ensures termination of the M-phase cycle by boosting Fzr1 expression to generate functional gametes.

    DOI

  • Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

    Federico Vaggi, James Dodgson, Archana Bajpai, Anatole Chessel, Ferenc Jordan, Masamitsu Sato, Rafael Edgardo Carazo-Salas, Attila Csikasz-Nagy

    PLOS COMPUTATIONAL BIOLOGY   8 ( 10 ) e100273  2012.10  [Refereed]

     View Summary

    The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify 'linker' proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted 'linkers' also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.

    DOI

  • Spatial segregation of polarity factors into distinct cortical clusters is required for cell polarity control.

    J. Dodgson, A. Chessel, F. Vaggi, S. Cox, M. Sato, A. Csikasz-Nagy, R. Carazo Salas

    MOLECULAR BIOLOGY OF THE CELL   23   1834  2012  [Refereed]

    DOI

  • A novel fission yeast mei4 mutant that allows efficient synchronization of telomere dispersal and the first meiotic division

    Yasutaka Kakui, Masamitsu Sato, Kayoko Tanaka, Masayuki Yamamoto

    YEAST   28 ( 6 ) 467 - 479  2011.06  [Refereed]

     View Summary

    The progression of meiosis is controlled by a number of gene-expression systems in the fission yeast Schizosaccharomyces pombe. A forkhead-type transcription factor Mei4 activates a number of genes essential for progression from the middle to late stages of meiosis, which include meiosis I, meiosis II and sporulation. The mei4-deletion mutant (mei4 Delta) arrests after meiotic prophase and does not enter meiosis I. To further analyse the Mei4 function, we isolated novel temperature-sensitive mei4 alleles. The two alleles isolated in the initial screen turned out to contain a substitution at N136 in the forkhead DNA-binding domain. Among site-directed mutants that carried a point mutation at this position, the mei4-N136A mutant showed the most severe temperature sensitivity. The mei4-N136A mutant arrested before meiosis I at the restrictive temperature, as did the mei4 Delta mutant. In fission yeast, the telomeres are clustered at the spindle pole body (SPB) in meiotic prophase and disperse from it at the onset of meiosis I. The mei4 Delta mutant was found to arrest with its telomeres clustered at the SPB, demonstrating a role for Mei4 in telomere dispersion. The mei4-N136A mutant also arrested with clustered telomeres at the restrictive temperature, and the clustering was synchronously resolved after a temperature down-shift, indicating that mei4-N136A is a reversible allele. Hence, the mei4-N136A mutant will be a unique tool to synchronize the meiotic cell cycle from meiosis I onwards and may facilitate analyses of cellular activities occurring during meiosis I. Copyright (C) 2011 John Wiley & Sons, Ltd.

    DOI

  • Space shuttling in the cell Nucleocytoplasmic transport and microtubule organization during the cell cycle

    Masamitsu Sato, Takashi Toda

    NUCLEUS-AUSTIN   1 ( 3 ) 231 - 236  2010.05  [Refereed]

     View Summary

    Microtubules form a multifunctional filamentous structure essential for the cell. In interphase, microtubules form networks in the cytoplasm and play pivotal roles in cell polarity and intracellular transport of various biomolecules. In mitosis, microtubules dramatically change their morphology to assemble the mitotic spindle, thereby pulling the chromosomes toward the spindle poles. One long-standing question is how microtubules are reorganized upon mitotic entry. Yeast cells undergo closed mitosis, in which the nuclear envelope persists, whereas higher eukaryotes undergo open mitosis, in which the nuclear envelope breaks down. Microtubule reorganization must be controlled by selective localization of microtubule-assembly factors. Recent findings in fission yeast indicate that several microtubule-associated proteins (MAPs) shuttle between the cytoplasm and the nucleus through regulation by Ran GTPase, the universal organizer of nucleocytoplasmic transport. Furthermore, the synergistic interplay of Ran and cyclin-dependent kinase (CDK) induces the critical spatiotemporal shift of modes in microtubule assembly from cytoplasmic arrays to nuclear spindles. A MAP complex Alp7/TACC-Alp14/TOG undergoes nucleocytoplasmic shuttling in interphase, whereas it is retained in the mitotic nucleus through a decrease of its nuclear export by CDK. Our understanding of how microtubules are reorganized during the cell cycle is beginning to emerge.

    DOI

  • Fission yeast Pcp1 links polo kinase-mediated mitotic entry to gamma-tubulin-dependent spindle formation

    Chii Shyang Fong, Masamitsu Sato, Takashi Toda

    EMBO JOURNAL   29 ( 1 ) 120 - 130  2010.01  [Refereed]

     View Summary

    The centrosomal pericentrin-related proteins play pivotal roles in various aspects of cell division; however their underlying mechanisms remain largely elusive. Here we show that fission-yeast pericentrin-like Pcp1 regulates multiple functions of the spindle pole body (SPB) through recruiting two critical factors, the gamma -tubulin complex (gamma-TuC) and polo kinase (Plo1). We isolated two pcp1 mutants (pcp1-15 and pcp1-18) that display similar abnormal spindles, but with remarkably different molecular defects. Both mutants exhibit defective monopolar spindle microtubules that emanate from the mother SPB. However, while pcp1-15 fails to localise the gamma-TuC to the mitotic SPB, pcp1-18 is specifically defective in recruiting Plo1. Consistently Pcp1 forms a complex with both gamma-TuC and Plo1 in the cell. pcp1-18 is further defective in the mitotic-specific reorganisation of the nuclear envelope (NE), leading to impairment of SPB insertion into the NE. Moreover pcp1-18, but not pcp1-15, is rescued by overproducing nuclear pore components or advancing mitotic onset. The central role for Pcp1 in orchestrating these processes provides mechanistic insight into how the centrosome regulates multiple cellular pathways. The EMBO Journal (2010) 29, 120-130. doi: 10.1038/emboj.2009.331; Published online 26 November 2009

    DOI

  • Nucleocytoplasmic transport of Alp7/TACC organizes spatiotemporal microtubule formation in fission yeast

    Masamitsu Sato, Naoyuki Okada, Yasutaka Kakui, Masayuki Yamamoto, Minoru Yoshida, Takashi Toda

    EMBO REPORTS   10 ( 10 ) 1161 - 1167  2009.10  [Refereed]

     View Summary

    Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled-coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during mitosis, although its underlying mechanism remains elusive. Here, we investigate the behaviour of Alp7 and its binding partner, Alp14/TOG, throughout the cell cycle. Interestingly, Alp7 enters the nucleus during interphase but is subsequently exported to the cytoplasm by the Exportin-dependent nuclear export machinery. The continuous nuclear export of Alp7 during interphase is essential for maintaining the array-like cytoplasmic microtubule structure. The mitosis-specific nuclear accumulation of Alp7 seems to be under the control of cyclin-dependent kinase (CDK). These results indicate that the spatiotemporal regulation of microtubule formation is established by the Alp7/TACC-Alp14/TOG complex through the coordinated interplay of Ran and CDK.

    DOI

  • [Is spindle formation in fission yeast specific to the species?: from the viewpoint of nuclear transport and spindle pole body].

    Masamitsu Sato, Mika Toya, Takashi Toda

    Tanpakushitsu Kakusan Koso   53 ( 3 ) 197 - 296  2008

  • Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast

    Masamitsu Sato, Takashi Toda

    NATURE   447 ( 7142 ) 334 - U9  2007.05  [Refereed]

     View Summary

    Microtubules are essential intracellular structures involved in several cellular phenomena, including polarity establishment and chromosome segregation(1). Because the nuclear envelope persists during mitosis ( closed mitosis) in fission yeast ( Schizosaccharomyces pombe), cytoplasmic microtubules must be reorganized into the spindle in the compartmentalized nucleus on mitotic entry(2). An ideal mechanism might be to take advantage of an evolutionarily conserved microtubule formation system that uses the Ran-GTPase nuclear transport machinery(3-5), but no targets of Ran for spindle formation have been identified in yeast. Here we show that a microtubule-associated protein, Alp7, which forms a complex with Alp14, is a target of Ran in yeast for spindle formation. The Ran-deficient pim1 mutant (pim1-F201S) failed to show mitosis-specific nuclear accumulation of Alp7. Moreover, this mutant exhibited compromised spindle formation and early mitotic delay. Importantly, these defects were suppressed by Alp7 that was artificially targeted to the nucleus by a Ran-independent and importin-alpha-mediated system. Thus, Ran targets Alp7 - Alp14 to achieve nuclear spindle formation, and might differentiate its targets depending on whether the organism undergoes closed or open mitosis.

    DOI

  • Mal3, the fission yeast EB1 homologue, cooperates with Bub1 spindle checkpoint to prevent monopolar attachment

    K Asakawa, M Toya, M Sato, M Kanai, K Kume, T Goshima, MA Garcia, D Hirata, T Toda

    EMBO REPORTS   6 ( 12 ) 1194 - 1200  2005.12  [Refereed]

     View Summary

    Bipolar microtubule attachment is central to genome stability. Here, we investigate the mitotic role of the fission yeast EB1 homologue Mal3. Mal3 shows dynamic inward movement along the spindle, initial emergence at the spindle pole body (SPB) and translocation towards the equatorial plane, followed by sudden disappearance. Deletion of Mal3 results in early mitotic delay, which is dependent on the Bub1, but not the Mad2, spindle checkpoint. Consistently, Bub1, but not Mad2, shows prolonged kinetochore localization. Double mutants between mal3 and a subset of checkpoint mutants, including bub1, bub3, mad3 and mph1, but not mad1 or mad2, show massive chromosome mis-segregation defects. In mal3bub1 mutants, both sister centromeres tend to remain in close proximity to one of the separating SPBs. Further analysis indicates that mis-segregated centromeres are exclusively associated with the mother SPB. Mal3, therefore, has a role in preventing monopolar attachment in cooperation with the Bub1/Bub3/Mad3/Mph1-dependent checkpoint.

    DOI

  • The roles of fission yeast Ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling

    A Yamashita, M Sato, A Fujita, M Yamamoto, T Toda

    MOLECULAR BIOLOGY OF THE CELL   16 ( 3 ) 1378 - 1395  2005.03  [Refereed]

     View Summary

    The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase. ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss. Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B. Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule-bundling factor. In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants. During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly. The Aurora kinase does not correctly localize to central spindles in the absence of Ase1. Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed. Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.

    DOI

  • Phosphorylation of Mei2 and Ste11 by Pat1 kinase inhibits sexual differentiation via ubiquitin proteolysis and 14-3-3 protein in fission yeast

    K Kitamura, S Katayama, S Dhut, M Sato, Y Watanabe, M Yamamoto, T Toda

    DEVELOPMENTAL CELL   1 ( 3 ) 389 - 399  2001.09  [Refereed]

     View Summary

    Fission yeast Pat1 kinase inhibits sexual differentiation by phosphorylating the meiotic inducer Mei2 and the transcription factor Ste11. Here, we show how Pat1 downregulates these proteins. Mei2 is degraded via a ubiquitin-proteasome pathway in a phosphorylation-dependent fashion. The E2 Ubc2 and the E3 Ubr1 are required for this proteolysis. In addition, Pat1 negatively regulates Ste11 via Rad24/14-3-3, thereby repressing mei2(+) transcription. The Pat1 phosphorylation sites of Ste11 match the consensus recognition sequence for 14-3-3. Rad24 binds preferentially to phosphorylated Ste11, and this binding results in inhibition of the transcriptional activation capacity of Ste11. Overall, therefore, these results show that Pat1 coordinates concerted molecular mechanisms that govern the sexual differentiation developmental decision.

  • The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling

    Masamitsu Sato, Satoko Shinozaki-Yabana, Akira Yamashita, Yoshinori Watanabe, Masayuki Yamamoto

    FEBS Letters   499 ( 3 ) 251 - 255  2001.06  [Refereed]

     View Summary

    Schizosaccharomyces pombe Mei2p is an RNA-binding protein that switches the cell cycle from mitotic to meiotic. Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non-coding RNA molecule termed meiRNA. Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling. Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus. Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus. © 2001 Federation of European Biochemical Societies.

    DOI PubMed

  • The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling

    M Sato, S Shinozaki-Yabana, A Yamashita, Y Watanabe, M Yamamoto

    FEBS LETTERS   499 ( 3 ) 251 - 255  2001.06  [Refereed]

     View Summary

    Schizosaccharomyces pombe Mei2p is an RNA-binding protein that switches the cell cycle from mitotic to meiotic, Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non-coding RNA molecule termed meiRNA. Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling. Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus. Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI

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Misc

  • 分裂酵母の減数分裂を司るRNA結合タンパク質Mei2の制御系

    山下朗, 佐藤政充, 島田忠之, 渡辺嘉典, 山本正幸

    日本分子生物学会年会プログラム・講演要旨集   23rd  2000

    J-GLOBAL

Awards

  • 酵母コンソーシアムフェロー

    2020.10   大隅基礎科学創成財団  

  • Waseda University Research Award

    2018.02  

    Winner: SATO, Masamitsu

  • Waseda University Teaching Award, The President Award

    2018   Waseda University  

  • Waseda University Teaching Award

    2016.12  

    Winner: SATO, Masamitsu

  • The Young Scientists’ Prize, The Commendation for Science and Technology by the Minister of Education, Culture, Sports, Science and Technology

    2012.04  

  • Human Frontier Science Program (HFSP) Young Investigator Grant

    2009.09  

  • JSPS Fellowship for Research Abroad

    2006.04  

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Research Projects

  • Regulatory mechanisms of meiosis

  • Cell cycle regulation specific to the interkinesis period between Meiosis I and Meiosis II

  • Analysis of novel functions of microtubules that bridge meiotic recombination and chromosome segregation

  • Regulatory Mechanisms of Meiosis in Fission Yeast

  • 減数分裂における細胞分裂装置の再編成機構

    科学研究費助成事業(東京大学)  科学研究費助成事業(特定領域研究)

  • 微小管の形成メカニズムと細胞内新機能の発見

    科学研究費助成事業(東京大学)  科学研究費助成事業(基盤研究(B))

  • シングルセル発現解析と核膜変異体ライブラリを用いた転写サイクル始動機構の解明

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(新学術領域研究(研究領域提案型))

  • Reconstitution of CENP-A nucleosomes and kinetochores of fission yeast

  • Functions and Control of Microtubules from cells to tissues

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Specific Research

  • 微小管の時空間的制御のメカニクス異常が原因で起きる疾患を組織レベルで追究する

    2020  

     View Summary

    微小管は細胞内に形成される繊維状の骨組み因子として知られる。微小管の機能は多岐にわたるが、未だに明らかにされていない機能があると考えられ、我々はそのような微小管の新規構造・新機能の発見を目指して研究を遂行している。これまでの研究の結果、微小管の制御が破綻することで、小腸上皮細胞の形態に異常が発生することが明らかにされている。そこで我々は、微小管結合タンパク質CAMSAP3の異常がどのようなメカニズムで細胞形態の異常を引き起こすのか、組織学的な解析をおこなった。解析の結果、微小管の異常が細胞小器官の配置異常を引き起こすことで、細胞の形態や機能に影響をもたらす可能性があると推測するに至った。

  • シングルセル・オミクス解析による、細胞が休眠から目覚める分子機構の解明

    2019  

     View Summary

    本研究では、休眠状態の細胞が、周囲の環境の変化に伴い休眠を打破して活動を開始する際に、どのような分子機構が働くのか、その解明を目指している。分裂酵母は減数分裂の結果として配偶子に相当する胞子を形成する。胞子は栄養に乏しい状態でも長期にわたり休眠状態を維持するが、周囲の栄養状態が改善すると、これを認識して発芽をおこない、その後通常見られるような体細胞分裂周期に入り、増殖を繰り返す。我々は休眠打破の分子機構を解明するために、遺伝子発現解析をおこなうことで、休眠から発芽にかけて、どのような遺伝子が発現変動するのかを調べた。

  • クロマチン動態解析のためのシングルセル・オミクス基盤技術の整備

    2018  

     View Summary

    本研究では、分裂酵母におけるシングルセルにもとづく遺伝子発現解析を可能とする実験手法を確立することを目的としている。本研究の成果として、栄養増殖状態にある分裂酵母の1個の細胞からRNAを抽出し、これをRNA-seq法により発現解析することをおこなった。その結果として得られた発現プロファイルは、既存の方法で作成された発現プロファイルと極めて高い相関性を示したため、本方法が有効であることが示された。

  • 高齢卵子における微小管異常と不妊との関連

    2017  

     View Summary

    出産の高齢化や不妊がおおきな社会問題となっている昨今,その原因を追究して不妊治療に応用する必要性が重視されてきている。しかしながら現段階では,これらの原因はじゅうぶんに追究されていない。そこで本研究では,ほ乳類の減数分裂(卵母細胞形成)に焦点を当てて,卵母細胞の経年劣化の原因の一端を探りたいと考えた。我々は紡錘体微小管に注目し,この異常が卵子の経年劣化や不妊の原因となっている可能性について実験をおこない,今後の研究の質的な基盤を固めたいと考えた。

  • 構造クラスタ分類によるncRNA新機能の発見

    2017  

     View Summary

    分裂酵母S. pombeにおいては,およそ1,800種類の非コードRNAが存在することが知られている。これらには細胞内でなんらかの機能を発揮するものがあると考えられるが,これまでのところ機能が解明されているものはごく一部のみであり,大部分は機能未定の状態である。そこで本研究では,これらの非コードRNAのなかから細胞内で何らかの機能を発揮するものを探索することを主目的としてスクリーンをおこなっている。

  • 減数分裂における染色体交叉の意義の解明と不稔不妊性との関連

    2015   新井邦生

     View Summary

    我々はすでに減数分裂の際に微小管が染色体配置を起こし、これによって染色体交叉の状態でも安全な染色体分配を促していることを発見してきた(Kakui et al. Nature Cell Biology 2013)。その際に微小管がどのように脱重合するかという点に焦点を当てて観察をおこなっている。その結果、当初予想されたキネシンKlp5/6ではなく、別の微小管結合タンパク質Dis1およびDam1が重要な働きをすることが分かった。現在はこれらの変異体を用いて、減数分裂特異的な微小管による染色体移動の分子原理を追究している。また、本研究では人工ミニ染色体を用いて、これが減数分裂でどのように振る舞うかも観察し、染色体の本数とそれが減数分裂に与える負担についても検証しているところである。

  • 微小管形成を人工的に誘導する実験系の確立 ~酵母からヒトへ~

    2014  

     View Summary

    微小管は細胞骨格のひとつであり様々な細胞内現象に不可欠な役割を担う。我々は第一に,微小管が細胞内で形成される分子機構を解明することを目標としている。分裂酵母をモデル生物として用いて,微小管の形成に重要な役割を担う因子を同定している。我々は次に,これらの因子を強制的に発現させると微小管を形成するだろうかを追求している。我々はこれらの因子を強制発現させることで,過剰な微小管を形成するか否かを検証する実験系を構築している。

  • 休眠状態の胞子から細胞周期と細胞骨格はどのように新規形成されるのか

    2014  

     View Summary

    従来の細胞周期・細胞骨格の研究は,増殖中の細胞のとある1サイクルの細胞状態に注目して解析が進められてきた。このような増殖中の細胞では,前回の細胞周期の「履歴」をもとに次の周期の分子機構が決まる。それでは,長らく休眠状態にある静止細胞は,どのように増殖を「ゼロから」始動するのであろうか。細胞周期の始動は,細胞が周囲の環境の変化を認識して遺伝子発現システムを起動させることによると考えられるが,その分子プログラムの実態は一切明らかにされていない。また,細胞骨格や染色体の構成は,休眠状態の細胞で維持されるのか,あるいは休眠が終わり細胞周期が新生する際に,これらの骨格が新生されるのかは,不明な点が多い。 そこで本研究では,(1)分裂酵母の休眠細胞,特に胞子(配偶子)が発芽する際の遺伝子発現プロファイルを作成すること,および(2)胞子および発芽時の細胞を顕微鏡観察することで,これらのメカニズムの解明を目指した。

  • ゲノムの多様性創出と継承を連携させる微小管システムの解明

    2013  

     View Summary

     本研究では、分裂酵母の微小管形成がどのようにおこなわれるかについて研究を継続している。体細胞分裂においては、複製された染色体を2個の娘細胞に分配するために、微小管からなる紡錘体が形成され、染色体の動原体部分を左右から引っ張ることで、染色体を1:1に正確に分離することができる。 紡錘体微小管形成の分子メカニズムを解明するために過去数多くの研究がおこなわれており、その成果として、多くの微小管結合タンパク質や分裂期キナーゼが必須の役割を担うことが明らかにされてきた。 しかしながら、特に紡錘体形成の初期にどのような分子が働くことが大切であるのか、既知の因子だけでは全体像に未だ到達できないのも事実であり、本研究では、分裂期の前期から中期にかけて、微小管形成に関わる因子を探索してきた。具体的には、我々がこれまで作製してきたGFP-tubulin、mCherry-核膜、CFP-中心体(SPB)の3色を発現する細胞株を作製し、これを野生型株として突然変異原処理をおこない、微小管の形態異常を示す変異体を検索するスクリーニングをおこなった。その結果、2,000を超える変異体が単離され、これを表現型ごとにクラス分けした。その中でも我々は、分裂中期以前に、微小管形成に異常を示すものに着目し、そのなかのひとつの変異体について重点的に解析を進めている。この変異体をkis1変異体と名付け、その原因遺伝子を調べたところ、未知のタンパク質をコードすることが分かった。 このkis1変異体が微小管形成のどのステップに異常を示すのかを解析したところ、前中期の紡錘体形成や、中期程度で紡錘体形成の維持に欠陥があることが分かった。 我々は、このkis1変異体では微小管形成中心であるSPBの機能に欠陥があると予想したが、これまでのところ、SPB構成因子の局在には問題が見つかっていない。これに対して、kis1変異体では、一部の動原体因子が正しく動原体に局在できていないことが分かった。これらの結果から、Kis1タンパク質は、動原体の構成維持に必須の新規因子であり、動原体の構成を介して紡錘体微小管を安定化させていると考えられる。

  • 学際的アプローチによる細胞極性制御メカニズムの解明

    2013  

     View Summary

     細胞が増殖・分化をおこなって組織を形成するにあたり、細胞の形態が重要な役割を担うことが一般的に知られている。細胞形態のなかでも、特に細胞が特定の方向に伸張する、いわゆる細胞の極性成長は、細胞の非対称分裂と分化、および3次元的にひろがっていく細胞の組織化において重要なファクターである。 しかしながら、細胞がどのように成長する極性を作り出しているのか、その分子メカニズムについては未知の部分が多い。そこで我々は、細長い極性をもって成長する分裂酵母をモデル生物として用いて、細胞極性が確立される分子メカニズムの解明を目指している。 多くの研究者がこの難題に取り組んだ成果として、これまでに100種類を超える「極性因子」が発見されており、これらの多くは、細胞が成長する末端に局在する性質を持つ(for review, Hachet et al., Curr Opin Cell Biol 2012)。我々は、これらの因子の中で、まずTea1とTea3という2つのタンパク質に注目した。Tea1もTea3も、通常の顕微鏡解析においては、細胞の末端に局在し、協調して働くと考えられていた(Mata and Nurse, Cell 1997; Arellano et al., Curr Biol. 2002)。 しかしながら、我々は、ケンブリッジ大学Carazo-Salas博士らとの共同研究により、細胞の末端の方向から細胞を顕微鏡観察するイメージング技法を用いると、驚くべき事にTea1とTea3は末端においてドット状に存在するが、お互いのドットは必ずしも共局在しないことが分かった。また、Tea1とTea3を強制的に共局在させると、細胞の極性成長に欠陥が生じることが分かり、これらの因子は協調して働くのではなく、細胞末端で個別に機能することで細胞極性を確立していることが示された。このような、極性因子のドット状局在は、生物種を超えてみられる普遍の原理であると考えられ、細胞極性因子の局在と活性に全く新しい知見をもたらすことに成功した。

  • 減数分裂においてみられる特殊な微小管の機能に迫る

    2013  

     View Summary

     当該研究では、体細胞分裂と減数分裂という目的が異なる2つの分裂様式において、細胞内の現象や分子機構がどのように異なっているのか、主に細胞骨格の微小管に焦点を当てて解析をおこなってきた。 研究をおこなうにあたり、人為的に減数分裂を誘導できる優れたモデル生物である分裂酵母を使用した。分裂酵母の微小管・動原体・中心体(SPB)をそれぞれGFP・mCherry・CFPという3つの異なる蛍光タンパク質でラベルした細胞を作製し、体細胞分裂および減数分裂を誘導することで、細胞の微小管構造がこれら2つの分裂様式においてどのように異なった挙動を示すのかが明らかになる。我々は既に、減数分裂が始まる直前に、体細胞分裂の開始前には見られない特殊な微小管構造が形成されることを発見していた。本研究では、この微小管の挙動がどのような因子によってなされているのかに焦点を当てて研究してきた。その結果、微小管結合タンパク質Dis1が、微小管の脱重合に関与する可能性が示唆された(Kakui et al. Nature Cell Biology, 2013)。 近年いくつかのグループがDis1の高等生物オーソログであるTOGタンパク質について、微小管を重合する活性があることを報告している。我々の発見は、この一般的に知られるTOGの機能とは異なり、分裂酵母の減数分裂においてはDis1/TOGが微小管を脱重合するという、全く正反対の機能を担うことを示している。そこで我々は、Dis1がどのように減数分裂微小管の脱重合を起こしているのか、主に次の2つの可能性から追究し、現在も研究を継続している。(1) Dis1が直接微小管を脱重合する可能性。(2) Dis1が何か別の因子を介して(相互作用因子などを介して)微小管を脱重合している可能性。 興味深いことに、Dis1と類似したパラログ分子であるAlp14は、他の生物におけるTOG同様に、分裂酵母の減数分裂では微小管の重合に関わるという結果を出している(同)。従って、この類似した2つの因子は、少なくとも減数分裂において、明確な機能の分離がなされていることが分かり、その原因について追及している。

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Syllabus

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