2024/10/10 更新

写真a

トヤ ミカ
戸谷 美夏
所属
理工学術院 国際理工学センター(理工学術院)
職名
准教授(任期付)
学位
博士(理学) ( 東京大学 )

所属学協会

  •  
     
     

    日本細胞生物学会

  •  
     
     

    日本分子生物学会

研究分野

  • 細胞生物学

研究キーワード

  • 細胞骨格

  • 微小管

  • 上皮細胞

  • 分裂酵母

  • 細胞構造・細胞形態と機能

 

論文

  • A germline-specific role for unconventional components of the γ-tubulin complex in Caenorhabditis elegans.

    Nami Haruta, Eisuke Sumiyoshi, Yu Honda, Masahiro Terasawa, Chihiro Uchiyama, Mika Toya, Yukihiko Kubota, Asako Sugimoto

    Journal of cell science   136 ( 13 )  2023年07月  [国際誌]

     概要を見る

    The γ-tubulin complex (γTuC) is a widely conserved microtubule nucleator, but some of its components, namely GCP4, GCP5 and GCP6 (also known as TUBGCP4, TUBGCP5 and TUBGCP6, respectively), have not been detected in Caenorhabditis elegans. Here, we identified two γTuC-associated proteins in C. elegans, GTAP-1 and GTAP-2, for which apparent orthologs were detected only in the genus Caenorhabditis. GTAP-1 and GTAP-2 were found to localize at centrosomes and the plasma membrane of the germline, and their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved γTuC component MZT-1 (also known as MOZART1 and MZT1) was essential for the localization of centrosomal γ-tubulin, depletion of GTAP-1 and/or GTAP-2 caused up to 50% reduction of centrosomal γ-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTAP-2 contributed to efficient recruitment of the γTuC to the plasma membrane. Depletion of GTAP-1, but not of GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure of the adult germline. We propose that GTAP-1 and GTAP-2 are unconventional components of the γTuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the γTuC to specific subcellular sites in a tissue-specific manner.

    DOI PubMed

    Scopus

    1
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  • Fission yeast Dis1 is an unconventional TOG/XMAP215 that induces microtubule catastrophe to drive chromosome pulling

    Yuichi Murase, Masahiko Yamagishi, Naoyuki Okada, Mika Toya, Junichiro Yajima, Takahiro Hamada, Masamitsu Sato

       2022年08月

     概要を見る

    Abstract

    The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry attached chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards the vicinity of spindle poles in meiocytes. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

    DOI

  • 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 - ar12  2021年10月  [査読有り]  [国際誌]

     概要を見る

    CAMSAP3, a protein that controls microtubule dynamics by binding to its minus-end, is localized at proximal regions of the axoneme in tracheal motile cilia. Its dysfunction results in a collapse of the central microtube pair, basal plate disorganization, and uncoordinated beating of multicilia.

    DOI PubMed

  • 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年06月  [査読有り]  [国際誌]

     概要を見る

    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

    Scopus

    1
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  • 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年04月  [査読有り]  [国際誌]

     概要を見る

    The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin.

    DOI PubMed

    Scopus

    6
    被引用数
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  • 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年04月  [査読有り]  [国際誌]

     概要を見る

    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<italic>Schizosaccharomyces pombe</italic>, 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

    Scopus

    13
    被引用数
    (Scopus)
  • 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年03月  [査読有り]  [国際誌]

    担当区分:責任著者

     概要を見る

    <title>Abstract</title>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<italic>Camsap3</italic>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

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    7
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  • Patronin Takes a Shot at Polarity

    Masatoshi Takeichi, Mika Toya

    DEVELOPMENTAL CELL   38 ( 1 ) 12 - 13  2016年07月  [招待有り]

     概要を見る

    To specify the anterior-posterior axis of Drosophila embryos, noncentrosomal microtubules grow out from cortical regions of the oocyte and help transport axis determinants. In this issue of Developmental Cell, Nashchekin et al. (2016) report a Shot- and Patronin-dependent mechanism by which the oocyte cortex produces polarized microtubule arrays.

    DOI PubMed

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    2
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  • CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells

    Mika Toya, Saeko Kobayashi, Miwa Kawasaki, Go Shioi, Mari Kaneko, Takashi Ishiuchi, Kazuyo Misaki, Wenxiang Meng, Masatoshi Takeichi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   113 ( 2 ) 332 - 337  2016年01月  [査読有り]

     概要を見る

    Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

    DOI PubMed

    Scopus

    105
    被引用数
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  • Organization of Non-centrosomal Microtubules in Epithelial Cells

    Mika Toya, Masatoshi Takeichi

    CELL STRUCTURE AND FUNCTION   41 ( 2 ) 127 - 135  2016年  [査読有り]  [招待有り]

     概要を見る

    Polarized epithelial cells contain a characteristic array of microtubules in which non-centrosomal microtubules are aligned along the apical-to-basal axis of the cell with their minus ends oriented towards the apical pole. Although this unique orientation of microtubules was discovered in the late 1980s, how this orientation is established remains unresolved partly because of limited information about molecular factors that regulate the minus ends of non-centrosomal microtubules. Recent studies, however, identified novel minus end-associated proteins, revealing mechanisms by which the polarized arrays of microtubules are established in epithelial cells. These studies have also demonstrated the importance of apico-basally orientated microtubules in intra-structural organization of cells. This review focuses on recent progress of our understanding of the molecular basis for epithelium-specific microtubule assembly and function.

    DOI PubMed

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    34
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  • The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A

    Elsa Kress, Francoise Schwager, Rene Holtackers, Jonas Seiler, Francois Prodon, Esther Zanin, Annika Eiteneuer, Mika Toya, Asako Sugimoto, Hemmo Meyer, Patrick Meraldi, Monica Gotta

    JOURNAL OF CELL BIOLOGY   201 ( 4 ) 559 - 575  2013年05月  [査読有り]

     概要を見る

    Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.

    DOI PubMed

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    23
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  • The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

    Virginie Hachet, Coralie Busso, Mika Toya, Asako Sugimoto, Peter Askjaer, Pierre Goenczy

    MOLECULAR BIOLOGY OF THE CELL   23 ( 16 ) 3111 - 3121  2012年08月  [査読有り]

     概要を見る

    Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference-based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.

    DOI PubMed

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    22
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  • Pob1 Ensures Cylindrical Cell Shape by Coupling Two Distinct Rho Signaling Events During Secretory Vesicle Targeting

    Kentaro Nakano, Mika Toya, Aki Yoneda, Yukiko Asami, Akira Yamashita, Naomi Kamasawa, Masako Osumi, Masayuki Yamamoto

    TRAFFIC   12 ( 6 ) 726 - 739  2011年06月  [査読有り]

     概要を見る

    Proper cell morphogenesis requires the co-ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1-664 has a curious lemon-like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin-polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon-like cell morphology similar to pob1-664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin-mediated vesicle transport and exocyst-mediated vesicle tethering during secretory vesicle targeting.

    DOI PubMed

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    26
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  • A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos

    Mika Toya, Masahiro Terasawa, Kayo Nagata, Yumi Tida, Asako Sugimoto

    NATURE CELL BIOLOGY   13 ( 6 ) 708 - U187  2011年06月  [査読有り]

     概要を見る

    The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity(1,2). Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of gamma-tubulin-independent microtubules in early embryos(3); however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the gamma-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.

    DOI PubMed

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    65
    被引用数
    (Scopus)
  • Caenorhabditis elegans ortholog of the p24/p22 subunit, DNC-3, is essential for the formation of the dynactin complex by bridging DNC-1/p150Glued and DNC-2/dynamitin

    Masahiro Terasawa, Mika Toya, Fumio Motegi, Miyeko Mana, Kuniaki Nakamura, Asako Sugimoto

    GENES TO CELLS   15 ( 11 ) 1145 - 1157  2010年11月  [査読有り]

     概要を見る

    Dynactin is a multisubunit protein complex required for the activity of cytoplasmic dynein. In Caenorhabditis elegans, although 10 of the 11 dynactin subunits were identified based on the sequence similarities to their orthologs, the p24/p22 subunit has not been detected in the genome. Here, we demonstrate that DNC-3 (W10G11.20) is the functional counterpart of the p24/p22 subunit in C. elegans. RNAi phenotypes and subcellular localization of DNC-3 in early C. elegans embryos were nearly identical to those of the known dynactin components. All other dynactin subunits were co-immunoprecipitated with DNC-3, indicating that DNC-3 is a core component of dynactin. Furthermore, the overall secondary structure of DNC-3 resembles to those of the mammalian and yeast p24/p22. We found that DNC-3 is required for the localization of the DNC-1/p150Glued and DNC-2/dynamitin, the two components of the projection arm of dynactin, to the nuclear envelope of meiotic nuclei in the adult gonad. Moreover, DNC-3 physically interacted with DNC-1 and DNC-2 and significantly enhanced the binding ability between DNC-1 and DNC-2 in vitro. These results suggest that DNC-3 is essential for the formation of the projection arm subcomplex of dynactin.

    DOI PubMed

    Scopus

    10
    被引用数
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  • Imaging of Mitotic Spindle Dynamics in Caenorhabditis elegans Embryos

    Mika Toya, Yumi Iida, Asako Sugimoto

    MICROTUBULES: IN VIVO   97   359 - 372  2010年  [査読有り]

     概要を見る

    Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.

    DOI PubMed

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    22
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  • 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年  [査読有り]

     概要を見る

    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

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    37
    被引用数
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  • [Is spindle formation in fission yeast specific to the species?: from the viewpoint of nuclear transport and spindle pole body].

    Sato M, Toya M, Toda T

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   53 ( 3 ) 197 - 206  2008年03月  [査読有り]

    PubMed CiNii

  • 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年06月  [査読有り]

     概要を見る

    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 PubMed

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    44
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  • 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年12月  [査読有り]

     概要を見る

    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 PubMed

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    23
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  • Identification and functional analysis of the gene for type I myosin in fission yeast

    M Toya, F Motegi, K Nakano, Mabuchi, I, M Yamamoto

    GENES TO CELLS   6 ( 3 ) 187 - 199  2001年03月  [査読有り]

     概要を見る

    Background: Type I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F-actin assembly has been inferred.
    Results: We cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F-actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1 Delta cells could mate, but heterothallic myo1 Delta cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck.
    Conclusions: Myo1p appears to control the redistribution of F-actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.

    DOI

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    33
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  • Fission yeast Pob1p, which is homologous to budding yeast Boi proteins and exhibits subcellular localization close to actin patches, is essential for cell elongation and separation

    M Toya, Y Iino, M Yamamoto

    MOLECULAR BIOLOGY OF THE CELL   10 ( 8 ) 2745 - 2757  1999年08月  [査読有り]

     概要を見る

    The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

    DOI

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    26
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  • Fission yeast protein kinase C gene homologues are required for protoplast regeneration: A functional link between cell wall formation and cell shape control

    Hiromi Kobori, Takashi Toda, Hiroko Yaguchi, Mika Toya, Mitsuhiro Yanagida, Masako Osumi

    Journal of Cell Science   107 ( 5 ) 1131 - 1136  1994年05月  [査読有り]

     概要を見る

    Two novel protein kinase C (n PKC) gene homologues, pck1+ and pck2+ were isolated from the fission yeast Schizosaccharomyces pombe. We examined the functional differences of pck1+ and pck2+ in cell wall formation and actin organization of S. pombe. Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+ (Δpck1) and pck2+ (Δpck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization. Protoplasts of the wild type strain and those of Δpck1 reverted to intact cells in osmotically stabilized liquid medium. A close spatial association between new cell wall formation and actin was observed in these two strains. In Δpck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed
    (2) actin was not reorganized
    and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts. These findings demonstrate that the pck2+ gene has a function essential for protoplast regeneration but the pck1+ gene does not. Involvement of n PKCs in cell wall formation and actin organization was also clarified. The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast.

    PubMed

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共同研究・競争的資金等の研究課題

  • 中心体に依存しない微小管の機能不全がひきおこす嚢胞腎形成の分子機構

    日本学術振興会  科学研究費助成事業

    研究期間:

    2020年04月
    -
    2023年03月
     

    戸谷 美夏

     概要を見る

    多くの臓器を構成する主要な成分である上皮細胞は、特徴的な微小管編成を保って機能する。我々は、この上皮微小管編成を司る微小管マイナス端結合タンパク質CAMSAP3の機能欠損マウス (Camsap3変異マウス)において、腎臓に多発性の嚢胞が形成されることを見出し、解析を進めている。昨年度までの研究から、Camsap3変異マウスで観察される嚢胞腎は、尿細管の特定の領域(近位尿細管)の上皮細胞で微小管編成が乱れることで、細胞の形態維持に支障を来し、その結果、メカニカルな力(原尿圧を推測)に抵抗できなくなった細胞が引き延ばされ、尿細管が拡張することにより形成されると推測された。Camsap3変異マウスでは、拡張した尿細管の上皮細胞で、細胞増殖や細胞形態制御に関わる転写共役因子YAPの活性化を示唆する核内局在が観察され、明らかな拡張を認めない近位尿細管においても、メカノセンサーチャネルであるPIEZO1の核局在が検出された。上皮微小管の構造異常がひきおこす近位尿細管拡張の分子機構を調べる目的で、腎組織の微細構造を識別して微小領域(110μmφ)を採取し、遺伝子発現解析に耐える品質のRNAが得られる試料を得た。
    今年度は、創薬等先端技術支援プラットフォーム (BINDS) を窓口とし、早大生命医科学科竹山研究室の支援を受けて、遺伝子発現解析を行うためのシークエンスデータの収集を行った。野生型マウスとCamsap3変異マウスそれぞれの腎組織切片から、組織・細胞の形態を指標に、糸球体、近位尿細管、集合管を、変異マウスではさらに、拡張の度合いの異なる尿細管の微小領域も採取し、それぞれのサンプルから、発現する遺伝子群と発現量の情報を得た。

  • 腎尿細管の構造と機能を制御する非中心体微小管の研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2017年04月
    -
    2020年03月
     

    戸谷 美夏

     概要を見る

    上皮細胞で、微小管細胞骨格は、細胞の頂端―基底軸に沿って配向する特徴的な編成を示す。この編成は、微小管マイナス端に結合するCAMSAP3タンパク質によって維持されるが、上皮機能への関与は、未だよく分かっていない。本研究におけるCamsap3変異マウスの解析から、上皮微小管編成の不全が、多発性嚢胞腎に似た腎臓の構造異常を引き起こすことが明らかになった。変異マウスでは、近位尿細管の拡張による、嚢胞が観察された。拡張した尿細管の上皮細胞は、引き延ばされるように扁平化し、それにともない、機械刺激に応答する制御因子が活性化されることによって、細胞増殖が亢進するという嚢胞の形成機序が示唆された。

  • 極性上皮細胞における微小管の編成機構とその役割

    日本学術振興会  科学研究費助成事業

    研究期間:

    2013年04月
    -
    2016年03月
     

    戸谷 美夏

     概要を見る

    上皮細胞は、多くの臓器の主成分で、上(頂端面)、下(基底面)という極性をもつ。微小管細胞骨格が、頂端-基底軸に沿って配向する特徴的な編成を示すことが知られていたが、その仕組みは不明であった。微小管は、それ自身がプラス端・マイナス端という極性をもつ。本研究では、微小管マイナス端結合タンパク質CAMSAP3に着目し、マウス小腸と、培養上皮細胞を用いた解析を行った。その結果、CAMSAP3が頂端面に局在してマイナス端を繋ぎとめることで、上皮細胞に特徴的な微小管編成を可能にしていること、そのような微小管編成は、核やゴルジ体などの細胞内小器官が正しく配置されるために必須の働きを示すことが明らかになった。

  • 線虫初期胚の紡錘体形成に関わる2つの微小管制御経路の解析

    日本学術振興会  科学研究費助成事業

    研究期間:

    2009年
    -
    2011年
     

    戸谷 美夏

     概要を見る

    微小管の形成起点として働く分子として、γチューブリンが広く知られているが、線虫初期胚の紡錘体形成時には、γチューブリン(TBG-1)に依存しない、オーロラキナーゼA(AIR-1)が担う微小管形成経路が存在する。本研究において、AIR-1は、そのキナーゼ活性が分裂期に重要な役割を果たすことに加えて、キナーゼ不活性型としても、紡錘体形成時に重要な役割を果たしている可能性を示した。機能的な紡錘体の形成には、中心体におけるγチューブリン複合体とAIR-1キナーゼ活性、微小管上で働くキナーゼ不活性型AIR-1とが協調して働く必要があることが示唆された。

 

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  • 理工学術院   大学院先進理工学研究科

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特定課題制度(学内資金)

  • 上皮微小管の制御がつかさどる尿細管拡張の分子機構

    2023年   佐藤 政充, 竹山 春子, 角井 康貢

     概要を見る

    上皮細胞は、腎臓や腸など多くの臓器を構成する主要な細胞で、細胞内の微小管は、特徴的な配向(上皮微小管編成)を示す。我々はこれまでに、微小管結合タンパク質Camsap3が上皮微小管編成の鍵となること、Camsap3変異マウスの腎臓に、多発性の嚢胞が形成されることを見出した。嚢胞腎は、その多くは良性であるが、遺伝性の疾患も知られる。遺伝性の嚢胞腎に関する多くの研究がなされているが、嚢胞形成のしくみについては、未だ多くのことがわかっていない。Camsap3変異マウスの腎嚢胞では、腎臓の最小機能単位であるネフロンを構成する尿細管の一部である近位尿細管が拡張する。拡張した尿細管では、上皮細胞の微小管編成が乱れ細胞が扁平化する。扁平化した細胞では、転写因子の局在変化が観察され、発現遺伝子の変動が示唆された。このことから、細胞の形態を指標に、腎組織切片から直径100μの微小領域を採取し、遺伝子発現解析を進めている。野生型とCamsap3ノックアウト(KO)マウスの腎組織から、それぞれ、糸球体、近位尿細管(PT: Proximal tubule)、集合管を、加えてKOマウスからは、嚢胞のサイズを区別した微小領域(KO-PTSC: KO-PT Small Cyst&nbsp;と、KO-PTLC: KO-PT Large Cyst)を採取し、mRNAを抽出した。本課題では、打ち抜いた組織片から得られた、発現遺伝子情報の解析を進めた。近位尿細管(PT)に着目すると、野生型マウスPTと比べて、KOマウスで形態変化を伴わないPTよりも、嚢胞形成初期(KO-PTSC)に、有意に発現変動する遺伝子がより多く検出された。これまで、腎組織を崩し細胞を分取しての発現解析は行われていたが、複雑な組織構造をもつ腎臓おいて、上皮微小管の編成が寄与する微細な細胞形態の変化にともなう遺伝子発現変動を検出することができたと考える。

  • 卵母細胞と体細胞を橋渡しする微小管の機能

    2023年   佐藤 政充

     概要を見る

    微小管は、細胞の極性や形態形成、物質の輸送など、細胞がもつ基本的な機能に重要な役割を果たす。微小管の制御については、in vitro実験や培養細胞を用いた多くの研究が進められているが、哺乳類の組織における、編成制御とその役割については、未だ多くのことがわかっていない。我々はこれまでに、微小管結合タンパク質Camsap3が、多くの臓器を構成する主要な細胞である上皮細胞において、微小管編成の鍵となる因子であることを見出した。Camsap3ノックアウト(KO)マウスを作出して解析を進める過程で、雌マウスが不妊となることに気づいた。Camsap3KO雌マウスは、性周期の進行は野生型と同等であるが、過排卵処理をおこなっても排卵が認められない。卵巣組織を調べたところ、Camsap3KOマウスでは、排卵に至る成熟した卵胞がほとんど観察されなかった。卵胞は、生殖細胞である卵母細胞と、それを取り囲む母体の体細胞(顆粒層細胞)から構成される。本研究課題では、Camsap3KOマウスにおける卵胞成熟過程について、解析を行った。その結果、1)&nbsp;Camsap3KOの卵巣では、一次卵胞、二次卵胞は、野生型と同様に観察されたが、胞状卵胞・グラーフ卵胞の数が有意に減少し、2)&nbsp;残存する胞状卵胞では、顆粒層細胞のアポトーシスが観察された。これらの観察は、二次卵胞以降の卵胞成熟過程に欠陥があり、卵胞の退縮がおこることを示唆する。二次卵胞では、卵母細胞を取り囲む透明帯の発達に伴って、顆粒層細胞から、透明帯をつきぬけて卵母細胞に至る細胞骨格系の突起Transzonal projection (TZP)が形成される。Camsap3KOでは、TZPの数が減少することがわかった。これまでTZPは、アクチン系の構造と考えられてきたが、その形成や維持にCamsap3が制御する微小管が重要な役割を果たすことがわかってきた。

  • 卵胞成熟における微小管制御タンパク質CAMSAP3の働き

    2022年   佐藤 政充

     概要を見る

    Microtubule cytoskeleton dynamically changes its organisation within cells and plays an important role in fundamental cellular functions, such as cell polarity and intracellular transport. Many studies have examined the regulation of microtubule organisation using in vitro experiments and model organisms; however, the regulation of microtubule organisation and its role in the organs and tissues in the mammalian body remains poorly understood. We have previously identified the microtubule-end-associated protein Camsap3 as a key regulator of microtubule organisation in epithelial cells, which are the major cellular component of many organs. In this research project, we focused on the process of follicle maturation in the ovaries as a contributing factor to the infertility of mice lacking Camsap3 function. Moreover, we analysed the transzonal projection, which is the structure of cytoskeletal bridges between the oocyte and the mother.&nbsp;

  • 上皮細胞の微小管が司る細胞小器官の構造と機能

    2021年   佐藤政充

     概要を見る

    上皮細胞は、臓器を構成する主要な細胞である。上皮細胞内で、微小管は、細胞の頂端―基底軸に沿って配向する特徴的な編成(上皮微小管編成)を示す。上皮微小管編成は、上皮細胞が正しく働くために重要であると考えられているが、その役割については、未だ多くのことが分かっていない。我々はこれまでに、微小管端結合タンパク質CAMSAP3が、上皮微小管編成の鍵となることを見出し、解析を行っている。CAMSAP3の機能が欠損すると、上皮微小管編成の乱れにともなって、細胞小器官の配置や形態が変化する。本研究課題では、培養上皮細胞を用いて、上皮微小管編成とミトコンドリアの形態、活性の関係を調べた。

  • 非中心体微小管が関わる腎尿細管構造の生理的役割

    2019年   佐藤政充, 竹市雅俊

     概要を見る

    腎臓は、体内の水分や塩分を調節する重要な役割をもつ。その構造は複雑で、皮質と髄質に分けられる2つの層に、糸球体と尿細管から成るネフロンと呼ばれる最小機能単位が、多数詰まっている。我々は、中心体に依存しない微小管に結合するCAMSAP3タンパク質の機能欠損マウスにおいて、腎臓の構造異常を見出した。組織化学解析から、Camsap3変異マウスでは、腎皮質に存在する尿細管の一部が拡張することがわかった。本研究課題では、尿および血液サンプルの生化学検査をおこない、CAMSAP3の機能欠損により生じる腎臓の構造異常が、生理的機能に与える影響を調べた。

  • 上皮細胞に特徴的な微小管編成が構築されるしくみ

    2018年  

     概要を見る

    上皮細胞は、臓器を構成する主要な成分であり、生体内で頂端―基底の極性をもつ。分化した上皮細胞では、微小管細胞骨格は、頂端―基底の軸に沿って規則正しく配向する。この特徴的な微小管編成は、上皮細胞が正しく機能するために重要であると考えられているが、編成構築のしくみについては、いまだ多くのことが分かっていない。本研究では、幹細胞領域と分化した細胞を同時に観察することが可能な、マウス小腸の上皮組織に着目した。絨毛に隣接して窪んだ、上皮幹細胞を含む領域(陰窩)と、分化した細胞から成る絨毛中央部に存在する細胞について、微小管編成を比較観察し、分化にともなう微小管編成の変化を調べた。

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