2022/10/01 更新

写真a

ウエダ タロウ
上田 太郎
所属
理工学術院 先進理工学部
職名
教授

兼担

  • 理工学術院   大学院先進理工学研究科

  • 附属機関・学校   グローバルエデュケーションセンター

学内研究所等

  • 2020年
    -
    2022年

    理工学術院総合研究所   兼任研究員

学歴

  •  
     
     

    東京大学  

  •  
     
     

    東京大学大学院  

  •  
     
     

    University of Tokyo  

  •  
     
     

    University of Tokyo  

学位

  • 理学博士

所属学協会

  •  
     
     

    生物物理学会

  •  
     
     

    アメリカ細胞生物学会

  •  
     
     

    The Biophysical Society of Japan

  •  
     
     

    The American Society for Cell Biology

  •  
     
     

    日本細胞性粘菌学会

 

研究分野

  • 生物物理学

研究キーワード

  • ミオシン 分子モーター 細胞運動 細胞質分裂

  • Cytokinesis

  • Cell motility

  • Molecular motor

  • Myosin

論文

  • Characterization of phalloidin-negative nuclear actin filaments in U2OS cells expressing cytoplasmic actin-EGFP.

    Akira Nagasaki, Kaoru Katoh, Masamichi Hoshi, Motomichi Doi, Chikashi Nakamura, Taro Q P Uyeda

    Genes to Cells : devoted to molecular & cellular mechanisms   27 ( 5 ) 317 - 330  2022年05月  [国際誌]

     概要を見る

    Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, β-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-β-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.

    DOI PubMed

  • Unidirectional cooperative binding of fimbrin actin-binding domain 2 to actin filament

    Naoki Hosokawa, Masahiro Kuragano, Atsuki Yoshino, Keitaro Shibata, Taro Q.P. Uyeda, Kiyotaka Tokuraku

    Biochemical and Biophysical Research Communications   552   59 - 65  2021年05月  [査読有り]  [国際誌]

     概要を見る

    Fimbrin forms bundles of parallel actin filaments in filopodia, but it remains unclear how fimbrin forms well-ordered bundles. To address this issue, we focused on the cooperative interaction between the actin-binding domain of fimbrin and actin filaments. First, we loosely immobilized actin filaments on a glass surface via a positively charged lipid layer and observed the binding of GFP-fused actin-binding domain 2 of fimbrin using fluorescence microscopy. The actin-binding domain formed low-density clusters with unidirectional growth along actin filaments. When the actin filaments were tightly immobilized to the surface by increasing the charge density of the lipid layer, cluster formation was suppressed. This result suggests that the propagation of cooperative structural changes of actin filaments evoked by binding of the actin-binding domain was suppressed by a strong physical interaction with the glass surface. Interestingly, binding of the fimbrin actin-binding domain shortened the length of loosely immobilized actin filaments. Based on these results, we propose that fimbrin-actin interactions accompanied by unidirectional long-range allostery help the formation of well-ordered parallel actin filament bundles.

    DOI PubMed

  • Long-Range and Directional Allostery of Actin Filaments Plays Important Roles in Various Cellular Activities.

    Kiyotaka Tokuraku, Masahiro Kuragano, Taro Q P Uyeda

    International journal of molecular sciences   21 ( 9 )  2020年05月  [査読有り]  [招待有り]  [国際誌]

     概要を見る

    A wide variety of uniquely localized actin-binding proteins (ABPs) are involved in various cellular activities, such as cytokinesis, migration, adhesion, morphogenesis, and intracellular transport. In a micrometer-scale space such as the inside of cells, protein molecules diffuse throughout the cell interior within seconds. In this condition, how can ABPs selectively bind to particular actin filaments when there is an abundance of actin filaments in the cytoplasm? In recent years, several ABPs have been reported to induce cooperative conformational changes to actin filaments allowing structural changes to propagate along the filament cables uni- or bidirectionally, thereby regulating the subsequent binding of ABPs. Such propagation of ABP-induced cooperative conformational changes in actin filaments may be advantageous for the elaborate regulation of cellular activities driven by actin-based machineries in the intracellular space, which is dominated by diffusion. In this review, we focus on long-range allosteric regulation driven by cooperative conformational changes of actin filaments that are evoked by binding of ABPs, and discuss roles of allostery of actin filaments in narrow intracellular spaces.

    DOI PubMed

  • Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

    Yuuki Hayakawa, Masak Takaine, Taiga Imai, Masafumi D. Yamada, Keiko Hirose, Kien Xuan Ngo, Noriyuki Kodera, Kiyotaka Tokuraku, Osamu Numata, Kentaro Nakano, Taro Q.P. Uyeda

       2020年04月

     概要を見る

    <title>Abstract</title>The contraction of contractile rings (CRs) depends on interaction between actin filaments and myosin II filaments. The rate of contraction in the fission yeast<italic>Schizosaccharomyces pombe</italic>is less than 1/120 of the velocity of acto-myosin II movement<italic>in vitro</italic>, but the mechanism of inhibition has not been described. Here, we found that the calponin-homology actin binding domain of fission yeast IQGAP Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on skeletal muscle myosin II fragments<italic>in vitro</italic>, even at a low ratio of bound Rng2CHD to actin protomers, reducing the sliding velocity to half when the binding ratio was 1/75. Rng2CHD also induced structural changes of actin filaments and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II carrying ADP. Intriguingly, actin-activated ATPase of S1 was only mildly inhibited, even by high concentrations of Rng2CHD. Moreover, the motility of actin filaments by myosin V was not inhibited by Rng2CHD. We propose a new regulatory mechanism for acto-myosin II movement that involves Rng2CHD-induced structural changes of actin filaments.

    DOI

  • CHLOROPLAST UNUSUAL POSITIONING 1 is a new type of actin nucleation factor in plants

    Sam-Geun Kong, Atsushi Shimada, Saku T. Kijima, Keiko Hirose, Kaoru Katoh, Jeongsu Ahn, Takeshi Higa, Akira Takano, Yuki Nakamura, Noriyuki Suetsugu, Daisuke Kohda, Taro Q. P. Uyeda, Masamitsu Wada

       2020年01月

     概要を見る

    <title>SUMMARY</title>Plants have evolved unique responses to fluctuating light conditions in their environment. One such response, chloroplast photorelocation movement, optimizes photosynthesis under weak light and prevents photodamage under strong light. CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) plays a pivotal role in the light-responsive chloroplast movements, which are driven by dynamic reorganization of chloroplast actin (cp-actin) filaments. In this study, we demonstrated that fluorescently tagged CHUP1 colocalized and was coordinately reorganized with cp-actin filaments during chloroplast movement in <italic>Arabidopsis thaliana</italic>. The resulting asymmetric distribution of CHUP1 was reversibly regulated by the blue light receptor phototropin. X-ray crystallography indicated that the CHUP1 C-terminal domain shares structural similarity with the formin homology 2 (FH2) domain, although there is no sequence similarity between the two domains. The CHUP1 C-terminal domain stimulated actin polymerization in the presence of profilin. We conclude that CHUP1 is a novel, plant-specific actin nucleator that functions in cp-actin-based chloroplast movement.

    <sec><title>Highlights</title><list list-type="order"><list-item>Blue light changes the distribution pattern of CHUP1

    </list-item><list-item>Formin FH2 and CHUP1 C-terminal domains are structurally similar but not homologous

    </list-item><list-item>CHUP1 nucleates and severs actin filaments in vitro

    </list-item><list-item>CHUP1 is a novel, plant-specific actin nucleator for chloroplast movement

    </list-item></list>

    </sec>

    DOI

  • Tree of Motility – A Proposed History of Motility Systems in the Tree of Life –

    Makoto MIYATA, Robert C ROBINSON, Taro QP UYEDA, Yoshihiro Fukumori, Shun-ichi Fukushima, Shin Haruta, Michio HOMMA, Kazuo Inaba, Masahiro Ito, Chikara Kaito, Kentaro KATO, Tsuyoshi KENRI, Yoshiaki Kinosita, Seiji Kojima, Tohru Minamino, Hiroyuki MORI, Shuichi Nakamura, Daisuke Nakane, Koji NAKAYAMA, Masayoshi NISHIYAMA, Satoshi SHIBATA, Katsuya Shimabukuro, Masatada Tamakoshi, Azuma TAOKA, Yosuke Tashiro, Isil Tulum, Hirofumi WADA, Ken-ichi WAKABAYASHI

    Genes to Cells   25 ( 1 ) 6 - 21  2020年01月  [査読有り]  [国際誌]

    担当区分:責任著者

    DOI PubMed

  • Dynamin-Like Protein B of Dictyostelium Contributes to Cytokinesis Cooperatively with Other Dynamins.

    Fujimoto K, Tanaka M, Rana AYKMM, Jahan MGS, Itoh G, Tsujioka M, Uyeda TQP, Miyagishima SY, Yumura S

    Cells   8 ( 8 )  2019年07月  [査読有り]

    DOI PubMed

  • K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins.

    Nobuhisa Umeki, Keitaro Shibata, Taro Q P Noguchi, Keiko Hirose, Yasushi Sako, Taro Q P Uyeda

    Scientific reports   9 ( 1 ) 5353 - 5353  2019年03月  [査読有り]  [国際誌]

     概要を見る

    Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion.

    DOI PubMed

  • Actin-binding domains mediate the distinct distribution of two Dictyostelium Talins through different affinities to specific subsets of actin filaments during directed cell migration.

    Masatsune Tsujioka, Taro Q P Uyeda, Yoshiaki Iwadate, Hitesh Patel, Keitaro Shibata, Tenji Yumoto, Shigenobu Yonemura

    PloS one   14 ( 4 ) e0214736  2019年  [査読有り]  [国際誌]

     概要を見る

    Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration.

    DOI PubMed

  • Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells

    Saku T. Kijima, Christopher J. Staiger, Kaoru Katoh, Akira Nagasaki, Kohji Ito, Taro Q. P. Uyeda

    Scientific Reports   8 ( 1 ) 4381  2018年12月  [査読有り]

     概要を見る

    Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions
    however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.

    DOI PubMed

  • A genome editing vector that enables easy selection and identification of knockout cells.

    Nagasaki A, Kato Y, Meguro K, Yamagishi A, Nakamura C, Uyeda TQP

    Plasmid    2018年09月  [査読有り]

    DOI PubMed

  • Uni-directional propagation of structural changes in actin filaments

    Taro Q.P. Uyeda, Kien Xuan Ngo, Noriyuki Kodera, Kiyotaka Tokuraku

    The Role of Water in ATP Hydrolysis Energy Transduction by Protein Machinery     157 - 177  2018年05月  [査読有り]

     概要を見る

    © Springer Nature Singapore Pte Ltd. 2018. When a protein molecule is bound with another, its structure is likely to change in one way or the other. The structure of a protein molecule in a protein complex is also likely to change when binding partner in the complex undergoes a conformational change. It is therefore no surprise that binding of an actin-binding protein to a protomer in an actin filament changes the structure of that actin pro-tomer, and that the resultant conformational change in the actin protomer affects the structure of the neighboring protomers in the same filament. Moreover, eukaryotic actin appears to have evolved to efficiently spread the conformational change in the actin protomer initially bound with actin-binding protein over a long distance along the filament (cooperative conformational change), as has been observed in the cases of cofilin- and myosin-induced cooperative conformational changes. We speculate that the high degree of cooperativity in conformational changes in actin filaments enables cooperative binding of actin-binding proteins, which is necessary for actin filaments to perform specific functions by selectively interacting with a subset of actin-binding proteins among the large number of actin-binding proteins present in the cell. Interestingly, cooperative conformational changes propagate to only one direction along the filament, at least in the cases of cofilin and myosin II-induced conformational changes. Functional significance of those uni-directional conformational changes in actin filaments is not known, but we propose that they play roles in directional signal transmission along one-dimensional polymer in cells, or in force generation by myosin.

    DOI

  • Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts.

    Masahiro Kuragano, Taro Q P Uyeda, Keiju Kamijo, Yota Murakami, Masayuki Takahashi

    Molecular biology of the cell   29 ( 8 ) 911 - 922  2018年04月  [査読有り]  [国際誌]

     概要を見る

    We demonstrated that myosin IIA and IIB are essential for the formation of transverse arcs and ventral stress fibers, respectively. Furthermore, we illustrated the roles of both isoforms in lamellar flattening and also raised the possibility that actin filaments in ventral stress fibers are in a stretched conformation.

    DOI PubMed

  • Measurement of enzymatic and motile activities of Arabidopsis myosins by using Arabidopsis actins

    Sa Rula, Takahiro Suwa, Saku T. Kijima, Takeshi Haraguchi, Shinryu Wakatsuki, Naruki Sato, Zhongrui Duan, Motoki Tominaga, Taro Q.P. Uyeda, Kohji Ito

    Biochemical and Biophysical Research Communications   495 ( 3 ) 2145 - 2151  2018年01月  [査読有り]

     概要を見る

    There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.

    DOI PubMed

  • Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy

    Rika Hirakawa, Yusuke Nishikawa, Taro Q. P. Uyeda, Kiyotaka Tokuraku

    CYTOSKELETON   74 ( 12 ) 482 - 489  2017年12月  [査読有り]

     概要を見る

    Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.

    DOI PubMed

  • Cell Mechanosensors and the Possibilities of Using Magnetic Nanoparticles to Study Them and to Modify Cell Fate

    Yajing Shen, Yu Cheng, Taro Q. P. Uyeda, Gustavo R. Plaza

    ANNALS OF BIOMEDICAL ENGINEERING   45 ( 10 ) 2475 - 2486  2017年10月  [査読有り]

     概要を見る

    The use of magnetic nanoparticles (MNPs) is a promising technique for future advances in biomedical applications. This idea is supported by the availability of MNPs that can target specific cell components, the variety of shapes of MNPs and the possibility of finely controlling the applied magnetic forces. To examine this opportunity, here we review the current developments in the use of MNPs to mechanically stimulate cells and, specifically, the cell mechanotransduction systems. We analyze the cell components that may act as mechanosensors and their effect on cell fate and we focus on the promising possibilities of controlling stem-cell differentiation, inducing cancer-cell death and treating nervous-system diseases.

    DOI PubMed

  • Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin

    Kohei Iwase, Masateru Tanaka, Keiko Hirose, Taro Q. P. Uyeda, Hajime Hondal

    PLOS ONE   12 ( 7 ) e0181171  2017年07月  [査読有り]

     概要を見る

    We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin) was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

    DOI PubMed

  • The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells

    Akira Nagasaki, Saku T. Kijima, Tenji Yumoto, Miku Imaizumi, Ayana Yamagishi, Hyonchol Kim, Chikashi Nakamura, Taro Q. P. Uyeda

    CELL STRUCTURE AND FUNCTION   42 ( 2 ) 131 - 140  2017年  [査読有り]

     概要を見る

    Actin, a major component of microfilaments, is involved in various eukaryotic cellular functions. Over the past two decades, actin fused with fluorescent protein has been used as a probe to detect the organization and dynamics of the actin cytoskeleton in living eukaryotic cells. It is generally assumed that the expression of fusion protein of fluorescent protein does not disturb the distribution of endogenous actin throughout the cell, and that the distribution of the fusion protein reflects that of endogenous actin. However, we noticed that EGFP-beta-actin caused the excessive formation of microfilaments in several mammalian cell lines. To investigate whether the position of the EGFP tag on actin affected the formation of filaments, we constructed an expression vector harboring a beta-actin-EGFP gene. In contrast to EGFP-beta-actin, cells expressing beta-actin-EGFP showed actin filaments in a high background from the monomer actin in cytosol. Additionally, the detergent insoluble assay revealed that the majority of the detergent-insoluble cytoskeleton from cells expressing EGFP-beta-actin was recovered in the pellet. Furthermore, we found that the expression of EGFP-beta-actin affects the migration of NBT-L2b cells and the mechanical stiffness of U2OS cells. These results indicate that EGFP fused to the N-terminus of actin tend to form excessive actin filaments. In addition, EGFP-actin affects both the cellular morphological and physiological phenotypes as compared to actin-EGFP.

    DOI PubMed

  • Elongated Nanoparticle Aggregates in Cancer Cells for Mechanical Destruction with Low Frequency Rotating Magnetic Field

    Yajing Shen, Congyu Wu, Taro Q. P. Uyeda, Gustavo R. Plaza, Bin Liu, Yu Han, Maciej S. Lesniak, Yu Cheng

    THERANOSTICS   7 ( 6 ) 1735 - 1748  2017年  [査読有り]

     概要を見る

    Magnetic nanoparticles (MNPs) functionalized with targeting moieties can recognize specific cell components and induce mechanical actuation under magnetic field. Their size is adequate for reaching tumors and targeting cancer cells. However, due to the nanometric size, the force generated by MNPs is smaller than the force required for largely disrupting key components of cells. Here, we show the magnetic assembly process of the nanoparticles inside the cells, to form elongated aggregates with the size required to produce elevated mechanical forces. We synthesized iron oxide nanoparticles doped with zinc, to obtain high magnetization, and functionalized with the epidermal growth factor (EGF) peptide for targeting cancer cells. Under a low frequency rotating magnetic field at 15 Hz and 40 mT, the internalized EGF-MNPs formed elongated aggregates and generated hundreds of pN to dramatically damage the plasma and lysosomal membranes. The physical disruption, including leakage of lysosomal hydrolases into the cytosol, led to programmed cell death and necrosis. Our work provides a novel strategy of designing magnetic nanomedicines for mechanical destruction of cancer cells.

    DOI PubMed

  • Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    Kien Xuan Ngo, Nobuhisa Umeki, Saku T. Kijima, Noriyuki Kodera, Hiroaki Ueno, Nozomi Furutani-Umezu, Jun Nakajima, Taro Q. P. Noguchi, Akira Nagasaki, Kiyotaka Tokuraku, Taro Q. P. Uyeda

    SCIENTIFIC REPORTS   6   35449  2016年10月  [査読有り]

     概要を見る

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 mu M ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

    DOI PubMed

  • Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin

    Shuxian Huang, Ryo Umemoto, Yuki Tamura, Yutaka Kofuku, Taro Q. P. Uyeda, Noritaka Nishida, Ichio Shimada

    SCIENTIFIC REPORTS   6   33690  2016年09月  [査読有り]

     概要を見る

    Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G-or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin beta 4 (T beta 4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the H-1-N-15 HSQC spectra of T beta 4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of T beta 4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/T beta 4 complex that shows great accordance with the reference structure.

    DOI PubMed

  • Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    Nobuhisa Umeki, Keiko Hirose, Taro Q. P. Uyeda

    SCIENTIFIC REPORTS   6   20406  2016年02月  [査読有り]

     概要を見る

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive.

    DOI PubMed

  • Distinct Biochemical Properties of Arabidopsis thaliana Actin Isoforms

    Saku T. Kijima, Keiko Hirose, Sam-Geun Kong, Masamitsu Wada, Taro Q. P. Uyeda

    PLANT AND CELL PHYSIOLOGY   57 ( 1 ) 46 - 56  2016年01月  [査読有り]

     概要を見る

    Plants and animals express multiple actin isoforms in a manner that is dependent on tissues, organs and the stage of development. Previous genetic analyses suggested that individual actin isoforms have specific roles in cells, but there is little biochemical evidence to support this hypothesis. In this study, we purified four recombinant Arabidopsis actin isoforms, two major vegetative actin isoforms, ACT2 and ACT7, and two major reproductive isoforms, ACT1 and ACT11, and characterized them biochemically. Phalloidin bound normally to the filaments of the two reproductive actins as well as to the filaments of skeletal muscle actin. However, phalloidin bound only weakly to ACT7 filaments and hardly at all to ACT2 filaments, despite the conserved sequence of the phalloidin-binding site. Polymerization and phosphate release rates among these four actin isoforms were also significantly different. Moreover, interactions with profilin (PRF) were also different among the four Arabidopsis actin isoforms. PRF1 and PRF2 inhibited the polymerization of ACT1, ACT11 and ACT7, while ACT2 was only weakly affected. Plant actin isoforms have different biochemical properties. This result supports the idea that actin isoforms play specific roles to achieve multiple cell functions.

    DOI PubMed

  • Actin binding domain of filamin distinguishes posterior from anterior actin filaments in migrating Dictyostelium cells.

    Keitaro Shibata, Akira Nagasaki, Hiroyuki Adachi, Taro Q P Uyeda

    Biophysics and physicobiology   13   321 - 331  2016年  [査読有り]  [国内誌]

     概要を見る

    Actin filaments in different parts of a cell interact with specific actin binding proteins (ABPs) and perform different functions in a spatially regulated manner. However, the mechanisms of those spatially-defined interactions have not been fully elucidated. If the structures of actin filaments differ in different parts of a cell, as suggested by previous in vitro structural studies, ABPs may distinguish these structural differences and interact with specific actin filaments in the cell. To test this hypothesis, we followed the translocation of the actin binding domain of filamin (ABDFLN) fused with photoswitchable fluorescent protein (mKikGR) in polarized Dictyostelium cells. When ABDFLN-mKikGR was photoswitched in the middle of a polarized cell, photoswitched ABDFLN-mKikGR rapidly translocated to the rear of the cell, even though actin filaments were abundant in the front. The speed of translocation (>3 μm/s) was much faster than that of the retrograde flow of cortical actin filaments. Rapid translocation of ABDFLN-mKikGR to the rear occurred normally in cells lacking GAPA, the only protein, other than actin, known to bind ABDFLN. We suggest that ABDFLN recognizes a certain feature of actin filaments in the rear of the cell and selectively binds to them, contributing to the posterior localization of filamin.

    DOI PubMed

  • The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

    Taro Q. P. Noguchi, Masatoshi Morimatsu, Atsuko H. Iwane, Toshio Yanagida, Taro Q. P. Uyeda

    PLOS ONE   10 ( 5 ) e0126262  2015年05月  [査読有り]

     概要を見る

    The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

    DOI PubMed

  • Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

    Kien Xuan Ngo, Noriyuki Kodera, Eisaku Katayama, Toshio Ando, Taro Q. P. Uyeda

    ELIFE   4  2015年02月  [査読有り]

     概要を見る

    High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was similar to 25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries.

    DOI PubMed

  • Identification of kinases and regulatory proteins required for cell migration using a transfected cell-microarray system

    Reiko Onuki-Nagasaki, Akira Nagasaki, Kazumi Hakamada, Taro Q. P. Uyeda, Masato Miyake, Jun Miyake, Satoshi Fujita

    BMC GENETICS   16   9  2015年02月  [査読有り]

     概要を見る

    Background: Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells.
    Results: We performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration.
    Conclusions: The genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.

    DOI PubMed

  • Study of the influence of actin-binding proteins using linear analyses of cell deformability

    Gustavo R. Plaza, Taro Q. P. Uyeda, Zahra Mirzaei, Craig A. Simmons

    SOFT MATTER   11 ( 27 ) 5435 - 5446  2015年  [査読有り]

     概要を見る

    The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, alpha-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of alpha-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over crosslinks during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actinbinding proteins, deformability and mechanosensing.

    DOI PubMed

  • Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Yuki Gomibuchi, Taro Q. P. Uyeda, Takeyuki Wakabayashi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   441 ( 4 ) 844 - 848  2013年11月  [査読有り]

     概要を見る

    Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 angstrom(2) to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostetium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V-max) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K-app) than that of the wild-type actin, with the 17 V-max being almost unchanged. The K-app and V-max of the Tyr143Phe-actin were similar to those of the wildtype actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because I/K-app reflects the affinity of F-actin for the myosin-ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release. (C) 2013 Elsevier Inc. All rights reserved.

    DOI PubMed

  • ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

    Nanami Shiozaki, Kentaro Nakano, Yasuharu Kushida, Taro Q. P. Noguchi, Taro Q. P. Uyeda, Dorota Wloga, Drashti Dave, Krishna Kumar Vasudevan, Jacek Gaertig, Osamu Numata

    EUKARYOTIC CELL   12 ( 8 ) 1080 - 1086  2013年08月  [査読有り]

     概要を見る

    ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

    DOI PubMed

  • Contraction speed of the actomyosin cytoskeleton in the absence of the cell membrane

    Gustavo R. Plaza, Taro Q. P. Uyeda

    Soft Matter   9 ( 17 ) 4390 - 4400  2013年05月  [査読有り]

     概要を見る

    The contraction of the actomyosin cytoskeleton, which is produced by the sliding of myosin II along actin filaments, drives important cellular activities such as cytokinesis and cell migration. To explain the contraction velocities observed in such physiological processes, we have studied the contraction of intact cytoskeletons of Dictyostelium discoideum cells after removing the plasma membrane using Triton X-100. The technique developed in this work allows for the quantitative measurement of contraction rates of individual cytoskeletons. The relationship of the contraction rates with forces was analyzed using three different myosins with different in vitro sliding velocities. The cytoskeletons containing these myosins were always contractile and the contraction rate was correlated with the sliding velocity of the myosins. However, the values of the contraction rate were two to three orders of magnitude slower than expected from the in vitro sliding velocities of the myosins, presumably due to internal and external resistive forces. The contraction process also depended on actin cross-linking proteins. The lack of α-actinin increased the contraction rate 2-fold and reduced the capacity of the cytoskeleton to retain internal materials, while the lack of filamin resulted in the ATP-dependent disruption of the cytoskeleton. Interestingly, the myosin-dependent contraction rate of intact contractile rings is also reportedly much slower than the in vitro sliding velocity of myosin, and is similar to the contraction rates of cytoskeletons (different by only 2-3 fold), suggesting that the contraction of intact cells and cytoskeletons is limited by common mechanisms. © 2013 The Royal Society of Chemistry.

    DOI

  • Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

    Nobuhisa Umeki, Jun Nakajima, Taro Q. P. Noguchi, Kiyotaka Tokuraku, Akira Nagasaki, Kohji Ito, Keiko Hirose, Taro Q. P. Uyeda

    JOURNAL OF BIOLOGICAL CHEMISTRY   288 ( 3 ) 1739 - 1749  2013年01月  [査読有り]

     概要を見る

    Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human alpha-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.

    DOI PubMed

  • G146V Mutation at the Hinge Region of Actin Reveals a Myosin Class-specific Requirement of Actin Conformations for Motility

    Taro Q. P. Noguchi, Tomotaka Komori, Nobuhisa Umeki, Noriyuki Demizu, Kohji Ito, Atsuko Hikikoshi Iwane, Kiyotaka Tokuraku, Toshio Yanagida, Taro Q. P. Uyeda

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 29 ) 24339 - 24345  2012年07月  [査読有り]

     概要を見る

    The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

    DOI PubMed

  • 2SE-03 アクチンフィラメントの構造多型と機能分化(2SE 運動超分子マシナリーが織りなす調和と多様性,新学術領域研究「運動超分子マシナリーが織りなす調和と多様性」共催,シンポジウム,日本生物物理学会第50回年会(2012年度))

    上田 太郎, Umeki Nobuhisa, Kijima Saku, Nishikawa Yusuke, Tokuraku Kiyotaka, Nagasaki Akira, Noguchi Taro Q.P.

    生物物理   52   S13  2012年

    DOI CiNii

  • Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    Taro Q. P. Uyeda, Yoshiaki Iwadate, Nobuhisa Umeki, Akira Nagasaki, Shigehiko Yumura

    PLOS ONE   6 ( 10 ) e26200  2011年10月  [査読有り]

     概要を見る

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

    DOI PubMed

  • Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors

    Nobuhisa Umeki, Taro Q. P. Uyeda

    ANALYTICAL BIOCHEMISTRY   414 ( 2 ) 309 - 311  2011年07月  [査読有り]

     概要を見る

    We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM-PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture. (C) 2011 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Structural Basis for Actin Assembly, Activation of ATP Hydrolysis, and Delayed Phosphate Release

    Kenji Murakami, Takuo Yasunaga, Taro Q. P. Noguchi, Yuki Gomibuchi, Kien X. Ngo, Taro Q. P. Uyeda, Takeyuki Wakabayashi

    CELL   143 ( 2 ) 275 - 287  2010年10月  [査読有り]

     概要を見る

    Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some alpha-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.

    DOI PubMed

  • Myosin-actin Interaction in Dictyostelium Cells Revealed by GFP-based Strain Sensor and Validated Linear Spectral Unmixing

    Sosuke Iwai, Taro Q. P. Uyeda

    CYTOMETRY PART A   77A ( 8 ) 743 - 750  2010年08月  [査読有り]

     概要を見る

    Myosin is an actin-based motor protein that is involved in a wide range of cellular motile processes. Although in vitro properties of the myosin-actin interaction have been extensively studied, the interaction in vivo remains poorly understood. Recently, we developed a GFP-based strain sensor termed PriSSM (PRIM-based strain sensor module), by using the proximity imaging (PRIM) technique, which detects spectral changes of two GFP molecules that are in direct contact. Using PriSSM-myosin II fusion proteins, the interaction between myosin II and F-actin can be detected in Dictyostelium cells. In the spectroscopic measurements of PriSSM, to decompose the measured spectra of the cells expressing the sensor proteins into the contributions from the sensor and the background autofluorescence, we applied the linear spectral unmixing approach, which was based on the assumption that the errors at each wavelength were independent. Cellular autofluorescence, however, often includes systematic errors, so that the unmixing procedures might lead to biased estimates. Here, to validate our spectral unmixing procedures, we estimate the possible maximum errors in the fluorescence ratio values that are obtained by unmixing spectra including such systematic errors. This estimation provided a general criterion to validate the results obtained by linear unmixing of spectra including serially correlated error terms. Using the proposed criterion and PriSSM-myosin II fusion proteins, we examined the interaction between myosin II and F-actin in Dictyostelium cells under several different conditions. The spectroscopic results, together with the microscopic observations of the cells expressing the proteins, suggest that the formation of myosin filaments through the tail region has only a slight effect on binding to F-actin but has significant effects on the cortical localization of myosin II. (C) 2010 International Society for Advancement of Cytometry

    DOI PubMed

  • Screening of novel dominant negative mutant actins using glycine targeted scanning identifies G146V actin that cooperatively inhibits cofilin binding

    Taro Q. P. Noguchi, Ryo Toya, Hironori Ueno, Kiyotaka Tokuraku, Taro Q. P. Uyeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   396 ( 4 ) 1006 - 1011  2010年06月  [査読有り]

     概要を見る

    A number of studies suggested that the structure of actin filaments changes by interaction with actin-binding proteins such as cofilin and myosin, and that the conformational changes of the actin subunits within a filament are cooperative. To understand the functions of these cooperative conformational changes induced by actin-binding proteins, we sought to obtain dominant negative mutant actins impaired in cooperative conformational changes. A series of mutant actin genes in which glycine residues in actin were systematically substituted by valine residues were constructed, and were expressed individually in yeast cells that carry a wild-type endogenous actin gene. Six dominant negative actin mutations were identified on the basis of growth inhibition. Among them, G146V mutation was chosen for further biochemical analysis because the Gly146 residue is located at the strategic hinge position connecting the large and small domains of an actin molecule. We found that G146V actin filaments hardly bind cofilin, consistent with a previous suggestion that cofilin binding causes conformational changes of actin around Gly146 (Galkin et al. [3]). Notably, copolymer that consists of 1:10 mixture of the mutant and wild-type actin molecules showed significantly reduced affinity for cofilin, suggesting that G146V mutant actin affects the conformation of neighboring wild-type actin within a filament, and inhibits cofilin binding. (C) 2010 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Photoregulated Assembly/Disassembly of DNA-Templated Protein Arrays Using Modified Oligonucleotide Carrying Azobenzene Side Chains

    You Hachikubo, Sosuke Iwai, Taro Q. P. Uyeda

    BIOTECHNOLOGY AND BIOENGINEERING   106 ( 1 ) 1 - 8  2010年05月  [査読有り]

     概要を見る

    DNA-templated self-assembly of nanomaterials provides great potential for applications including biosensors, nanoelectronics, and programmable and autonomous molecular machines. To switch or regulate the activities of those nanobiotechnological devices, non-invasive methods to assemble and disassemble specific nanoscale components are needed. Here, we describe photocontrol of assembly of DNA-templated protein arrays in solution, by using photo-controlled duplex formation of oligonucleotides carrying azobenzene. As a proof of concept prototype, we designed a one-dimensional protein array system that consists of a scaffold of DNA and two kinds of anchor DNA that were conjugated with fluorescent proteins (CFP and YFP, respectively). The scaffold DNA was modified to carry multiple azobenzene side chains so that the hybridization involving the scaffold DNA is regulated by photoirradiation through conformational changes of the azobenzene moieties. Melting temperatures of duplex made of the modified DNA scaffold and an anchor oligonucleotide were shifted significantly and reversibly by UV and visible photoirradiation (difference of T(m) was 34.8 degrees C in 150 mM potassium acetate). Measurements of Forster resonance energy transfer between CFP and YFP showed that the assembly of the protein array system was also changed by photoirradiation. Such non-invasive and reversible method to control assembly/disassembly of multiple, specific proteins in a DNA-templated protein array system would provide many functions for nanobiotechnological devices such as on/off switches and the ability to change the configuration reversibly. Biotechnol. Bioeng. 2010;106: 1-8. (C) 2010 Wiley Periodicals, Inc.

    DOI PubMed

  • Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana

    Noriyuki Suetsugu, Noboru Yamada, Takatoshi Kagawa, Hisashi Yonekura, Taro Q. P. Uyeda, Akeo Kadota, Masamitsu Wada

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 19 ) 8860 - 8865  2010年05月  [査読有り]

     概要を見る

    Organelle movement is essential for efficient cellular function in eukaryotes. Chloroplast photorelocation movement is important for plant survival as well as for efficient photosynthesis. Chloroplast movement generally is actin dependent and mediated by blue light receptor phototropins. In Arabidopsis thaliana, phototropins mediate chloroplast movement by regulating short actin. laments on chloroplasts (cp-actin filaments), and the chloroplast outer envelope protein CHUP1 is necessary for cp-actin. lament accumulation. However, other factors involved in cp-actin. lament regulation during chloroplast movement remain to be determined. Here, we report that two kinesin-like proteins, KAC1 and KAC2, are essential for chloroplasts to move and anchor to the plasma membrane. A kac1 mutant showed severely impaired chloroplast accumulation and slow avoidance movement. A kac1kac2 double mutant completely lacked chloroplast photorelocation movement and showed detachment of chloroplasts from the plasma membrane. KAC motor domains are similar to those of the kinesin-14 subfamily (such as Ncd and Kar3) but do not have detectable microtubule-binding activity. The C-terminal domain of KAC1 could interact with F-actin in vitro. Instead of regulating microtubules, KAC proteins mediate chloroplast movement via cp-actin. laments. We conclude that plants have evolved a unique mechanism to regulate actin-based organelle movement using kinesin-like proteins.

    DOI PubMed

  • Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

    Taro Q. P. Noguchi, Yuki Gomibuchi, Kenji Murakami, Hironori Ueno, Keiko Hirose, Takeyuki Wakabayashi, Taro Q. P. Uyeda

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 7 ) 4337 - 4347  2010年02月  [査読有り]

     概要を見る

    Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca2+. Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca2+ sensitivity in the presence of tropomyosin-troponin.

    DOI PubMed

  • Utilization of Myosin and Actin Bundles for the Transport of Molecular Cargo

    Hideyo Takatsuki, Kevin M. Rice, Shinichi Asano, B. Scott Day, Mizuki Hino, Kazuhiro Oiwa, Ryoki Ishikawa, Yuichi Hiratsuka, Taro Q. P. Uyeda, Kazuhiro Kohama, Eric R. Blough

    SMALL   6 ( 3 ) 452 - 457  2010年02月  [査読有り]

     概要を見る

    The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F-actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin-propelled filaments, transport molecules across a motor-patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.

    DOI PubMed

  • G146V mutant actin is defective in conformational changes, accompanied by impaired motility with skeletal myosin.

    Noguchi TPQ, Morimatsu M, Komori T, Iwane AH, Yanagida T, Uyeda TQP

    Biophysical Journal   98 ( 3 ) 158a  2010年01月

    DOI

  • Stabilization of anaphase midzone microtubules is regulated by Rho during cytokinesis in human fibrosarcoma cells

    Masamitsu Kanada, Akira Nagasaki, Taro Q. P. Uyeda

    EXPERIMENTAL CELL RESEARCH   315 ( 16 ) 2705 - 2714  2009年10月  [査読有り]

     概要を見る

    The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly Understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone Structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but More severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was Sufficient to maintain the midzone Structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J.. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, R DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529-9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during Mitosis and cytokinesis. (C) 2009 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Cell adhesion molecules regulate contractile ring-independent cytokinesis in Dictyostelium discoideum

    Akira Nagasaki, Masamitsu Kanada, Taro Q. P. Uyeda

    CELL RESEARCH   19 ( 2 ) 236 - 246  2009年02月  [査読有り]

     概要を見る

    To investigate the roles of substrate adhesion in cytokinesis, we established cell lines lacking paxillin (PAXB) or vinculin (VINA), and those expressing the respective GFP fusion proteins in Dictyostelium discoideum. As in mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like complexes on the cell bottom. paxB(-) cells in suspension grew normally, but on substrates, often failed to divide after regression of the furrow. The efficient cytokinesis of paxB(-) cells in suspension is not because of shear forces to assist abscission, as they divided normally in static suspension culture as well. Double knockout strains lacking mhcA, which codes for myosin II, and paxB or vinA displayed more severe cytokinetic defects than each single knockout strain. In mitotic wild-type cells, GFP-PAXB was diffusely distributed on the basal membrane, but was strikingly condensed along the polar edges in mitotic mhcA(-) cells. These results are consistent with our idea that Dictyostelium displays two forms of cytokinesis, one that is contractile ring-dependent and adhesion-independent, and the other that is contractile ring-independent and adhesion-dependent, and that the latter requires PAXB and VINA. Furthermore, that paxB(-) cells fail to divide normally in the presence of substrate adhesion suggests that this adhesion molecule may play additional signaling roles.

    DOI PubMed

  • Novel Mode of Cooperative Binding between Myosin and Mg2+-actin Filaments in the Presence of Low Concentrations of ATP

    Kiyotaka Tokuraku, Rika Kurogi, Ryo Toya, Taro Q. P. Uyeda

    JOURNAL OF MOLECULAR BIOLOGY   386 ( 1 ) 149 - 162  2009年02月  [査読有り]

     概要を見る

    Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca2+-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg2+-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg2+-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI PubMed

  • Visible-Light Photocontrol of (E)/(Z) Isomerization of the 4-(Dimethylamino)azobenzene Pseudo-Nucleotide Unit Incorporated into an Oligonucleotide and DNA Hybridization in Aqueous Media

    Takashi Kamei, Haruhisa Akiyama, Hisayuki Morii, Nobuyuki Tamaoki, Taro Q. P. Uyeda

    NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS   28 ( 1 ) 12 - 28  2009年  [査読有り]

     概要を見る

    We demonstrate significant photoresponsivity in aqueous media to visible light of pseudo-oligonucleotides possessing 4-(dimethylamino)azobenzene (4-DMAzo) side chains. The spectrum of the 4-DMAzo moiety during 436 nm light irradiation at pH 9 was clearly different from that of the all (E)-form, indicating the presence of the (Z)-form. Thermal (Z)-to-(E) recovery isomerization was faster at pH 9 (kZ-E = 101 s-1) than at pH 11; however, addition of 50% ethanol significantly slowed the thermal recovery isomerization at pH 9 (kZ-E = 2 s-1) and increased the magnitude of the spectral changes. Significant photoregulation of DNA hybridization by visible light was demonstrated under this condition.

    DOI PubMed

  • Correlated Waves of Actin Filaments and PIP3 in Dictyostelium Cells

    Yukako Asano, Akira Nagasaki, Taro Q. P. Uyeda

    CELL MOTILITY AND THE CYTOSKELETON   65 ( 12 ) 923 - 934  2008年12月  [査読有り]

     概要を見る

    Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to gain insight into roles PIP3 plays in those spontaneous motilities. In keratocyte-like cells, PIP3 showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP3 repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP3 waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP3 waves by coupling steady lamellipodial protrusions to the PIP3 waves. Simultaneous live observation of actin filaments and PIP3 in wild type or stalled amiB(-)cells indicated that the PIP3 waves were correlated with wave-like distributions of actin filaments. Most notably, PIP3 waves often followed actin waves, suggesting that PIP3 induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP3 was often correlated with local retraction of the periphery. We propose that the waves of PIP3 and actin filaments are loosely coupled with each other and play important roles in generating spontaneous Cell polarity. Cell Motil. Cytoskeleton 65: 923-934, 2008. (C) 2008 Wiley-Liss, Inc.

    DOI PubMed

  • Chemotaxis-Mediated Scission Contributes to Efficient Cytokinesis in Dictyostelium

    Akira Nagasaki, Taro Q. P. Uyeda

    CELL MOTILITY AND THE CYTOSKELETON   65 ( 11 ) 896 - 903  2008年11月  [査読有り]

     概要を見る

    Interphase amoeba of Entamoeba invadens are attracted to the furrowing region of a neighboring dividing cell to assist with the division. A seemingly similar behavior has been observed in Dictyostelium discoideum, but in this case, it has not been shown whether the movements were truly directed toward the furrowing region region or whether they have any relevance. We thus used myosin II-null cells, which spend more time than wild type cells in cytokinesis, and successfully demonstrated that nearly half of the division events involve the attraction of a neighbor cell to the furrowing region. Cells lacking the beta submit of the trimeric G protein (G beta), which are incapable of chemotaxis, did not show such midwifery. Culturing wild type cells flattened under agarose sheets also slowed the cytokinesis process, and this allowed us to demonstrate that phosphatidylinositol trisphosphate was enriched in the anterior region of midwifing cells, consistent with the view that midwifery in D. discoideum is also chemotaxis. On substrates, while only 3.6% of wild type cells were multinucleate, 8.1% of G beta-null cells were multinucleate, and this was reduced to 3.4% when they were surrounded by wild type cells. Conversely, multinucleated wild type cells increased to 6.8% when they were surrounded by G beta-null cells. Thus, G beta-null cells frequently fail to divide because they cannot assist each other's division and midwifery ensures successful cytokinesis in Dictyostelium discoideum. Cell Motil. Cytoskeleton 65: 896-903, 2008. (C) 2008 Wiley-Liss, Inc.

    DOI PubMed

  • Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor

    Sosuke Iwai, Taro Q. P. Uyeda

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   105 ( 44 ) 16882 - 16887  2008年11月  [査読有り]

     概要を見る

    Many proteins have been shown to undergo conformational changes in response to externally applied force in vitro, but whether the force-induced protein conformational changes occur in vivo remains unclear. To reveal the force-induced conformational changes, or strains, within proteins in living cells, we have developed a genetically encoded fluorescent "strain sensor," by combining the proximity imaging (PRIM) technique, which uses spectral changes of 2 GFP molecules that are in direct contact, and myosin-actin as a model system. The developed PRIM-based strain sensor module (PriSSM) consists of the tandem fusion of a normal and circularly permuted GFP. To apply strain to PriSSM, it was inserted between 2 motor domains of Dictyostelium myosin II. In the absence of strain, the 2 GFP moieties in PriSSM are in contact, whereas when the motor domains are bound to F-actin, PriSSM has a strained conformation, leading to the loss of contact and a concomitant spectral change. Using the sensor system, we found that the position of the lever arm in the rigor state was affected by mutations within the motor domain. Moreover, the sensor was used to visualize the interaction between myosin II and F-actin in Dictyostelium cells. In normal cells, myosin was largely detached from F-actin, whereas ATIP depletion or hyperosmotic stress increased the fraction of myosin bound to F-actin. The PRIM-based strain sensor may provide a general approach for studying force-induced protein conformational changes in cells.

    DOI PubMed

  • On-chip screening method for cell migration genes based on a transfection microarray

    Reiko Onuki-Nagasaki, Akira Nagasaki, Kazumi Hakamada, Taro Q. P. Uyeda, Satoshi Fujita, Masato Miyake, Jun Miyake

    LAB ON A CHIP   8 ( 9 ) 1502 - 1506  2008年09月  [査読有り]

     概要を見る

    Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy However, the traditional methods used for analysis of cell migration cannot easily be scaled up for high-throughput screening. In this study, we have attempted to develop a novel simple method for high-throughput phenotypic screening for the identification of genes that are required for cell migration. As the appropriate cell line for the method, we found NBT-L2b cells that would be suitable for screening of migration-related genes in our method without influence by other cellular processes. Moreover, the idea for printing both the labeled fibronectin, for identification of the starting region of a cell, and the green fluorescent protein (GFP) expression vector, for identification of cells that had been transfected with siRNA and of the end point of migration, brings a rapid and efficient high-throughput screening procedure. Our new method will lead to an enhanced understanding of cell migration.

    DOI PubMed

  • Overlapping functions of the two talin homologues in Dictyostelium

    Masatsune Tsujioka, Kunito Yoshida, Akira Nagasaki, Shigenobu Yonemura, Annette Mueller-Taubenberger, Taro Q. P. Uyeda

    EUKARYOTIC CELL   7 ( 5 ) 906 - 916  2008年05月  [査読有り]

     概要を見る

    Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA(-) cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB(-) cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA(-) talB(-) cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA(-) cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB(-) cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA(-) talB(-) cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.

    DOI PubMed

  • Screening of genes involved in cell migration in Dictyostelium

    Akira Nagasaki, Taro Q. P. Uyeda

    EXPERIMENTAL CELL RESEARCH   314 ( 5 ) 1136 - 1146  2008年03月  [査読有り]

     概要を見る

    A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed qfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration. (c) 2007 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Loading and unloading of molecular cargo by DNA-conjugated microtubule

    Shu Taira, Yong-Zhong Du, Yuich Hiratsuka, Taro Q. P. Uyeda, Noboru Yumoto, Masato Kodaka

    BIOTECHNOLOGY AND BIOENGINEERING   99 ( 3 ) 734 - 739  2008年02月  [査読有り]

     概要を見る

    We developed a novel method to load and unload molecular cargos to and from microtubules (MTs) that move on kinesin-coated surfaces. Quantum dots (Qds) (molecular cargo) connected to 21-mer DNA can be selectively loaded on DNA-conjugated MTs through DNA hybridization. The average velocity of the Qd-loaded MTs (0.43 +/- 0.06 mu m s(-1) at 25 degrees C) was comparable to that of control MTs. In addition, MTs conjugated with two types DNA sequences can achieve multiloading of Qds. To unload Qd molecular cargos from MTs, the DNA double helix connecting Qds to MTs were cleaved by an appropriate restriction enzyme. This biomolecular motors-based transport system should enable us to construct nanometer-scale devices such as nanobiosensor, nanofluidic system, or nanomachine.

    DOI PubMed

  • How well can an amoeba climb?

    Yoshio Fukui, Taro Q. P. Uyeda, Chikako Kitayama, Shinya Inoué

    Collected Works of Shinya Inoue: Microscopes, Living Cells, and Dynamic Molecules     819 - 824  2008年01月  [査読有り]

     概要を見る

    We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were "stalled" or no longer able to "climb up." The "apparent weight" of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only In low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod Is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense Indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II.

    DOI

  • Novel functions of Ect2 in polar lamellipodia formation and polarity maintenance during "Contractile Ring-Independent" cytokinesis in adherent cells

    Masamitsu Kanada, Akira Nagasaki, Taro Q. P. Uyeda

    MOLECULAR BIOLOGY OF THE CELL   19 ( 1 ) 8 - 16  2008年01月  [査読有り]

     概要を見る

    Some mammalian cells are able to divide via both the classic contractile ring-dependent method ( cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.

    DOI PubMed

  • Phospholipase D Is Essential for Keratocyte-like Migration of NBT-II Cells

    Akira Nagasaki, Kimiko Inotsume, Masamitsu Kanada, Taro Q. P. Uyeda

    CELL STRUCTURE AND FUNCTION   33 ( 1 ) 27 - 33  2008年  [査読有り]

     概要を見る

    NBT-II cells on collagen-coated substrates move rapidly and persistently, maintaining a semi-circular shape with a large lamellipodium, in a manner similar to fish keratocytes. The inhibitor of phospholipase D (PLD), n-butanol, completely blocked the migration and disturbed the characteristic localization of actin along the edge of lamellipodia. To investigate the functional difference between the two isozymes of PLD (PLD1 and PLD2), we transfected NBT-II cells with vectors expressing shRNA to deplete PLD1 or PLD2. Depletion of both PLD1 and 2 by RNA interference reduced the velocity of the migration, but depletion of PLD2 inhibited motility more severely than that of PLD1. Furthermore, GFP-PLD2 was localized to the protruding regions of lamellipodia in migrating cells. Thus, PLD is essential for the maintenance of keratocyte-like locomotion of NBT-II cells, presumably by regulating the actin cytoskeleton.

    DOI PubMed

  • A novel system for expressing toxic actin mutants in Dictyostelium and purification and characterization of a dominant lethal yeast actin mutant

    Taro Q. P. Noguchi, Noriko Kanzaki, Hironori Ueno, Keiko Hirose, Taro Q. P. Uyeda

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 38 ) 27721 - 27727  2007年09月  [査読有り]

     概要を見る

    We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin 6 via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin beta moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wildtype actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E2016A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3-4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.

    DOI PubMed

  • A novel shRNA vector that enables rapid selection and identification of knockdown cells

    Akira Nagasaki, Masamitsu Kanada, T'aro Q. P. Uyeda

    PLASMID   58 ( 2 ) 190 - 194  2007年09月  [査読有り]

     概要を見る

    Small interference RNA (siRNA) is a powerful tool for disrupting expression of specific genes in a variety of cells. We have developed a vector, piMARK, which mediates expression of both small hairpin RNA (shRNA) and the blasticidin resistance (Bsr) protein fused with enhanced green fluorescent protein (EGFP), enabling rapid selection and identification of knockdown cells. Using this vector, we targeted Ect2, a gene encoding a guanine nucleotide exchange factor for several small GTPases, in human cell lines. Incubation in the presence of 10 mu g/ml blasticidin S rapidly killed untransfected cells, so that after 24 h &gt;90% of surviving HeLa S3 cells emitted green fluorescence and &gt;70% were binucleate as a result of the frequent failure of cell division. The GFP-Bsr fluorescence enabled easy identification of individual knockdown cells under a fluorescence microscope, which in turn enabled unambiguous assessment of the morphological consequences of silencing Ect2. Moreover, because untransfected cells rapidly died and detached from the substrate, they were easily removed by simply rinsing the culture dishes. It thus should be possible to analyze the biochemical consequences of gene silencing en masse in the absence of a background of untransfected cells. (c) 2007 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Visible-light photoresponsivity of a 4-(dimethylamino)azobenzene unit incorporated into single-stranded DNA: Demonstration of a large spectral change accompanying isomerization in DMSO and detection of rapid (Z)-to-(E) isomerization in aqueous solution

    Takashi Kamei, Masabumi Kudo, Haruhisa Akiyama, Momoyo Wada, Jun'ichi Nagasawa, Masahiro Funahashi, Nobuyuki Tamaoki, Taro Q. P. Uyeda

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY   ( 11 ) 1846 - 1853  2007年04月  [査読有り]

     概要を見る

    We demonstrate significant visible-light photoresponsivity in a synthesized oligonucleotide containing a built-in pseudonucleotide possessing a 4-(dimethylamino)azobenzene (4-DMAzo) side chain. In dry DMSC) as solvent, two clearly distinguishable spectra corresponding to the (E) and (Z) forms of the 4-DMAzo moiety tethered to the oligonucleotide were recorded with a conventional spectrophotometer before and after irradiation with 420 nm wavelength light, which induced (E)-to-(Z) isomerization. In addition, (Z)-to-(E) isomerization was accelerated by irradiation with either visible (lambda = 550 nm) or UV (lambda = 350 nm) light, demonstrating reversible photoresponsivity of the pseudo-oligonucleotide. In aqueous solutions the (Z)-to-(E) thermal isomerization of the photoresponsive pseudo-oligonucleotide was very rapid and was only detectable by laser flash photolysis. (c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007).

    DOI

  • 3P310 GFPの相互作用による蛍光変化を用いたタンパク質構造変化の検出(バイオイメージング,ポスター発表,第45回日本生物物理学会年会)

    岩井 草介, 上田 太郎

    生物物理   47   S280  2007年

    DOI CiNii

  • Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein

    Shu Taira, Yong-Zhong Du, Yuichi Hiratsuka, Kaoru Konishi, Tai Kubo, Taro Q. P. Uyeda, Noboru Yumoto, Masato Kodaka

    BIOTECHNOLOGY AND BIOENGINEERING   95 ( 3 ) 533 - 538  2006年10月

     概要を見る

    DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis. (c) 2006 Wiley Periodicals, Inc.

    DOI PubMed CiNii J-GLOBAL

  • A microrotary motor powered by bacteria

    Yuichi Hiratsuka, Makoto Miyata, Tetsuya Tada, Taro Q. P. Uyeda

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   103 ( 37 ) 13618 - 13623  2006年09月  [査読有り]

     概要を見る

    Biological molecular motors have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures, as is seen in muscle sarcomeres and bacterial and eukaryotic flagella. The development of an appropriate interface between such biological materials and synthetic devices should enable us to realize useful hybrid micromachines. Here we describe a microrotary motor composed of a 20-mu m-diameter silicon dioxide rotor driven on a silicon track by the gliding bacterium Mycoplasma mobile. This motor is fueled by glucose and inherits some of the properties normally attributed to living systems.

    DOI PubMed J-GLOBAL

  • Genetic engineering of a Ca2+ dependent chemical switch into the linear biomotor kinesin

    K Konishi, TQP Uyeda, T Kubo

    FEBS LETTERS   580 ( 15 ) 3589 - 3594  2006年06月

     概要を見る

    Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca2+-calmodulin, the association of the engineered kinesin with microtubutes showed a steep Ca2+-dependency in ATPase activity at Ca2+ concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca2+-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca2+ -calmodulin) and, thus, can be used as a model system for a bio/nano-actuator. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI PubMed CiNii J-GLOBAL

  • Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability

    Miki Hirabayashi, Shu Taira, Suzuko Kobayashi, Kaoru Konishi, Kaoru Katoh, Yuichi Hiratsuka, Masato Kodaka, Taro Q. P. Uyeda, Noboru Yumoto, Tai Kubo

    BIOTECHNOLOGY AND BIOENGINEERING   94 ( 3 ) 473 - 480  2006年06月

     概要を見る

    We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel. (c) 2006 Wiley Periodicals, Inc.

    DOI PubMed CiNii J-GLOBAL

  • Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides

    H Yonekura, A Nomura, H Ozawa, Y Tatsu, N Yumoto, TQP Uyeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   343 ( 2 ) 420 - 427  2006年05月

     概要を見る

    We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect. (c) 2006 Elsevier Inc. All rights reserved.

    DOI PubMed CiNii J-GLOBAL

  • Intermolecular interaction of actin revealed by a dynamic light scattering technique

    N Kanzaki, TQP Uyeda, K Onuma

    JOURNAL OF PHYSICAL CHEMISTRY B   110 ( 6 ) 2881 - 2887  2006年02月

     概要を見る

    The intermolecular interaction force of actin was studied by a dynamic light scattering technique. The Mutual diffusion coefficients (D) of monomeric actin were accurately determined in a G-buffer with a low concentration of KCl from 0 to 10 mM. The translational diffusion coefficient was obtained as D-0 = (87 +/- 3) x 10(-12) m(2)center dot s(-1) at 25 degrees C and pH 7.4, which gives a hydrodynamic radius of monomeric actin of r(H) = 2.8 +/- 0.1 nm. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, assuming electrostatic and van der Waals potentials, failed to describe the change in interaction parameter (gimel) with KCl concentration, but the extended DLVO theory succeeded if an additional repulsive potential was assumed. The Hamaker constant of actin in the Ca2+-ATP bound state was determined for the first time as A(H) = 10.4 +/- 0.6 k(B)T.

    DOI PubMed CiNii J-GLOBAL

  • Photo-control of kinesin-microtubule motility using caged peptides derived from the kinesin C-terminus domain

    Akiko Nomura, Taro Q. P. Uyeda, Noboru Yumoto, Yoshiro Tatsu

    CHEMICAL COMMUNICATIONS   ( 34 ) 3588 - 3590  2006年

     概要を見る

    To design a nanoscale biodevice that can be controlled by an external stimulus, we have introduced photochemical switching peptides derived from the kinesin C-terminus domain into the kinesin-microtubule in vitro motility system.

    DOI J-GLOBAL

  • Cooperative structural change of actin filaments interacting with activated myosin motor domain, detected with copolymers of pyrene-labeled actin and acto-S1 chimera protein

    MSP Siddique, G Mogami, T Miyazaki, E Katayama, TQP Uyeda, M Suzuki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   337 ( 4 ) 1185 - 1191  2005年12月

     概要を見る

    Acto-SI chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin 11, and have MaATPase activity close to that of natural Diciryostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda. M. Suzuki, Evidence against essential roles Of subdomain I of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474-481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480 nm using 365- nm excitation. The relative increases of fluorescence intensity at 390 run were 14%, 46%, and 77% for the copolymer Filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP IS molecule appears to affect three actin molecules in SA-CP18 copolymers. (c) 2005 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • Multiple myosin II heavy chain kinases: Roles in filament assembly control and proper cytokinesis in Dictyostelium

    S Yumura, M Yoshida, Betapudi, V, LS Licate, Y Iwadate, A Nagasaki, TQP Uyeda, TT Egelhoff

    MOLECULAR BIOLOGY OF THE CELL   16 ( 9 ) 4256 - 4266  2005年09月

     概要を見る

    Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B-/C- triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.

    DOI J-GLOBAL

  • Adhesion-dependent and contractile ring-independent equatorial furrowing during cytokinesis in mammalian cells

    M Kanada, A Nagasaki, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   16 ( 8 ) 3865 - 3872  2005年08月

     概要を見る

    Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 mu M blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 mu M), which inhibits Rho-kinase, was similar to 30 mu M blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 mu M blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 mu M blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

    DOI J-GLOBAL

  • Evidence against essential roles for subdomain 1 of actin in actomyosin sliding movements

    MSP Siddique, T Miyazaki, E Katayama, TQP Uyeda, M Suzuki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   332 ( 2 ) 474 - 481  2005年07月

     概要を見る

    We have engineered acto-Slchimera proteins carrying the entire actin inserted in loop 2 of the motor domain of Dictyostelium myosin 11 with 24 or 18 residue-linkers (CP24 and CP18, respectively). These proteins were capable of self-polymerization as well as copolymerization with skeletal actin and exhibited rigor-like structures. The MgATPase rate of CP24-skeletal actin copolymer was 1.06 s(-1), which is slightly less than the V-max Of Dictyostelium S 1. Homopolymer filaments of skeletal actin, CP24, and CP 18 moved at 4.7 &PLUSMN; 0.6, 2.9 &PLUSMN; 0.6, and 4.1 &PLUSMN; 0.8 &mu; m/s (mean &PLUSMN; SD), respectively, on coverslips coated with skeletal myosin at 27&DEG; C. Statistically thermodynamic considerations suggest that the S1 portion of chimera protein mostly resides on subdomain 1 (SD-1) of the actin portion even in the presence of ATP. This and the fact that filaments of CP18 with shorter linkers moved faster than CP24 filaments suggest that SD-1 might not be as essential as conventionally presumed for actomyosin sliding interactions. (C) 2005 Elsevier Inc. All rights reserved.

    DOI J-GLOBAL

  • 運動タンパク質を用いたナノバイオマシンの構築

    平塚 祐一, 上田 太郎

    生物物理   45 ( 3 ) 134 - 139  2005年05月

     概要を見る

    さまさまな魅力的な特徴をもつタンパク質ナノマシンをいかに利用するか?成功の鍵は, これらの分子の機能を.利用しやすい形で生体外で再構成できるかどうかにかかっている.タンパク質分子モーターの分野では, 微細加工技術との融合でその道筋がつきつつある, さらに新たな挑戦として.優れた運動性を有する細胞等を利用し, 遺伝子レベルで内側から改造していく方法が提案されはじめている.

    DOI CiNii

  • Living microtransporter by uni-directional gliding of Mycoplasma along microtracks

    Y Hiratsuka, M Miyata, TQP Uyeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   331 ( 1 ) 318 - 324  2005年05月

     概要を見る

    The gliding bacterium Mycoplasma mobile adheres to plastic surfaces and mows around rigorously. However, it has not been possible to control the direction of movements on plain Surfaces. Here we report that oil patterned lithographic substrates, M. mobile cells are unable to climb tall walls and move along the bottom edge of the walls. This property, to move persistently along walls enabled us to design patterns that control direction of movements, resulting ill uni-directional circling or one-way gating between two areas. Furthermore, cells loaded with streptavidin beads following biotinylation of surface proteins moved at normal speeds. These bacteria could be useful as living microtransporters. carrying cargo around within micrometer-scale spaces. &COPY; 2005 Elsevier Inc. All rights reserved.

    DOI PubMed CiNii J-GLOBAL

  • A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa

    F Rivero, T Muramoto, AK Meyer, H Urushihara, TQP Uyeda, C Kitayama

    BMC GENOMICS   6  2005年03月

     概要を見る

    Background: Formins are multidomain proteins defined by a conserved FH2 ( formin homology 2) domain with actin nucleation activity preceded by a proline-rich FH1 ( formin homology 1) domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes.
    Results: We present a detailed sequence analysis of the 10 formins ( ForA to J) identified in the genome of the social amoeba Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells.
    Conclusion: Our analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/ FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.

    DOI J-GLOBAL

  • 2P167 ミオシンS1はアクチンのサブドメイン1の上を運動中に通らない?(筋肉(筋蛋白・収縮)))

    鈴木 誠, Siddique Md.S.P., 宮崎 崇, 片山 栄作, 上田 太郎

    生物物理   45   S161  2005年

    DOI CiNii

  • 3P323 マイコプラズマ・モービレにより駆動する微小回転モーター(バイオエンジニアリング))

    平塚 祐一, 宮田 真人, 上田 太郎

    生物物理   45   S284  2005年

    DOI CiNii

  • Dielectric and Fluoroscopic Study on the Dynamic Effects of Myosin-S1 with/without ATP on the Hyper-Mobile Water around Actin Filaments

    Makoto Suzuki, Md Shahjahan, P Siddique, Takashi Miyazaki, George Mogami, Eisaku Katayama, Takao Kodama, Taro Q P Uyeda

    FEBS JOURNAL   272   336  2005年

  • Motor protein nano-biomachine powered by self-supplying ATP

    YZ Du, Y Hiratsuka, S Taira, M Eguchi, TQP Uyeda, N Yumoto, M Kodaka

    CHEMICAL COMMUNICATIONS   ( 16 ) 2080 - 2082  2005年

     概要を見る

    A new nano-biomachine has been created from microtubules (MTs) and hetero-bifunctional polymer particles bearing pyruvate kinase, which is propelled on glass surfaces coated with kinesin by use of self-supplying ATP.

    DOI J-GLOBAL

  • Keratocyte-like locomotion in amiB-null Dictyostelium cells

    Y Asano, T Mizuno, T Kon, A Nagasaki, K Sutoh, TQP Uyeda

    CELL MOTILITY AND THE CYTOSKELETON   59 ( 1 ) 17 - 27  2004年09月

     概要を見る

    Starved Dictyostelium amoebae continuously change their shape and they are elongated along the front-rear axis during locomotion. In contrast, we found that disruption of the amiB gene, which had been identified as a gene required for the aggregation process during development, caused these cells to move in a manner similar to fish keratocytes. Starved amiB(-) cells were elongated laterally and had one large lamelli-podium along the front side arc of the cell. These cells moved unidirectionally for long distances maintaining the half-moon shape, and this movement followed the predictions of the graded radial extension model, which was originally developed to describe the keratocyte movements. Furthermore, the distributions of actin, Arp2, and myosin H in amiB(-) cells were similar to those in keratocytes. Therefore, locomotion by keratocytes and amiB(-) cells appears to be driven by similar mechanisms of cytoskeletal regulation. Double knockout cells lacking both AmiB and myosin II were still able to move unidirectionally in a keratocyte-like manner, although the frequency of those movements was lower. Thus, myosin II is dispensable for the unidirectional movement, though it likely functions in the maintenance of the characteristic half-moon shape. This mutant cell can be a useful tool for further molecular genetic analysis of the mechanism of cell locomotion. (C) 2004 Wiley-Liss, Inc.

    DOI J-GLOBAL

  • DWWA, a novel protein containing two WW domains and an IQ motif, is required for scission of the residual cytoplasmic bridge during cytokinesis in Dictyostelium

    A Nagasaki, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   15 ( 2 ) 435 - 446  2004年02月

     概要を見る

    We have identified a novel gene, dwwA, which is required for cytokinesis of Dictyostelium cells on solid surfaces. its product, Dd WW domain containing protein A (DWWA), contains several motifs, including two WW domains, an IQ motif, a C2 domain, and a proline-rich region. On substrates, cells lacking dwwA were multinucleated and larger and flatter than wild-type cells due to their frequent inability to sever the cytoplasmic bridge connecting daughter cells after mitosis. When cultured in suspension, however, dwwA-null cells seemed to carry out cytokinesis normally via a process not driven by the shearing force arising from agitation of the culture. GFP-DWWA localized to the cell cortex and nucleus; analysis of the distributions of various truncation mutants revealed that the N-terminal half of the protein, which contains the C2 domain, is required for the cortical localization of DWWA. The IQ motif of DWWA binds calmodulin in vitro. Given that the scission process is also defective in calmodulin knockdown cells cultured on substrates (Liu et al., 1992), we propose that DWWA's multiple binding domains enable it to function as an adaptor protein, facilitating the scission process through the regulation of Ca2+/calmodulin-mediated remodeling of the actin cytoskeleton and/or modulation of membrane dynamics.

    DOI J-GLOBAL

  • 2P319 マイクロパターンを用いたMyocoplasma mobileの運動方向制御(バイオエンジニアリング)

    平塚 祐一, 宮田 真人, 上田 太郎

    生物物理   44   S189  2004年

    DOI CiNii

  • 2P161 基質の微細線状パターンを認識した細胞運動の方向制御(細胞生物的課題 : 接着・運動・骨格・伝達・膜)

    浅野 由香子, 平塚 祐一, 上田 太郎

    生物物理   44   S150  2004年

    DOI CiNii

  • Regulatory mechanism of Dictyostelium myosin light chain kinase A

    H Tokumitsu, N Hatano, H Inuzuka, Y Ishikawa, TQP Uyeda, JL Smith, R Kobayashi

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 1 ) 42 - 50  2004年01月

     概要を見る

    In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr(166) by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an similar to 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr(289) and that, subsequent to Thr(166) phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr(289) is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283 - 288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr(166) phosphorylation. Simple removal of this region resulted in a significant increase in the k(cat) of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr(166) phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.

    DOI PubMed J-GLOBAL

  • Dictyostelium discoideum talin A is crucial for myosin II-independent and adhesion-dependent cytokinesis

    HIBI M, NAGASAKI A, TAKAHASHI M, YAMAGISHI A, UYEDA Tqp

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   25 ( 2 ) 127 - 140  2004年

    DOI J-GLOBAL

  • Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

    K Ito, TQP Uyeda, Y Suzuki, K Sutoh, K Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 33 ) 31049 - 31057  2003年08月

     概要を見る

    Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.

    DOI PubMed CiNii J-GLOBAL

  • ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development in Dictyostelium discoideum

    C Kitayama, TQR Uyedat

    JOURNAL OF CELL SCIENCE   116 ( 4 ) 711 - 723  2003年02月

     概要を見る

    Formins are highly conserved regulators of cytoskeletal organization and share three regions of homology: the FH1, FH2 and FH3 domains. Of the nine known formin genes or pseudogenes carried by Dictyostelium, forC is novel in that it lacks an FH1 domain. Mutant Dictyostelium lacking forC (DeltaforC) grew normally during the vegetative phase and, when starved, migrated normally and formed tight aggregates. Subsequently, however, DeltaforC cells made aberrant fruiting bodies with short stalks and sori that remained unlifted. DeltaforC aggregates were also unable to migrate as slugs, suggesting forC is involved in mediating cell movement during multicellular stages of Dictyostelium development. Consistent with this idea, expression of forC was increased significantly in aggregates of wild-type cells. GFP-ForC expressed in DeltaforC cells was localized at the crowns, which are macropinocytotic structures rich in F-actin, suggesting that, like other formin isoforms, ForC functions in close relation with the actin cytoskeleton. Truncation analysis of GFP-ForC revealed that the FH3 domain is required for ForC localization; moreover, localization of a truncated GFP-ForC mutant at the site of contacts between cells on substrates and along the cortex of cells within a multicellular culminant suggests that ForC is involved in the local actin cytoskeletal reorganization mediating cell-cell adhesion.

    DOI PubMed CiNii J-GLOBAL

  • 蛋白質の三次元配置化法

    平塚 祐一, 多田 哲也, 上田 太郎

    生物物理   43   S230  2003年

    DOI CiNii

  • Novel myosin heavy chain kinase involved in disassembly of myosin II filaments and efficient cleavage in mitotic Dictyostelium cells

    Akira Nagasaki, Go Itoh, Shigehiko Yumura, Taro Q.P. Uyeda

    Molecular Biology of the Cell   13 ( 12 ) 4333 - 4342  2002年12月  [査読有り]

     概要を見る

    We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, the mhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

    DOI PubMed

  • Design and functional analysis of actomyosin motor domain chimera proteins

    K Yokoyama, Y Hiratuka, E Akimaru, K Hirose, TQP Uyeda, M Suzuki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   299 ( 5 ) 825 - 831  2002年12月

     概要を見る

    To gain more structural and functional information on the actomyosin complexes, we have engineered chimera proteins carrying the entire Dictyostelium actin in the loop 2 sequence of the motor domain of Dictyostelium myosin II. Although the chimera proteins were unable to polymerize by themselves, addition of skeletal actin promoted polymerization. Electron microscopic observation demonstrated that the chimera proteins were incorporated into actin filaments, when copolymerized with skeletal actin. Copolymerization with skeletal actin greatly enhanced the MgATPase, while the chimera proteins without added skeletal actin hydrolyzed ATP at a very low rate. These results indicate that the actin part and the motor domain part of the chimera proteins are correctly folded, but the chimera proteins are structurally stressed so that efficient polymerization is inhibited. (C) 2002 Elsevier Science (USA). All rights reserved.

    DOI PubMed CiNii J-GLOBAL

  • A novel myosin II heavy chain kinase (MHCK C) involved in disassembly of myosin II filaments in contractile rings in mitotic Dictyostelium cells

    A Nagasaki, G Itoh, S Yumura, T Uyeda

    MOLECULAR BIOLOGY OF THE CELL   13 ( 12 ) 447A - 447A  2002年11月

    DOI J-GLOBAL

  • Confirmation by FRET in individual living cells of the absence of significant amyloid beta-mediated caspase 8 activation

    R Onuki, A Nagasaki, H Kawasaki, T Baba, TQP Uyeda, K Taira

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   99 ( 23 ) 14716 - 14721  2002年11月

     概要を見る

    When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (t) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (it) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.

    DOI PubMed CiNii J-GLOBAL

  • Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development

    S Shu, XN Liu, CA Parent, TQP Uyeda, ED Korn

    JOURNAL OF CELL SCIENCE   115 ( 22 ) 4237 - 4249  2002年11月

     概要を見る

    Dictyostelium lacking myosin 11 cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin 11 heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin 11 is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin 11 and there may be no specific sequence required for localization of myosin to the cleavage furrow.

    DOI PubMed CiNii J-GLOBAL

  • Amino acids 519-524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change

    TQP Uyeda, B Patterson, L Mendoza, Y Hiratsuka

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   23 ( 7-8 ) 685 - 695  2002年10月

     概要を見る

    Residues 519 - 524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (Delta519 - 524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the Delta519-524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519 - 524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for Delta519-524/+A and those for L596S showed complementary charge patterns. In ATPase assays, Delta519 - 524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, Delta519 - 524/+A myosin released actin. laments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin. laments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that Delta519 - 524/+A S1 is not only slower than wild type to bind actin. laments, but also slower to dissociate from actin. laments. Based on these results, we concluded that the 519 - 524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.

    DOI J-GLOBAL

  • Evidence for a novel, strongly bound acto-S1 complex carrying ADP and phosphate stabilized in the G680V mutant of Dictyostelium myosin II

    TQP Uyeda, K Tokuraku, K Kaseda, MR Webb, B Patterson

    BIOCHEMISTRY   41 ( 30 ) 9525 - 9534  2002年07月

     概要を見る

    Gly 680 of Dictyostelium myosin II sits at a critical position within the reactive thiol helices. We have previously shown that G680V mutant subfragment 1 largely remains in strongly actin-bound states in the presence of ATP. We speculated that acto-G680V subfragment 1 complexes accumulate in the A(.)M(.)ADP(.)P(i) state on the basis of the biochemical phenotypes conferred by mutations which suppress the G680V mutation in vivo [Wu, Y., et al. (1999) Genetics 153, 107-116]. Here, we report further characterization of the interaction between actin and G680V subfragment 1. Light scattering data demonstrate that the majority of G680V subfragment 1 is bound to actin in the presence of ATP. These acto-G680V subfragment 1 complexes in the presence of ATP do not efficiently quench the fluorescence of pyrene-actin, unlike those in rigor complexes or in the presence of ADP alone. Kinetic analyses demonstrated that phosphate release, but not ATP hydrolysis or ADP release, is very slow and rate limiting in the acto-G680V subfragment 1 ATPase cycle. Single turnover kinetic analysis demonstrates that, during ATP hydrolysis by the acto-G680V subfragment 1 complex, quenching of pyrene fluorescence significantly lags the increase of light scattering. This is unlike the situation with wild-type subfragment 1, where the two signals have similar rate constants. These data support the hypothesis that the main intermediate during ATP hydrolysis by acto-G680V subfragment 1 is an acto-subfragment 1 complex carrying ADP and P-i, which scatters light but does not quench the pyrene fluorescence and so has a different conformation from the rigor complex.

    DOI PubMed CiNii J-GLOBAL

  • Genetic and morphological evidence for two parallel pathways of cell-cycle-coupled cytokinesis in Dictyostelium

    A Nagasaki, EL de Hostos, TQP Uyeda

    JOURNAL OF CELL SCIENCE   115 ( 10 ) 2241 - 2251  2002年05月

     概要を見る

    Myosin-II-null cells of Dictyostelium discoideum cannot divide in suspension, consistent with the dogma that myosin II drives constriction of the cleavage furrow and, consequently, cytokinesis (cytokinesis A). Nonetheless, when grown on substrates, these cells exhibit efficient, cell-cycle-coupled division, suggesting that they possess a novel, myosin-II-independent, adhesion-dependent method of cytokinesis (cytokinesis B). Here we show that double mutants lacking myosin II and either AmiA or coronin, both of which are implicated in cytokinesis B, are incapable of cell-cycle-coupled cytokinesis. These double mutants multiplied mainly by cytokinesis C, a third, inefficient, method of cell division, which requires substrate adhesion and is independent of cell cycle progression. In contrast, double mutants lacking AmiA and coronin were no sicker than each of the single mutants, indicating that the severe defects of myosin II-/AmiA(-) or myosin II-/coronin(-) mutants are not simple additive effects of two mutations. We take this as genetic evidence for two parallel pathways both of which lead to cell-cycle-coupled cytokinesis. This conclusion is supported by differences in morphological changes during cytokinesis in the mutant cell lines.

  • 3R1400 酵素により駆動される微小運動素子の創製(28.バイオエンジニアリング,一般講演,日本生物物理学会第40回年会)

    平塚 祐一, 多田 哲也, 金山 敏彦, 上田 太郎

    生物物理   42 ( 2 ) S208  2002年

    DOI CiNii

  • Actin plays an important role in the processive 36nm steps of class V and VI myosin.

    Katayama E, Uyeda TQP, Iwane AH, Ikebe M, Yanagida T

    Biophysical Journal   82 ( 1 ) 15a - 16a  2002年01月

  • Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks

    Y Hiratsuka, T Tada, K Oiwa, T Kanayama, TQP Uyeda

    BIOPHYSICAL JOURNAL   81 ( 3 ) 1555 - 1561  2001年09月  [査読有り]

     概要を見る

    Motor proteins are able to move protein filaments in vitro. However, useful work cannot be extracted from the existing in vitro systems because filament motions are in random directions on two-dimensional surfaces. We succeeded in restricting kinesin-driven movements of microtubules along linear tracks by using micrometer-scaled grooves lithographically fabricated on glass surfaces. We also accomplished the extraction of unidirectional movement from the bidirectional movements along the linear tracks by adding arrowhead patterns on the tracks. These "rectifiers" enabled us to construct microminiturized circulators in which populations of microtubules rotated in one direction, and to actively transport microtubules between two pools connected by arrowheaded tracks in the fields of micrometer scales.

  • 3P118ミオシン構造変化におけるスイッチIIからコンバーターへの情報伝達経路

    伊藤 光二, 上田 太郎, 鈴木 良和, 佐々木 直哉, 須藤 和夫, 山本 啓一

    生物物理   41   S188  2001年

    DOI CiNii

  • Functional characterization of vertebrate nonmuscle myosin IIB isoforms using Dictyostelium chimeric myosin II

    M Takahashi, K Takahashi, Y Hiratsuka, K Uchida, A Yamagishi, TQP Uyeda, M Yazawa

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 2 ) 1034 - 1040  2001年01月  [査読有り]

     概要を見る

    The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(BE), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the BE-inserted form of loop 2 from human MHC-IIB(BE), The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin, We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin, In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain.

    DOI PubMed

  • Phalloidin affects the myosin-dependent sliding velocities of actin filaments in a bound-divalent cation dependent manner

    K Tokuraku, TQP Uyeda

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   22 ( 4 ) 371 - 378  2001年

     概要を見る

    We examined sliding velocities in vitro of four types of actin filaments, that is, filaments with Ca2+ or Mg2+ bound at the high affinity metal binding site, each with rhodamine phalloidin bound with a high or low stoichiometry. When surfaces coated with a high density of heavy meromyosin (HMM) were used, high stoichiometric concentrations of rhodamine phalloidin reduced sliding velocities of only Ca2+-actin filaments, by 40%. As the HMM density on surfaces was reduced, continuous movement of actin filaments became dependent on the presence of methylcellulose and sliding velocities of all four types became progressively slower. Interestingly, Ca2+-actin filaments with a high stoichiometric concentration of rhodamine phalloidin were the fastest among the four types of filaments on sparse HMM surfaces. In contrast, phalloidin did not affect steady state ATPase activities of HMM in the presence of Ca2+- or Mg2+-actin filaments. We speculate that the reversal of the order of sliding velocities among the four types of actin filaments between high and low densities of HMM relates with different axial elasticity of the actin filaments, so that stiffer filaments move slower on dense HMM surfaces, but faster on sparse surfaces, than elastic ones.

    DOI J-GLOBAL

  • Controlling the directions of kinesin-driven microtubule movements along microlithographic tracks.

    Y Hiratsuka, T Tada, K Oiwa, T Kanayama, TQP Uyeda

    BIOPHYSICAL JOURNAL   80 ( 1 ) 72A - 72A  2001年01月

  • How well can an amoeba climb?

    Y Fukui, TQP Uyeda, C Kitayama, S Inoue

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   97 ( 18 ) 10020 - 10025  2000年08月  [査読有り]

     概要を見る

    We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were "stalled" or no longer able to "climb up." The "apparent weight" of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only in low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that th is pseudopod is very dense indeed, This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II.

    DOI PubMed

  • Involvement of tail domains in regulation of Dictyostelium myosin II

    Liu, X, K Ito, RJ Lee, TQP Uyeda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   271 ( 1 ) 75 - 81  2000年04月  [査読有り]

     概要を見る

    The actin-dependent ATPase activity of Dictyostelium myosin II filaments is regulated by phosphorylation of the regulatory light chain. Four deletion mutant myosins which lack different parts of subfragment 2 (S2) showed phosphorylation-independent elevations in their activities. Phosphorylation-independent elevation in the activity was also achieved by a double point mutation to replace conserved Glu932 and Glu933 in S2 with Lys. These results suggested that inhibitory interactions involving the head and S2 are required for efficient regulation. Regulation of wild-type myosin was not affected by copolymerization with a S2 deletion mutant myosin in the same filaments. Furthermore, the activity linearly correlated with the fraction of phosphorylated molecules in wild-type filaments. These latter two results suggest that the inhibitory head-tail interactions are primarily intramolecular, (C) 2000 Academic Press.

    DOI PubMed

  • Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications

    TQP Uyeda, C Kitayama, S Yumura

    CELL STRUCTURE AND FUNCTION   25 ( 1 ) 1 - 10  2000年02月  [査読有り]

     概要を見る

    Similar to higher animal cells, ameba tells of the cellular slime mold Dirtyostelium discoideum form contractile rings containing filaments of myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells both on substrates and in suspension. In suspension, mutant cells lacking the single myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates, Detailed analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"), Cytokinesis A, B, and C lose their function and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities in telophase. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells.

    DOI PubMed

  • 1B1700 キネシン分子で駆動される微小管運動を操る

    平塚 祐一, 多田 哲也, 大岩 和弘, 金山 敏彦, 上田 太郎

    生物物理   40   S17  2000年

    DOI CiNii

  • 3I1030 粘菌/車軸藻キメラミオシンのストップドフローによる速度論的解析

    伊藤 光二, 樫山 拓, 八久保 有, 上田 太郎, 山本 啓一

    生物物理   40   S200  2000年

    DOI CiNii

  • Molecular circulator driven by motor proteins

    Y Hiratsuka, T Taka, K Oiwa, T Kanayama, TQP Uyeda

    MICROPROCESSES AND NANOTECHNOLOGY 2000, DIGEST OF PAPERS     296 - 297  2000年  [査読有り]

  • Migration forces in Dictyostelium measured by centrifuge DIC microscopy

    Y Fukui, TQP Uyeda, C Kitayama, S Inoue

    BIOLOGICAL BULLETIN   197 ( 2 ) 260 - 262  1999年10月  [査読有り]

  • Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium

    S Shu, RJ Lee, JM LeBlanc-Straceski, TQP Uyeda

    JOURNAL OF CELL SCIENCE   112 ( 13 ) 2195 - 2201  1999年07月  [査読有り]

     概要を見る

    Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: My Delta 824-941, My Delta 943-1464, My Delta 943-1194 and My Delta 1156-1464, Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow All these deletion and chimeric mutant myosins, except for My Delta 943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.

    PubMed

  • Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction

    MLW Knetsch, TQP Uyeda, DJ Manstein

    JOURNAL OF BIOLOGICAL CHEMISTRY   274 ( 29 ) 20133 - 20138  1999年07月  [査読有り]

     概要を見る

    The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 mu M actin decreased ADP affinity from 0.6 to 34 mu M for M765NL and from 1.6 to 39 mu M for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s(-1)) was 3-fold larger than for M765 (0.06 s(-1)), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and P-i release, and they confirm that loop 2 is involved in high affinity actin binding.

    DOI PubMed

  • Effect of internal deletion of Myosin II on growth and development of Dictyostelium discoideum

    S Shu, TQP Uyeda, AX Liu, GQ Liu, LF Yen

    CHINESE SCIENCE BULLETIN   44 ( 9 ) 811 - 816  1999年05月  [査読有り]

     概要を見る

    Four deletion mutant Dictyostelium myosin II heavy chain genes, My Delta 824-941 (Delta 1/3S2), My Delta 934-1454 (Delta S2), My Delta 934-1194 (Delta S2-1) and My Delta 1157-1454 (Delta S2-2), were transformed by standard electroporation into mhcA- cells (T-null), a mutant Dictyostelium cell devoid of endogenous myosin II heavy chain gene. The growth, development and formation of fruiting bodies of cells expressing those mutant myosin II s under suspension culture were investigated by comparison with the wild type cell. The results indicate that internal deletion of myosin II affects the growth and development of Dictyostelium. Furthermore, the longer the length of deletion, the more serious the defect in phenotype.

  • Cooperativity between two heads of Dictyostelium myosin II in in vitro motility and ATP hydrolysis

    K Ito, Liu, X, E Katayama, TQP Uyeda

    BIOPHYSICAL JOURNAL   76 ( 2 ) 985 - 992  1999年02月  [査読有り]

     概要を見る

    To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 mu M actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax, These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity.

    DOI PubMed

  • 2PD005 非筋ミオシンII B2に特異的に挿入されたアミノ酸配列の役割

    高橋 慶太, 平塚 祐一, 矢澤 道生, 内田 恵司, 高橋 正行, 上田 太郎, 山岸 晧彦

    生物物理   39   S138  1999年

    DOI CiNii

  • 2PD013 粘菌DictyosteliumミオシンIIのアクチン結合面欠失変異(Δ519-524/+A)とその抑制性変異(L596S)の解析

    上田 太郎, 平塚 祐一, Patterson Bruce

    生物物理   39   S140  1999年

    DOI CiNii

  • Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

    Liu, X, K Ito, S Morimoto, A Hikkoshi-Iwane, T Yanagida, TQP Uyeda

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   95 ( 24 ) 14124 - 14129  1998年11月  [査読有り]

     概要を見る

    Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, My Delta C1225 and;My Delta C1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only,weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin LI requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

    DOI PubMed

  • Transport of myosin II to the equatorial region without its own motor activity in mitotic Dictyostelium cells

    S Yumura, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   8 ( 10 ) 2089 - 2099  1997年10月  [査読有り]

     概要を見る

    Fluorescently labeled myosin moved and accumulated circumferentially in the equatorial region of dividing Dictyostelium cells within a time course of 4 min, followed by contraction of the contractile ring. To investigate the mechanism of this transport process, we have expressed three mutant myosins that cannot hydrolyze ATP in myosin null cells. Immunofluorescence staining showed that these mutant myosins were also correctly transported to the equatorial region, although no contraction followed. The rates of transport, measured using green fluorescent protein-fused myosins, were indistinguishable between wild-type and mutant myosins. these observations demonstrate that myosin is passively transported toward the equatorial region and incorporated into the forming contractile ring without its own motor activity.

    DOI PubMed

  • Myosin II can be localized to the cleavage furrow and to the posterior region of Dictyostelium amoebae without control by phosphorylation of myosin heavy and light chains

    S Yumura, TQP Uyeda

    CELL MOTILITY AND THE CYTOSKELETON   36 ( 4 ) 313 - 322  1997年  [査読有り]

     概要を見る

    To elucidate the role of phosphorylation in regulation of intracellular distribution of myosin II, we have characterized mutant Dictyostelium cells expressing myosin II that could not be regulated by the phosphorylation on the mapped heavy chain sites, the light chain site, or both sites. Immunofluorescence microscopy demonstrated that all three mutant myosin IIs were localized in the furrow region of dividing cells and in the tail region of migrating cells, similar to wild-type cells. Thus, regulation by phosphorylation is not required to direct myosin II toward the furrow region and the tail region in Dictyostelium. However, myosins that were deficient in heavy chain phosphorylation were distributed only in the cortical region of interphase cells, whereas some myosin IIs were present throughout the endoplasm in wild-type cells. Video microscopy showed that the rate of cell migration was significantly lower in cells that were deficient in heavy chain phosphorylation- than in light chain phosphorylation-deficient cells, myosin null cells and wild-type cells. Chemotactic behavior of cells that were deficient in heavy chain phosphorylation was also retarded. These results suggest that loss of regulation by heavy chain phosphorylation results in excessive myosin in the cortex, which leads to retarded motility. (C) 1997 Wiley-Liss, Inc.

    DOI PubMed

  • The neck region of the myosin motor domain acts as a lever arm to generate movement

    TQP Uyeda, PD Abramson, JA Spudich

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   93 ( 9 ) 4459 - 4464  1996年04月  [査読有り]

     概要を見る

    The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-lever model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The Delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The Delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

    DOI PubMed

  • Myosin structure and function

    JA Spudich, J Finer, B Simmons, K Ruppel, B Patterson, T Uyeda

    COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY   60   783 - 791  1995年  [査読有り]

  • 3 RECENT BREAKTHROUGHS IN MOLECULAR MOTOR RESEARCH - RECOMBINANT MYOSIN, MONOMOLECULAR IN-VITRO MOTILITY ASSAY AND ATOMIC-STRUCTURE OF S1

    TQP UYEDA

    MATERIALS SCIENCE & ENGINEERING C-BIOMIMETIC AND SUPRAMOLECULAR SYSTEMS   2 ( 1-2 ) 1 - 11  1994年12月  [査読有り]

     概要を見る

    Muscle contraction results from ATP-dependent sliding between actin filaments and S1 domain of myosin, but its molecular mechanism of chemomechanical energy transformation is still elusive despite decades of extensive research. There were three major breakthroughs in this field during the last few years, however; recombinant myosin technology, monomolecular in vitro motility assay, and atomic structure of S1. This article briefly reviews what is currently under dispute in this field, and introduces how molecular biologists, with these new tools in hand, are approaching those questions.

  • ROLE OF HIGHLY CONSERVED LYSINE-130 OF MYOSIN MOTOR DOMAIN - IN-VIVO AND IN-VITRO CHARACTERIZATION OF SITE-SPECIFICALLY MUTATED MYOSIN

    KM RUPPEL, TQP UYEDA, JA SPUDICH

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 29 ) 18773 - 18780  1994年07月  [査読有り]

     概要を見る

    We have created a mutant Dictyostelium myosin II heavy chain gene in which a highly conserved lysine residue (Lys-130) is changed to leucine. Lys-130 is a residue that is known to be trimethylated in skeletal muscle myosin and had been thought to play an integral role in the interaction of myosin with ATP during the actomyosin chemomechanical cycle. We report here the first in vivo and in vitro characterization of an engineered missense mutation in the motor domain of myosin. Expression of the K130L myosin in a Dictyostelium strain that lacks the myosin II heavy chain gene is sufficient to restore the ability of that cell line to undergo cytokinesis and multicellular development, processes that require functional myosin. The K130L myosin purified from these cells displays maximal actin-activated ATPase activities and promotes maximal sliding velocities of actin filaments in an in vitro motility assay that are comparable with those of wild type myosin. These results demonstrate that this lysine residue is not required for the enzymatic or motile activities of myosin. However, the mutant protein exhibits a 4-fold increase in K-m for ATP over wild-type myosin, indicating that this residue participates in the interaction of myosin with its nucleotide substrate.

    PubMed

  • ENZYMATIC-ACTIVITIES CORRELATE WITH CHIMERIC SUBSTITUTIONS AT THE ACTIN-BINDING FACE OF MYOSIN

    TQP UYEDA, KM RUPPEL, JA SPUDICH

    NATURE   368 ( 6471 ) 567 - 569  1994年04月  [査読有り]

     概要を見る

    MYOSINS are a functionally divergent group of mechanochemical enzymes involved in various motile activities in cells1. Despite a high degree of conservation in the amino-acid sequence of the 130K motor domain2,3 (head region) of the molecule, there are large differences in the enzymatic and motile activities (Tables 1 and 2) of myosins from diverse species and cell types. However, the degree of conservation is not uniform throughout the head sequence4; therefore, one reasonable hypothesis is that the functional differences between myosins derive from the poorly conserved areas. The most prominent divergent region occurs at the 50K/20K junction, a region of the molecule sensitive to proteolytic digestion5 and a binding site for actin6-12. We have now constructed chimaeras of this region of myosin by substituting the 9-amino-acid Dictyostelium junction region with those from myosins from other species and find that the actin-activated ATPase correlates well with the activity of the myosin from which the junction region was derived. Our results suggest that this region, likely to be part of the myosin head that interacts directly with actin10,13,14, is important in deter mining the enzymatic activity of myosin.

    DOI PubMed

  • A FUNCTIONAL RECOMBINANT MYOSIN-II LACKING A REGULATORY LIGHT-CHAIN BINDING-SITE

    TQP UYEDA, JA SPUDICH

    SCIENCE   262 ( 5141 ) 1867 - 1870  1993年12月

     概要を見る

    Myosin II, which converts the energy of adenosine triphosphate hydrolysis into the movement of actin filaments, is a hexamer of two heavy chains, two essential light chains, and two regulatory light chains (RLCs). Dictyostelium myosin II is known to be regulated in vitro by phosphorylation of the RLC. Cells in which the wild-type myosin II heavy chain was replaced with a recombinant form that lacks the binding site for RLC carried out cytokinesis and almost normal development, processes known to be dependent on functional myosin II. Characterization of the purified recombinant protein suggests that a complex of RLC and the RLC binding site of the heavy chain plays an inhibitory role for adenosine triphosphatase activity and a structural role for the movement of myosin along actin.

    DOI J-GLOBAL

  • A DICTYOSTELIUM MYOSIN-II LACKING A PROXIMAL 58-KDA PORTION OF THE TAIL IS FUNCTIONAL INVITRO AND INVIVO

    EW KUBALEK, TQP UYEDA, JA SPUDICH

    MOLECULAR BIOLOGY OF THE CELL   3 ( 12 ) 1455 - 1462  1992年12月  [査読有り]

     概要を見る

    We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 mum/s at 30-degrees-C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

    DOI PubMed

  • QUANTIZED VELOCITIES AT LOW MYOSIN DENSITIES IN AN INVITRO MOTILITY ASSAY

    TQP UYEDA, HM WARRICK, SJ KRON, JA SPUDICH

    NATURE   352 ( 6333 ) 307 - 311  1991年07月

     概要を見る

    An in vitro motility assay has been developed in which single actin filaments move on one or a few heavy meromyosin (HMM) molecules. This movement is slower than when many HMM molecules are involved, in contrast to analogous experiments with microtubules and kinesin. Frequency analysis shows that sliding speeds distribute around integral multiples of a unitary velocity. This discreteness may be due to differences in the numbers of HMM molecules interacting with each actin filament, where the unitary velocity reflects the activity of one HMM molecule. The value of the unitary velocity predicts a step size of 5-20 nm per ATP, which is consistent with the conventional swinging crossbridge model for myosin function.

    DOI J-GLOBAL

  • CHARACTERIZATION AND INTRACELLULAR-DISTRIBUTION OF PEA PHYTOCHROME-I POLYPEPTIDES EXPRESSED IN ESCHERICHIA-COLI

    K TOMIZAWA, N ITO, Y KOMEDA, TQP UYEDA, K TAKIO, M FURUYA

    PLANT AND CELL PHYSIOLOGY   32 ( 1 ) 95 - 102  1991年01月  [査読有り]

     概要を見る

    The pea phytochrome I (PI) cDNA clone, pPP1001, was expressed in E. coli. The plasmid pPP1001 contains pea PI cDNA which covers the entire coding region with the Shine-Dalgarno consensus sequence joined upstream of the cDNA in an expression vector pNUT6. The pPP1001 transformants formed typical inclusion bodies when cultured at 32-degrees-C. However, when cultured at 37-degrees-C or in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) at 32-degrees-C, the bacteria lysed before inclusion body formation. Immuno-staining with anti-PI monoclonal antibody, mAP5, of transformants fixed by cold methanol showed that stainable materials were distributed in whole cytoplasmic region. When the inclusion bodies were observed clearly, the regions corresponding to the inclusion bodies became difficult to stain. Western blot analysis, however, showed that a ca. 100 kDa PI polypeptide was detected in the fraction from inclusion bodies and a ca. 90 kDa PI polypeptide from the soluble fraction. The amino acid sequence analysis of purified 100 kDa PI sample indicated that its amino terminus is blocked. However, minor signals in one experiment yielded a sequence corresponding to the expected amino terminus of pea PI except for the initiation methionine. One of the anti-pea PI monoclonal antibodies, mAP9, that recognizes the near N-terminus of pea phytochrome was reactive to the 100 kDa polypeptide.

  • AN APPROACH TO RECONSTITUTING MOTILITY OF SINGLE MYOSIN MOLECULES

    SJ KRON, TQP UYEDA, HM WARRICK, JA SPUDICH

    JOURNAL OF CELL SCIENCE     129 - 133  1991年  [査読有り]

     概要を見る

    Over the last five years, the value of in vitro motility assays as probes of the mechanical properties of the actin-myosin interaction has been amply demonstrated. Motility assays in which single fluorescent actin filaments are observed moving over surfaces coated with myosin or its soluble fragments are now used in many laboratories. They have been applied to a wide range of problems including the study of structure-function relationships in the myosin molecule and measurement of fundamental properties of the myosin head. However, one limitation of these assays has been uncertainty over the number of myosin heads interacting with each sliding filament, that frustrates attempts to determine properties of individual heads. In order to address this limitation, we have modified the conditions of the actin sliding filament assay to reduce the number of heads interacting with each filament. Our goal is to establish an assay in which the motor function of a single myosin head can be characterized from the movement of a single actin filament.

  • MYOSIN STEP SIZE - ESTIMATION FROM SLOW SLIDING MOVEMENT OF ACTIN OVER LOW-DENSITIES OF HEAVY-MEROMYOSIN

    TQP UYEDA, SJ KRON, JA SPUDICH

    JOURNAL OF MOLECULAR BIOLOGY   214 ( 3 ) 699 - 710  1990年08月  [査読有り]

    DOI PubMed

  • EVIDENCE FOR ACTIVE INTERACTIONS BETWEEN MICROFILAMENTS AND MICROTUBULES IN MYXOMYCETE FLAGELLATES

    TQP UYEDA, M FURUYA

    JOURNAL OF CELL BIOLOGY   108 ( 5 ) 1727 - 1735  1989年05月

    DOI J-GLOBAL

  • Destruction of organelle nuclei during spermatogenesis in Chara corallina examined by staining with DAPI and anti-DNA antibody

    Ge-Hong Sun, Taro Q.P. Uyeda, Tsuneyoshi Kuroiwa

    Protoplasma   144 ( 2-3 ) 185 - 188  1988年06月  [査読有り]

     概要を見る

    The behavior of plastid and mitochondrial nuclei (synonymous with nucleoids) during spermatogenesis in Chara corallina was examined by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI). These organelle nuclei, which were present in internode cells and cells at the early spermatid stage, disappeared during spermatogenesis. This conclusion was confirmed by immunofluorescence microscopy using of a monoclonal anti-DNA antibody. The pattern of fluorescence obtained using the antibody coincided with that obtained by staining with DAPI. These results suggest that the disappearance of nuclei from male-derived organelles is most likely to result in maternal inheritance in Chara corallina. © 1988 Springer-Verlag.

    DOI

  • PURIFICATION OF MYXAMEBAL FRAGMIN, AND SWITCHING OF MYXAMEBAL FRAGMIN TO PLASMODIAL FRAGMIN DURING DIFFERENTIATION OF PHYSARUM-POLYCEPHALUM

    TQP UYEDA, S HATANO, K KOHAMA, M FURUYA

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   9 ( 3 ) 233 - 240  1988年06月  [査読有り]

    PubMed

  • INVOLVEMENT OF MYXAMEBAL FRAGMIN IN THE CA-2+-INDUCED REORGANIZATION OF THE MICROFILAMENTOUS CYTOSKELETON IN FLAGELLATES OF PHYSARUM-POLYCEPHALUM

    TQP UYEDA, S HATANO, M FURUYA

    CELL MOTILITY AND THE CYTOSKELETON   10 ( 3 ) 410 - 419  1988年  [査読有り]

  • MYOSIN SWITCHING DURING AMOEBO-PLASMODIAL DIFFERENTIATION OF SLIME-MOLD, PHYSARUM-POLYCEPHALUM

    TQP UYEDA, K KOHAMA

    EXPERIMENTAL CELL RESEARCH   169 ( 1 ) 74 - 84  1987年03月  [査読有り]

    DOI PubMed

  • EFFECTS OF LOW-TEMPERATURE AND CALCIUM ON MICROFILAMENT STRUCTURE IN FLAGELLATES OF PHYSARUM-POLYCEPHALUM

    TQP UYEDA, M FURUYA

    EXPERIMENTAL CELL RESEARCH   165 ( 2 ) 461 - 472  1986年08月  [査読有り]

    DOI PubMed

  • CA2+-BINDING LIGHT CHAIN OF PHYSARUM MYOSIN CONFERS INHIBITORY CA2+-SENSITIVITY ON ACTIN-MYOSIN-ATP INTERACTION VIA ACTIN

    K KOHAMA, TQP UYEDA, H TAKANOOHMURO, T TANAKA, T YAMAGUCHI, K MARUYAMA, T KOHAMA

    PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES   61 ( 10 ) 501 - 505  1985年12月  [査読有り]

  • CYTOSKELETAL CHANGES VISUALIZED BY FLUORESCENCE MICROSCOPY DURING AMOEBA-TO-FLAGELLATE AND FLAGELLATE-TO-AMOEBA TRANSFORMATIONS IN PHYSARUM-POLYCEPHALUM

    TQP UYEDA, M FURUYA

    PROTOPLASMA   126 ( 3 ) 221 - 232  1985年  [査読有り]

  • CYCLOHEXIMIDE INSENSITIVE AMEBO-FLAGELLATE TRANSFORMATION IN STARVED AMEBAS OF PHYSARUM-POLYCEPHALUM

    TQP UYEDA, M FURUYA

    DEVELOPMENT GROWTH & DIFFERENTIATION   26 ( 2 ) 121 - 128  1984年  [査読有り]

    DOI

  • SEARCH FOR BACTERIAL STRAINS IN LABORATORY STOCKS USING A MICROCOMPUTER

    TQP UYEDA, S HARAYAMA

    JAPANESE JOURNAL OF GENETICS   58 ( 3 ) 251 - 255  1983年  [査読有り]

    DOI

  • Glycan polymerase with no penicillin binding activity in Escherichia coli.

    Hara H, Ueda T, Suzuki H

    The target of penicillin     583 - 588  1983年  [査読有り]

▼全件表示

Misc

  • GFPアクチン融合タンパク質におけるGFP導入位置によるアクチンフィラメント形成への影響

    長崎晃, 貴嶋紗久, 湯本天嗣, 金賢徹, 今泉美玖, 中村史, 中村史, 上田太郎

    日本分子生物学会年会プログラム・要旨集(Web)   39th   ROMBUNNO.2P‐0340 (WEB ONLY)  2016年

    J-GLOBAL

  • Structural polymorphism of actin detected by intramolecular FRET in vivo and in vitro.

    T. Noguchi, M. Okazaki, S. Kijima, M. Morimatsu, A. Nagasaki, Y. Iwadate, T. Yanagida, T. Uyeda

    MOLECULAR BIOLOGY OF THE CELL   25  2014年12月

    研究発表ペーパー・要旨(国際会議)  

  • Video Imaging of Cofilin-Induced Actin Filament Severing by High Speed AFM

    Kien Xuan Ngo, Noriyuki Kodera, Akira Nagasaki, Toshio Ando, Taro Q. P. Uyeda

    BIOPHYSICAL JOURNAL   106 ( 2 ) 163A - 163A  2014年01月

    研究発表ペーパー・要旨(国際会議)  

  • Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    Onuki-Nagasaki R, Nagasaki A, Hakamada K, Uyeda TQ, Fujita S, Miyake M, Miyake J

    Methods in molecular biology (Clifton, N.J.)   629   193 - 203  2010年  [査読有り]

    DOI PubMed CiNii

  • 細胞運動関連遺伝子群のゲノムワイドスクリーニング法の開発

    長崎晃, 長崎玲子, 藤田聡史, 上田太郎

    生化学   81 ( 5 ) 381 - 386  2009年05月

    J-GLOBAL

  • Genome-wide screening methods for genes involved in cell migration

    Akira Nagasaki, Reilo Nagasaki, Satoshi Fujita, Taro Q.P. Uyeda

    Seikagaku   81 ( 5 ) 381 - 386  2009年  [査読有り]

    書評論文,書評,文献紹介等  

    PubMed

  • Visible-light photoresponsivity of a 4-(dimethylamino)azobenzene unit incorporated into single-stranded DNA: Demonstration of a large spectral change accompanying isomerization in DMSO and detection of rapid (Z)-to-(E) isomerization in aqueous solution (April, pg 1846, 2007)

    Takashi Kamei, Masabumi Kudo, Haruhisa Akiyama, Momoyo Wada, Jun'ichi Nagasawa, Masahiro Funahashi, Nobuyuki Tamaoki, Taro Q. P. Uyeda

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY   ( 18 ) 3053 - 3053  2007年06月

    その他  

    DOI

  • 1P314 4-ジメチルアミノアゾベンゼンを側鎖として組み込んだ合成DNAの可視光応答性(バイオエンジニアリング,ポスター発表,第45回日本生物物理学会年会)

    亀井 敬, 秋山 陽久, 工藤 成史, 和田 百代, 長沢 順一, 舟橋 正浩, 玉置 信之, 上田 太郎

    生物物理   47 ( 0 ) S102  2007年

    DOI CiNii

  • Revised behavior of myosin crossbridges during in vitro actin sliding

    Eisaku Katayama, Yoshitaka Kimori, Masaforni D. Yamada, Taro Eguchi, Takeshi Sasak, Shinsaku Maruta, Norio Baba, Taro Q. P. Uyeda

    BIOPHYSICAL JOURNAL     479A - 479A  2007年01月

    研究発表ペーパー・要旨(国際会議)  

  • Three approaches to assembling nano-bio-machines using molecular motors

    Yuichi Hiratsuka, Takashi Kamei, Noboru Yumoto, Taro Q. P. Uyeda

    Nanobiotechnology   2 ( 3-4 ) 101 - 115  2006年09月

    書評論文,書評,文献紹介等  

     概要を見る

    Efforts to use protein molecular motors as nanoactuators are making rapid progress. For instance, it is now possible to carry out directional transport of small cargo along microtracks or microchannels using kinesin-microtubule systems, which could be the basis of micro-conveyor belts or molecular shuttles. However, the applicability of protein-based devices is limited by their poor stability in artificial environments. In addition, assembly of complex, intelligent microdevices or systems will likely require bottom-up self-assembly, and we still do not have sufficient knowledge to rationally design self-assembling protein-based microdevices or systems. One approach to solving the problems associated with protein-based systems is to use DNA-based nanodevices, which are amenable to rational design. Indeed, ingenious design has enabled realization of DNA-based nanoactuators and self-assembled micropatterns of various shapes. One also could use cells, organelles, or tissues as preassembled motile units, and several motile devices have already been realized using this approach. In addition to being less prone to the assemaly problems, cell-based microdevices have the advantage that the motile units reproduce themselves, and genetically encoded functional modifications can be replicated effortlessly. These protein-based, DNA-based, and cell-based systems each have distinct advantages and disadvantages, so that hybrid devices combining the best characteristics of all three would seem the most likely to succeed. Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved.

    DOI

  • Paxillin is required for contractile ring-independent cytokinesis in Dictyostelium discoideum

    Akira Nagasaki, Taro Uyeda

    CELL STRUCTURE AND FUNCTION   30   54 - 54  2005年06月

    研究発表ペーパー・要旨(国際会議)  

  • Adhesion-dependent and contractile ring-independent equatorial farrowing during cytokinesis in mammalian cells

    Masamitsu Kanada, Akira Nagasaki, Taro Q. P. Uyeda

    CELL STRUCTURE AND FUNCTION   30   109 - 109  2005年06月

    研究発表ペーパー・要旨(国際会議)  

  • Incorporation of photochromic molecule into the functional region of myosin motor domain

    T Yoshizawa, MD Yamada, TQP Uyeda, E Katayama, S Maruta

    BIOPHYSICAL JOURNAL   88 ( 1 ) 633A - 633A  2005年01月

    研究発表ペーパー・要旨(国際会議)  

  • Adhesion-dependent and contractile ring-independent furrowing during cytokinesis of mammalian cells.

    M Kanada, A Nagasaki, TQP Uyeda

    BIOPHYSICAL JOURNAL   88 ( 1 ) 491A - 491A  2005年01月

    研究発表ペーパー・要旨(国際会議)  

  • 2P188 ミオシン変異体G680Vとアクチンの協同的結合(分子モーター))

    黒木 梨加, 上田 太郎, 徳楽 清孝

    生物物理   45 ( 0 ) S166  2005年

    DOI CiNii

  • Function and intracellular dynamics of aurora kinase in Dictyostelium discoideum

    Go Itoh, Akira Nagasaki, Taro Q. P. Uyeda, Shivehiko Yumura

    CELL STRUCTURE AND FUNCTION   29   34 - 34  2004年05月

    研究発表ペーパー・要旨(国際会議)  

  • Cell migration and polarization in Dictyoslelium Cells: PIP3 involvement and effects of micropatterned substrate

    O. Yukako Asano, Yuichi Hiratsuka, Akira Nagasaki, Taro Uyeda

    CELL STRUCTURE AND FUNCTION   29   26 - 26  2004年05月

    研究発表ペーパー・要旨(国際会議)  

  • Variations on a theme: the many modes of cytokinesis.

    Uyeda TQ, Nagasaki A

    Current opinion in cell biology   16 ( 1 ) 55 - 60  2004年02月  [査読有り]

    DOI PubMed

  • 3P169 GFP融合HMMを用いたアクチン-ミオシン協同的結合の解析(分子モーター)

    伊知地 香奈, 黒木 梨加, 上田 太郎, 徳楽 清孝

    生物物理   44 ( 0 ) S232  2004年

    DOI CiNii

  • Multiple parallelisms in animal cytokinesis

    TQP Uyeda, A Nagasaki, S Yumura

    INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL. 240   240   377 - +  2004年  [査読有り]

    書評論文,書評,文献紹介等  

     概要を見る

    The process of cytokinesis in animal cells is usually presented as a relatively simple picture: A cleavage plane is first positioned in the equatorial region by the astral microtubules of the anaphase mitotic apparatus, and a contractile ring made up of parallel filaments of actin and myosin 11 is formed and encircles the cortex at the division site. Active sliding between the two filament systems constricts the perimeter of the cortex, leading to separation of two daughter cells. However, recent studies in both animal cells and lower eukaryotic model organisms have demonstrated that cytokinesis is actually far more complex. It is now obvious that the three key processes of cytokinesis, cleavage plane determination, equatorial furrowing, and scission, are driven by different mechanisms in different types of cells. In some cases, moreover, multiple pathways appear to have redundant functions in a single cell type. In this review, we present a novel hypothesis that incorporates recent observations on the activities of mitotic microtubules and the biochemistry of Rho-type GTPase proteins and postulates that two different sets of microtubules are responsible for the two known mechanisms of cleavage plane determination and also for two distinct mechanisms of equatorial furrowing.

    DOI PubMed

  • [Motor proteins as nano actuators].

    Hiratsuka Y, Uyeda TQ

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   48 ( 11 Suppl ) 1586 - 1594  2003年08月  [査読有り]

    PubMed

  • アクチンミオシン結合における協同性の方向性の解析

    友行 眞美子, 横山 慶一, 鈴木 誠, 上田 太郎, 徳樂 清孝

    生物物理   43 ( 0 ) S99  2003年

    DOI CiNii

  • Myosins and cell dynamics in cellular slime molds

    S Yumura, TQP Uyeda

    INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL 224   224   173 - 225  2003年  [査読有り]

    書評論文,書評,文献紹介等  

    DOI PubMed

  • 細胞質分裂 収縮環の収縮に依存しない新規細胞質分裂機構

    上田太郎, 長崎晃

    生体の科学   53 ( 3 ) 204 - 208  2002年05月

    DOI CiNii J-GLOBAL

  • 細胞性粘菌におけるミオシンII非依存型の細胞周期同調性細胞質分裂に関わる遺伝子の同定

    日比慎, 日比慎, 長崎晃, 高橋正行, 上田太郎

    日本細胞生物学会大会講演要旨集   55th  2002年

    J-GLOBAL

  • Periodic binding of G680V mutant mosin II along one side of actin filaments at 36 nm iintervals in the presence of ATP.

    T Uyeda, E Katayama

    BIOPHYSICAL JOURNAL   82 ( 1 ) 373A - 374A  2002年01月

    研究発表ペーパー・要旨(国際会議)  

  • Genetic approaches to dissect the mechanisms of two distinct pathways of cell cycle-coupled cytokinesis in Dictyostelium

    A Nagasaki, M Hibi, Y Asano, TQP Uyeda

    CELL STRUCTURE AND FUNCTION   26 ( 6 ) 585 - 591  2001年12月  [査読有り]

    書評論文,書評,文献紹介等  

     概要を見る

    Dictyostelium discoideum is a unique experimental organism which allows genetic analysis of the mechanism of cytokinesis of the animal type, and a number of mutations which affect cytokinesis in one way or other have been identified. Myosin II filaments accumulate in the equatorial region, and myosin II-null cells cannot divide in suspension, indicating that active, myosin II-dependent constriction of the cleavage furrow contributes to bisection of the cell. We refer to this method of cytokinesis as cytokinesis A. On substrates, however, myosin II-null cells divide efficiently in a cell cycle-coupled manner. This adhesion-dependent but myosin II-independent division method, which we termed cytokinesis B. is carried out by a pathway that is genetically distinct from that of cytokinesis A. Morphological analyses suggested that cytokinesis B is driven by radial traction forces generated along polar peripheries, which indirectly cause furrow ingression. Identification of two redundant pathways have allowed us to search genes involved in either pathway by mutagenizing cells which are already defective in one of the pathways. This approach enabled us to identify a number of novel cytokinesis-related genes, as well as to reclassify known genes as cytokinesis-related.

    DOI PubMed

  • Studies on the functional properties of the coiled-coil helical tails of class II myosins

    S Shu, TQP Uyeda, ED Korn

    MOLECULAR BIOLOGY OF THE CELL   12   41A - 41A  2001年11月

    研究発表ペーパー・要旨(国際会議)  

  • Genetic and morphological evidence for two parallel pathways of cell cycle-coupled cytokinesis in Dictyostelium discoideum

    A Nagasaki, EL de Hostos, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   12   410A - 410A  2001年11月

    研究発表ペーパー・要旨(国際会議)  

  • An extended prestroke A.S1.ADP.Pi state of G680V mutant Dictyostelium S1 in the presence of ATP.

    TQP Uyeda, E Katayama, K Kaseda, K Tokuraku, B Patterson

    BIOPHYSICAL JOURNAL   80 ( 1 ) 81A - 82A  2001年01月

    研究発表ペーパー・要旨(国際会議)  

  • 3P116アクチン-ミオシン結合における協同性に方向性はあるか!?

    友行 眞美子, 上田 太郎, 徳樂 清孝

    生物物理   41 ( 0 ) S187  2001年

    DOI CiNii

  • Crawling ability of Dictyostelium amoebae measured with centrifuge polarizing microscope

    Y Fukui, TQP Uyeda, C Kitayama, S Inoue

    MOLECULAR BIOLOGY OF THE CELL   11   383A - 383A  2000年12月

    研究発表ペーパー・要旨(国際会議)  

  • Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium

    TQP Uyeda, S Yumura

    MICROSCOPY RESEARCH AND TECHNIQUE   49 ( 2 ) 136 - 144  2000年04月  [査読有り]

     概要を見る

    The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including,parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. Microsc. Res. Tech. 49:136-144, 2000. (C) 2000 Wiley-Liss, Inc.

    DOI PubMed

  • 3I1145 Ca2+, Mg2+, Phalloidinのアクトミオシン滑り速度への影響

    徳楽 清孝, 上田 太郎

    生物物理   40 ( 0 ) S201  2000年

    DOI CiNii

  • Characterization of a mutant Dictyostellium myosin II lacking a portion of the actin binding face and its suppressor.

    TQP Uyeda, L Mendoza, Y Hiratsuka, B Patterson

    MOLECULAR BIOLOGY OF THE CELL   10   31A - 31A  1999年11月

    研究発表ペーパー・要旨(国際会議)  

  • 2PD006 アクチン-ミオシン結合における協同性

    徳楽 清孝, 上田 太郎

    生物物理   39 ( 0 ) S138  1999年

    DOI CiNii

  • Role of myosin II tail sequences di function and localization to the cleavage furrow in Dictyostelium.

    S Shu, RJ Lee, J Straceski-Leblanc, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   9   400A - 400A  1998年11月

    研究発表ペーパー・要旨(国際会議)  

  • Generation and phenotypic analysis of a myosin triple mutant (myosin II-/myoa(-)/B-)

    C Kitayama, J Dai, HP Ting-Beall, MA Titus, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   9   387A - 387A  1998年11月

    研究発表ペーパー・要旨(国際会議)  

  • ミオシンS2 coiled-coil欠失による双頭の位置関係の変化とアクチン滑り運動への影響

    伊藤 光二, 劉 態, 上田 太郎

    生物物理   38 ( 2 ) S129  1998年09月

    CiNii

  • Cooperativity between two heads of Dictyostelium myosin II

    K Ito, Liu, X, E Katayama, T Uyeda

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   19 ( 4 ) 456 - 457  1998年05月

    研究発表ペーパー・要旨(国際会議)  

  • Kinetic characterisation of a Dictyostelium myosin head fragment with truncated 50/20 K junction.

    MLW Knetsch, TQP Uyeda, DJ Manstein

    MOLECULAR BIOLOGY OF THE CELL   8   917 - 917  1997年11月

    研究発表ペーパー・要旨(国際会議)  

  • Filament structure as the essential factor for efficient regulation of Dictyostelium myosin by RLC phosphorylation

    Liu, X, K Ito, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   8   918 - 918  1997年11月

    研究発表ペーパー・要旨(国際会議)  

  • Cooperative interaction between two heads of Dictyostelium myosin II

    K Ito, Liu, X, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   8   919 - 919  1997年11月

    研究発表ペーパー・要旨(国際会議)  

  • 分子生物学的手法により作成した粘菌単頭ミオシン

    伊藤 光二, 劉 熊, 上田 太郎

    生物物理   37   S216  1997年10月

    CiNii

  • Restoration of Dictyostelium myosin assembly and cellular function by a rat skeletal myosin assembly domain.

    RJ Lee, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   7   200 - 200  1996年12月

    研究発表ペーパー・要旨(国際会議)  

  • The role of nonconserved surface loops in myosin function

    CT Murphy, TQP Uyeda, JA Spudich

    MOLECULAR BIOLOGY OF THE CELL   7   1141 - 1141  1996年12月

    研究発表ペーパー・要旨(国際会議)  

  • Specific tail domains required for regulation of Dictyostelium myosin by RLC phosphorylation

    Liu, X, K Ito, RJ Lee, TQP Uyeda

    MOLECULAR BIOLOGY OF THE CELL   7   198 - 198  1996年12月

    研究発表ペーパー・要旨(国際会議)  

  • ROLE OF MYOSIN NECK REGION AS A LEVER ARM TO GENERATE MOVEMENT

    TQP UYEDA, PD ABRAMSON, JA SPUDICH

    MOLECULAR BIOLOGY OF THE CELL   6   850 - 850  1995年11月

    研究発表ペーパー・要旨(国際会議)  

  • MUTATION ANALYSIS OF THE MOLECULAR MOTOR MYOSIN

    KM RUPPEL, TQP UYEDA, JA SPUDICH

    BIOPHYSICAL JOURNAL   64 ( 2 ) A2 - A2  1993年02月

    研究発表ペーパー・要旨(国際会議)  

  • MOLECULAR GENETIC APPROACHES TO DICTYOSTELIUM-MOTILITY

    TT EGELHOFF, RJ LEE, B PATTERSON, KM RUPPEL, TQP UYEDA, JA SPUDICH

    JOURNAL OF CELLULAR BIOCHEMISTRY     124 - 124  1993年02月

    研究発表ペーパー・要旨(国際会議)  

  • INVIVO AND INVITRO STUDIES ON THE STRUCTURE AND FUNCTION OF MYOSIN

    TT EGELHOFF, RJ LEE, B PATTERSON, KM RUPPEL, TQP UYEDA, JA SPUDICH

    JOURNAL OF CELLULAR BIOCHEMISTRY     6 - 6  1993年02月

    研究発表ペーパー・要旨(国際会議)  

  • IN-VITRO METHODS FOR MEASURING FORCE AND VELOCITY OF THE ACTIN-MYOSIN INTERACTION USING PURIFIED PROTEINS

    HM WARRICK, RM SIMMONS, JT FINER, TQP UYEDA, S CHU, JA SPUDICH

    METHODS IN CELL BIOLOGY, VOL 39   39   1 - 21  1993年  [査読有り]

    書評論文,書評,文献紹介等  

    PubMed

  • APPROACHES TO CHARACTERIZE MOLECULAR FACETS OF THE ACTOMYOSIN INTERACTION USING INVITRO MOTILITY ASSAYS

    TQP UYEDA, JT FINER, RM SIMMONS, HM WARRICK, JA SPUDICH

    FASEB JOURNAL   6 ( 1 ) A140 - A140  1992年01月

    研究発表ペーパー・要旨(国際会議)  

  • An approach to reconstituting motility of single myosin molecules.

    Kron SJ, Uyeda TQ, Warrick HM, Spudich JA

    Journal of cell science. Supplement   14   129 - 133  1991年  [査読有り]

    PubMed

  • ASSAYS FOR ACTIN SLIDING MOVEMENT OVER MYOSIN-COATED SURFACES

    SJ KRON, YY TOYOSHIMA, TQP UYEDA, JA SPUDICH

    METHODS IN ENZYMOLOGY   196   399 - 416  1991年  [査読有り]

    書評論文,書評,文献紹介等  

    DOI PubMed

  • SINGLE MYOSIN HEAD MOTILITY

    SJ KRON, TQP UYEDA, HM WARRICK, JA SPUDICH

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   11 ( 6 ) 542 - 542  1990年12月

    研究発表ペーパー・要旨(国際会議)  

  • ESTIMATE OF MYOSIN STEP SIZE FROM SLOW SLIDING MOVEMENT AT LOW HEAD DENSITIES IN AN INVITRO ASSAY

    TQP UYEDA, SJ KRON, JA SPUDICH

    BIOPHYSICAL JOURNAL   57 ( 2 ) A337 - A337  1990年02月

    研究発表ペーパー・要旨(国際会議)  

  • [Actin binding proteins from physarum and the organization of the actin cytoskeleton].

    Furuhashi K, Hatano S, Uyeda TQ

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   34 ( 12 Suppl ) 1524 - 1532  1989年09月  [査読有り]

    PubMed

  • MYOSIN SWITCHING DURING AMEBOPLASMOIDAL DIFFERENTIATION OF SLIME-MOLD, PHYSARUM-POLYCEPHALUM

    TQP UYEDA, K KOHAMA

    JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY   8 ( 3 ) 287 - 287  1987年06月

    研究発表ペーパー・要旨(国際会議)  

  • ATP-INDUCED RELATIVE MOVEMENT BETWEEN MICROFILAMENTS AND MICROTUBULES IN MYXOMYCETE FLAGELLATES

    TQP UYEDA, M FURUYA

    PROTOPLASMA   140 ( 2-3 ) 190 - 192  1987年

    記事・総説・解説・論説等(学術雑誌)  

  • EXCHANGE IN MYOSIN SUBUNITS DURING AMEBO-PLASMODIAL TRANSITION IN THE LIFE-CYCLE OF PHYSARUM-PLASMODIUM

    K KOHAMA, H TAKANOOHMURO, TQP UYEDA, T TANAKA, T YAMAGUCHI, T KOHAMA

    JAPANESE JOURNAL OF PHARMACOLOGY   40   P142 - P142  1986年

    研究発表ペーパー・要旨(国際会議)  

  • MYOSIN GENE SWITCHING DURING AMEBOPLASMODIAL DIFFERENTIATION OF SLIME-MOLD, PHYSARUM

    TQP UYEDA, K KOHAMA

    DEVELOPMENT GROWTH & DIFFERENTIATION   28 ( 4 ) 409 - 409  1986年

    研究発表ペーパー・要旨(国際会議)  

  • Calcium-binding light chain of Pysarum myosin confers inhibitory calcium-sensitivity on actin-myosin-ATP interaction via actin. ,

    Kohama,K, Uyeda,T.Q.P, Takano-Ohmuro, H, Tanaka, H, Yamaguchi, T. Maruyama, K, Kohama, T

    Proc. Japan Acad.   61巻 ( ser.B ) 501-503  1985年12月

    記事・総説・解説・論説等(その他)  

     概要を見る

    粘菌フィザルムのアメーバ体と変形体からアクトミオシン(収縮蛋白質であるアクチン、ミオシン以外に収縮調節蛋白質を含む)画分を調製し、アメーバ体と変形体に共通の軽鎖が存在し、その軽鎖はカルシウム結合蛋白質であることを示した。また、アクトミオシンATPase活性は現在まで報告されている生物では、カルシウムが活性化に働くが、粘菌では、抑制的に働いている可能性を示した。論文17ではさらに詳細に検討を加え、証明を行っている。(生化学的解析を主に担当))

    DOI

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共同研究・競争的資金等の研究課題

  • 1. 分子モーター 2. 細胞運動

特定課題研究

  • 疎らに結合したアクチン結合タンパク質がアクチン線維全体の機能を調節するメカニズム

    2021年  

     概要を見る

    Rng2は分裂酵母の収縮環構成タンパク質である。われわれはこれまでに、そのアクチン結合ドメインであるRng2CHDがin vitroで骨格筋ミオシンIIによるアクチン線維の運動を強く阻害することを示していた。今年度はRng2CHD存在下のアクチン線維を高速AFMで詳細に計測したところ、6%程度の結合比でらせんピッチの有意な短縮がおき、この構造変化は結合比30%程度で飽和した。したがって、Rng2CHDはアクチン線維に対して協同的な構造変化を誘起することが実験的に証明され、Rng2CHDの疎らな結合がアクチン線維の広範な領域に協同的な構造変化を誘起し、その結果、ミオシンIIとの相互作用が阻害されるというモデルが確実となった。

  • アクチン繊維の長距離アロステリー:その構造的基盤と生理的機能

    2020年  

     概要を見る

    Rng2CHDとよばれるアクチン結合ドメインが、骨格筋ミオシンIIによるアクチンフィラメントの運動を強く阻害することを示していたが、この結果を論文にまとめ、BioRxivにアップロードしたうえで投稿した。しかし査読者から追加実験を求められ、その対応を行ったところ、非筋ミオシンII(細胞性粘菌のミオシンII)による運動は強く阻害されないことが明らかとなり、想定される阻害メカニズムの再検討を迫られた。また、この阻害機構には、見かけ上きわめて強い協同性が認められることがその特徴であるが、この協同性に、アクチンフィラメントの協同的な構造変化が関与するのか、記憶効果が関与するのかを実験的に区別するための実験の準備を進めた。

  • アクチン繊維の長距離アロステリー:その構造的基盤と細胞内機能

    2019年  

     概要を見る

    われわれは最近、収縮環の構成タンパク質であるRng2のアクチン結合ドメイン(Rng2CHD)が、アクチン線維とミオシンの相互作用を強く阻害すること、とくに、線維中のアクチン20分子に1分子のRng2CHDが結合した条件で、ミオシンの運動活性が90%阻害されることを見出していたが、どのような分子機構で1分子のRng2CHDが多数のアクチン分子を阻害できるのか分かっていなかった。本研究において、ミオシンとRng2CHDはそれぞれアクチン線維に構造変化を誘起するが、そうしたアクチン線維の構造変化はヒステリシスを持つことが示された。この結果は、Rng2CHDの一過的な結合によって引き起こされたアクチン分子の構造変化が長時間持続するため、Rng2CHDが異なるアクチン分子と結合解離を繰り返すことによって結果的に大半のアクチン分子が阻害状態になるためミオシンによる運動が強く阻害されるという「メモリー効果モデル」支持する。

  • アクチンフィラメントの協同性の物理機構

    2019年  

     概要を見る

    Actin filaments (AF)は、真核細胞内で多様な機能を果たしているが、われわれはAF内部のactin分子間の長距離に及ぶ協同的な構造多型性、すなわち長距離アロステリーがこうしたAFの多機能性に寄与すると考え、これを支持する状況証拠を提示してきた。本特定課題では、上記と関連していくつかの新知見が得られたが、紙面の都合から以下にそのうち二項目を記す。*DrebrinはAFと協同的に結合しクラスターを形成することが先行研究で示されていたが、われわれは全反射蛍光顕微鏡を用いて、Drebrinクラスターがアクチンフィラメントにそって成長する様子を初めて可視化することに成功した。今後は、構造変化に方向性があるかを検討する。*Phalloidinに結合させたローダミンの蛍光が、AFの構造変化を反映して変化し、さらに構造変化がAFにそって伝播する様子をリアルタイムで観察することに成功した。

  • 生体運動システムの自律性とデザイン原理の総括

    2018年   山崎陽祐, 湯本天嗣

     概要を見る

    アクチン繊維に、in vitroで張力をかけたりアクチン結合タンパク質が結合すると、繊維の原子構造が長距離にわたって協同的に変化し、さらに、それによってアクチン結合タンパク質との親和性が制御される(長距離アロステリー)。これによってアクトミオシンの力発生や、コフィリンによるアクチン系細胞骨格の再構築も調節を受けるため、アクチン繊維の長距離アロステリーが細胞運動の自律的制御に重要な機能を果たすのではないかと考えている。本研究では、(1)in vitroで、2次元面上に押しつけたアクチン繊維のメッシュワークにミオシンIIとコフィリンを加えたときに、長距離アロステリーに基づいてコフィリンとミオシンIIが自発的に分かれた領域を形成することを見いだした。(2)われわれが以前同定した酵母細胞内でのアクチン機能を阻害するアクチン変異と、その抑制性変異を用いて、標的となるアクチン結合タンパク質の生化学的な探索を進めた。

  • アクチン繊維の長距離アロステリーの生理的意義と構造的基盤

    2018年   鈴木真耶, 早川悠貴, 小野知徳, 金田龍一

     概要を見る

     われわれは、アクチン繊維は、協同的に構造変化することで、多様な機能を発揮すると提唱してきた。このテーマに関して二つの研究を行った。研究1:Rng2という収縮環タンパク質のアクチン結合ドメイン(Rng2CHD)がアクチン繊維に結合し、ミオシンIIとの運動を強く阻害する。このとき、フィラメント中のアクチン分子20 分子に対してわずか1分子のRng2CHDが結合した状態で運動速度が90%阻害され、極めて強い協同性が示された。高速原子間力顕微鏡により、Rng2CHD存在下でのアクチン繊維の構造変化がリアルタイム観察された。研究2:アクチン繊維と強く結合するphalloidinのrhodamine標識化合物(RhPh)を用いて、Rhの蛍光強度を指標に、アクチン繊維の構造変化のリアルタイム観察系の確立に努めた。その結果、二つの異なる時定数でアクチン繊維の構造が協同的に揺らぐことが示唆された。

  • 古細菌アクチンの精製と解析 - 真核生物アクチンの起源を求めて-

    2018年   柳瀬雄太

     概要を見る

     真核生物の起源は極めて興味深い生物学的な問題であるが、真核進化にともなって生じた様々な変化のうち、配列保存性が高くかつ多機能という真核型アクチンの出現は、その最初期に起きた重要なイベントであろうと推測した。 またこの点に実験的にアプローチするため、真核生物の起源に近いと考えられる古細菌Lokiarchaeumのアクチン(Loki actin)の構造と機能解析を行っている。その前提としてLoki actinを生化学的に調製する必要があるが、われわれは先行研究において、大腸菌を用いた発現系でLoki actinを発現しても、正しく折りたたまれないことを見いだしていた。そこで真核細胞発現系として細胞性粘菌をを用いたところ、発現レベルが低く、生化学的解析に必要な量を得ることができなかった。この結果を受け、昆虫細胞の発現系を試みることとし、発現用のバキュロウイルスを作成した。

  • 古細菌アクチンの精製と解析-真核生物アクチンの起源を求めて-

    2016年  

     概要を見る

    配列が多様でそれぞれが単機能という原核アクチンから、配列はほぼ単一でありながら多機能という真核アクチンがどのように進化したのかは、真核生物の起源を考えるうえできわめて興味深い問題である。最近、真核生物の起源に近いと考えられる古細菌Lokiarchaeumのゲノムが発見され、Loki actinの配列は、原核アクチンでありながら真核アクチンのそれときわめて類似していた。そこで本研究では、このLoki actinを組換えタンパク質として調製し、その性質を生化学的、生物物理学的に解析することで、原核アクチンと真核アクチンの関のギャップを埋め、真核アクチンの起源を探る。予備的実験において、大腸菌を用いた発現系でLoki actinを発現しても不溶性となってしまうことが分かっていたが、本研究で細胞性粘菌を使った真核細胞発現系を用いたところ、可溶性タンパク質として精製することができた。今後はさらに発現系をリファインし、生化学的・構造生物学的な解析に進みたい。&nbsp; &nbsp;&nbsp;

  • 蛍光顕微鏡によるアクチンフィラメントの構造ゆらぎのリアルタイム観察

    2016年  

     概要を見る

    Fluorescence intensity of rhodamine (Rh) isaffected by its local environment, and the fluorescence intensity of Rh-phalloidin(RhPh) is enhanced by binding to actin filaments. During the initial phase ofthe binding process of RhPh to actin filaments, fluorescence intensity wasnon-uniform and punctate along the filaments. Time-lapse observationdemonstrated that punctate fluorescence intensity fluctuates over time, and moreover,the fluorescent puncta apparently moved along the filaments. When filamentsfully bound with RhPh were partially bleached, the fluorescence was again punctate,and the puncta moved along the filaments. When filaments were labeled by an Alexadye on the surface of each actin molecule by chemical crosslinking, fluctuationof fluorescence intensity was suppressed. The intensity fluctuations of punctatefluorescence of actin-bound RhPh were also suppressed when filaments were fixedwith glutaraldehyde, suggesting that the intensity fluctuations reflectconformational dynamics of actin filaments that affect the local environment ofRh.&nbsp;

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